Total Submitted Abstracts: 286


Submitted for: Graduate Student Presentation Okay to Post to Web: Yes

 

  Molecular mechanisms of innate immune activation and accelerated liver pathology in HCV/HIV coinfection
  Presenter: Lauren Aarreberg
  All Authors:Lauren Aarreberg, Amina Negash, Michael Gale, Jr.
  University of Washington
   
 

Hepatic inflammation is a hallmark feature of pathogenesis during HCV/HIV coinfection, but the processes that propagate this outcome are not understood. Coinfection results more commonly in inflammatory liver disease than monoinfection of either virus, with a quicker progression to end-stage disease. HCV causes significant liver inflammation by triggering potent inflammasome activation and IL-1β release after phagocytic uptake by hepatic macrophages. Also known as “Kupffer cells”, these CD4+ cells are targets of HIV infection. Here, we are evaluating differential modulation of innate immune signaling in a hepatocyte/macrophage co-culture model of HCV/HIV coinfection. Furthermore, investigations of the macrophage as a hub of inflammatory signaling have revealed a hereto unappreciated role for IL-1 signaling in the regulation of interferon and antiviral response genes. We therefore hypothesize that the pro-inflammatory signaling molecule IL-1β acts to synergize innate immune activation in response to viral insult in the liver, and that the resulting maladaptive, non-resolving response to chronic infection underlies hepatic injury and accelerated disease in HCV/HIV coinfection.

   



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  Interleukin-1[b] induces innate antiviral gene expression via activation of IRF3 and STAT signaling
  Presenter: Lauren Aarreberg
  All Authors:Lauren Aarreberg, Courtney Wilkins, Hilario Ramos, Adriana Forero, Justin Roby, Michael Gale, Jr.
  University of Washington
   
 

Defense against microbial infection requires rapid detection of the pathogen and an appropriate cellular response. This process is driven by the recognition of pathogen- or danger-associated molecular patterns (PAMPs or DAMPs) by pattern recognition receptors to initiate intracellular signaling pathways that culminate in the expression of host defense molecules. These may include cytokines like interferon (IFN) and/or interleukin-1β (IL-1β). IFN acts to induce hundreds of genes that promote an antiviral state in infected and bystander cells. IL-1β, a product of inflammasome activation, is a potent inducer of NF-κB-responsive genes, whose products mediate inflammatory and immunomodulatory actions. IL-1 receptor (IL-1R) signaling also induces activation of the kinase TBK1, though a physiological role for this kinase in IL-1R signaling has not been elucidated. Here we report that exogenous IL-1β induces TBK1-mediated IRF3 activation and IRF3-dependent innate immune response genes in mouse myeloid cells, and in human myeloid and epithelial cells. IL-1β-induced IRF3 activation is dependent upon the essential DNA sensing pathway adaptor, stimulator of interferon genes (STING). We provide evidence that IL-1β-mediated STING activation likely occurs through the recognition of mitochondrial DNA in the cytosol. We demonstrate that exogenous IL-1β potentiates PAMP-induced IFN production and STAT signaling to amplify the innate immune response. Additionally, we show that IL-1R is required for maximal IRF3-mediated IFN production and immune activation in response to various PAMPs and RNA virus infection. These studies identify a new role for IL-1R signaling in the onset and/or enhancement of the actions of IFN and IFN-stimulated genes, with exciting new implications for the role of STING in integrating antiviral and inflammatory cues for host defense at barrier surfaces.

   



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  All Authors:abwtua abwtua, abwtua
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  Screening of TLR4 inhibiting peptides using phage display technique
  Presenter: Asma Achek
  All Authors:Asma Achek, Hyeon-Jun Shin and Sangdun Choi
  Department of Molecular Science and Technology, Ajou University, Suwon, Korea
   
 

Toll-like receptors (TLRs) are type I transmembrane proteins that are among the most important receptors in innate immunity. They play a crucial role in inflammatory response upon infection. Since the activation of these receptors protects the body from invading pathogens, several studies have focused on the screening of TLRs-targeting molecules. In the present study, we screened multiple libraries of random peptides against TLR4/MD2 complex and identified several peptides. Among these, one peptide (Ajou-2) that antagonizes TLR4 can be a good candidate for TLR4-mediated diseases. The antagonistic effects of Ajou-2 were confirmed through the inhibition of LPS-induced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, the suppression of MAPKs (mitogen-activated protein kinases) activation as well as the inhibition of pro-inflammatory cytokines (interleukin 6 and tumor necrosis factor-α). This peptide also inhibited the oxidative stress mediators by downregulating inducible nitric oxide synthase and cyclooxygenase 2 and decreasing the intracellular production of NO and reactive oxygen species. This peptide did not affect TLR2/1, TLR2/6 and TLR7/8/9-mediated inflammatory responses in RAW264.7 cells when co-treated with their cognate ligands. Our results showed that Ajou-2 may be a promising and specific therapeutic option for TLR4-mediated inflammatory diseases.

   



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  [b]2 adrenergic receptor signaling prevents hyperinflammation through early release of IL-10
  Presenter: Didem Agac
  All Authors:Didem Agac, Leonardo D. Estrada, J. David Farrar
  University of Texas Southwestern Medical Center
   
 

Primary and secondary lymphoid tissues are innervated by catecholaminergic nerve endings, which allow the sympathetic nervous system to communicate with immune cells through the release of norepinephrine (NE). Additionally, immune cells express adrenergic receptors, which can be activated by NE. We found that NE can suppress pro-inflammatory cytokine secretion from bone marrow-derived macrophages and dendritic cells in response to TLR signaling in vitro. NE exerts its effects by signaling through the beta2-adrenergic receptor (ADRB2). This suppressive effect depends on the early induction of the anti-inflammatory cytokine IL-10, where neutralizing autocrine IL-10 signaling restores inflammatory cytokine secretion. We assessed the in vivo role of this pathway. Upon challenging with sub-lethal LPS injection, ADRB2 knockout (KO) animals succumbed to death. Additionally, we have observed dysregulation of serum cytokines, where the serum levels of TNFα was significantly higher in ADRB2 KO animals. On contrary, the serum levels of IL-10 was significantly lower in ADRB2 KO animals. Additionally, the lethality in the ADRB2 KO animals can be rescued by administering exogenous IL-10. These observations suggest that ADRB2 signaling is critical for controlling inflammation. We have shown that ADRB2 signaling is a negative regulator of TLR signaling and suggest that this pathway plays an important role in immune activation and homeostasis. Understanding this pathway will provide new insights to crosstalk between nervous and immune systems. Research supported by R01-AI056222-07 and AAI Careers in Immunology Fellowship Program

   



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  Differential effect of Leukocyte associated immunoglobulin-like receptor 1 (LAIR1) ligation by C1q in a dendritic cell based Th1 response and a monocyte based Th17 response
  Presenter: Vrushali Agashe
  All Authors:Vrushali Agashe, Jeremy Sullivan, Ewa Jankowska-Gan, William Burlingham
  University of Wisconsin Madison
   
 

Background: Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR1) is a transmembrane receptor expressed by a variety of cells of the immune system. LAIR1 ligands include collagens and collagenous domain containing proteins such as C1q. LAIR1 ligation is though to induce inhibitory signals in Natural Killer cells, T cells and B cells. Recent evidence from our lab demonstrates that LAIR1 ligation through its natural ligand inhibits a Th1 (tetanus) response but augments a Th17 (Collagen type V) based response. Hypothesis: The differential regulation of the Th1 and Th17 response lies in the selective use of APCs: dendritic cells (Th1) vs monocytes (Th17). Results and Conclusions: Pre-incubation of PBMCs with C1q, led to an increase in the number of monocytes expressing IL1(b) and TNF(a), after an overnight stimulation with Collagen Type V, whereas stimulation with tetanus failed to induce either cytokine. Current experiments are underway to study the effect of C1q on DCs. Subsequent experiments will also aim to look at the effect of C1q on Th1 and the Th17 cells.

   



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  All Authors:aguouhmhjx aguouhmhjx, aguouhmhjx
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  The impact of TIGIT on innate and adaptive responses during Toxoplasma gondii infection
  Presenter: Daniel Aldridge
  All Authors:Daniel Aldridge, Anthony Phan, Rene de Wall Maefyt, Christopher Hunter
  University of Pennsylvania
   
 

Resistance to the parasite Toxoplasma gondii is mediated by NK and T cell production of IFN-γ, but failure to contract this response can lead to sever immunopathology. While cytokines, such as IL-10 and IL-27, are critical in limiting the immune response during infection, the role of inhibitory receptors in this process is unclear. The inhibitory receptor TIGIT is expressed on both NK and T cells during infection, while its ligand, CD155, is expressed on monocytes, macrophages, and dendritic cells. Furthermore, the co-stimulatory molecule CD226, which can compete with TIGIT in cis for CD155 binding, is also expressed on NK and T cells during infection. This suggests that the TIGIT signaling axis may be operational and relevant in limiting NK and T cell responses during toxoplasmosis. To investigate this, wildtype (WT) and TIGIT KO mice were infected with T. gondii. In this model, WT and TIGIT KO mice show comparable parasite burdens and similar NK and T cell responses at one week and four weeks post-infection. However, eight weeks post-infection TIGIT KO T cells show enhanced cytokine responses to the parasite. This suggests that during early stages of infection, TIGIT-independent mechanisms restrain the immune response, and a hierarchy of suppressive mechanisms may then exist during infection, with TIGIT eventually playing a role at later stages.

   



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  Delineating the Distinct Cellular and Molecular Properties of Myeloid-Derived Suppressor Cells in Breast Cancer using Single Cell RNA Sequencing
  Presenter: Hamad Alshetaiwi(1,2,3)
  All Authors:Hamad Alshetaiwi(1,2,3), Nicholas Pervolarakis(4,3), Laura L. McIntyre(5), Dennis Ma(1,3), Quy Nguyen(1,3), Kevin Nee(1,3), Jan Rath(1), Katrina Evans(1,2,6), Leona Torosian(7), Anushka Silva(7), Craig Walsh(5), Kai Kessenbrock(1,2,3)
  (1)Chao Family Comprehensive Cancer Center, (2)Institute for Immunology, (3)Department of Biological Chemistry, School of Medicine, (4)Center for Complex Biological Systems, (5)Department of Molecular Biology & Biochemistry, (6)Department of Physiology & Biophysics, (7)Health Sciences and Pharmaceutical Sciences Schools. University of California, Irvine.
   
 

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent immune suppressive activity. These cells accumulate in pathological conditions such as cancer. MDSCs in breast cancer promote angiogenesis, tumor invasion and metastasis. In addition, MDSCs regulate immune response by suppressing T cell (CD4/CD8) proliferation and INFγ production. Multiple mechanisms of immune suppression by MDSCs exist. For instance, the production of reactive oxygen and nitrogen species (ROS, NO) and arginase (Arg). MDSCs, normal neutrophil granulocytes, and monocytes are defined by the same flow cytometry markers, CD11b+Gr1+. MDSCs can be further classified into granulocytic MDSC (G-MDSC) and monocytic MDSC (M-MDSC). Here, we use a breast cancer mouse model (MMTV-PyMT) to study the cellular and molecular properties of MDSCs in single cell resolution. To test MDSCs capacity to inhibit immune responses, CD11b+Gr1+ cells from tumor-bearing PyMT mice and control WT mice were sorted by fluorescence-activated cell sorting (FACS) from bone marrow, lung, and spleen, and then subjected to a T cell activation assay in co-culture with T cells. We found that exclusively CD11b+Gr1+ cells from spleen of tumor-bearing PyMT mice significantly suppresses CD4 and CD8 T cell proliferation and INFγ production in CD8 T cells, whereas CD11b+Gr1+ cells from bone marrow and lung showed no effect on T cell activation. These data indicate that CD11b+Gr1+ cells sorted from spleen from tumor bearing mice are MDSCs because of their ability to suppress T cell function. Ongoing experiments utilize single cell RNA sequencing (ScRNAseq) to directly compare breast cancer derived MDSCs to the respective cell populations harvested from normal, non-tumor bearing animals. Results revealed a distinct G-MDSC cluster from normal neutrophils and a distinct M-MDSC cluster from normal monocytes. Our studies may provide crucial insights into the biology of MDSCs, which may ultimately form the basis for novel marker and therapeutic avenues to improve cancer immunotherapy.

   



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  The Role of the GPI Anchor in B-cell Mediated Immune Responses to Toxoplasma gondii
  Presenter: Julia Alvarez
  All Authors:Julia Alvarez, Scott P Souza, Jessica N Wilson, Kirk DC Jensen
  UC Merced
   
 

The development of an effective treatment or vaccine against parasitic infections like Toxoplasma gondii requires more understanding about the battle between host and pathogen. What allows for some mouse strains to survive while others succumb? What antigens elicit the strongest antibody response? We discovered through a genetic screen of recombinant inbred resistant A/J and susceptible C57BL/6J mice a gene that correlates to survival of secondary infections of T. gondii. This gene, Nfκbid, is an atypical regulator of NF-κB. Mice lacking Nfκbid have complete loss of the B-1 cell compartment, diminished antibody responses, and 100% death in secondary infections of T. gondii. This led us to investigate the antibody responses to T. gondii. Since B-1 cells are responsible primarily for T-Independent antigen responses, we investigated antibody reactivity to non-protein antigens of T. gondii, like glycophosphatidylinositols (GPI) that cover the surface of most protozoan parasites. We have found that antibody reactivity to known GPI anchored proteins is lost after cleavage of the GPI lipid moiety with PI-PLC treatment, and that the resistant A/J mice have higher reactivity to T. gondii GPI’s. We have created T. gondii mutants of genes predicted to modify the side-chain of the GPI to further investigate its role, and that of various glycoforms, in antibody responses to T. gondii and parasite virulence. T. gondii GPI’s are known to trigger TLR2 and TLR4 activation, and we are also examining the role of TLR’s in the production of protective antibody responses, and in survival to secondary infections of T. gondii. We have found that similar to the Nfκbid null mice, parasite specific IgG1 responses are diminished in TLR2 -/- and TLR4 -/- mice. The model we are entertaining is that TLR-driven B cell responses are required for antibody reactivity to GPI-anchored proteins of T. gondii. Ongoing results will be discussed.

   



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  The gut commensal Akkermansia muciniphila induces homeostatic T-dependent IgG1 antibodies and T follicular helper responses
  Presenter: Eduard Ansaldo
  All Authors:Eduard Ansaldo, Leianna Slayden, Krystal Ching, Natalie Wolf, Meghan Koch, James Moon, Gregory Barton
  University of California, Berkeley
   
 

Despite the great diversity of commensal antigens present in the lumen of the intestine, only a small subset of commensal species have been identified to date that lead to homeostatic adaptive immune responses. T-dependent IgA and commensal-specific CD4 T-regulatory and TH17 responses target a small subset of identified commensal species, while T-independent IgA and IgG antibodies and other innate immune effector functions target a more diverse fraction of the microbiota. Understanding which commensal species lead to adaptive immune responses at steady state, the signals that are important in their induction and regulation, how these responses can change in the context of infection or inflammation and their effects on host physiology remain important questions in the field. Here we unravel a new arm of the T-dependent antibody response against commensal bacteria, T-dependent IgG1, and show that Akkermansia muciniphila is one of the main targets of this response. T cell responses to A. muciniphila are mostly limited to T follicular helper cells in the Peyer’s patches at steady state, but the response can be skewed towards the induction of pro-inflammatory T cell fates that home to the lamina propria in certain contexts. Adaptive immune responses to A. muciniphila are thus distinct compared to the homeostatic immune responses against Segmented Filamentous bacteria (SFB) or Helicobacter spp., other commensal species that have been shown to induce T cell responses in the intestine. Akkermansia muciniphila has been previously shown to positively influence host metabolism and responses to checkpoint blockade therapy, and this work could help reveal the mechanisms by which it mediates these effects on host physiology.

   



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  To Model Immune Homeostasis Under Normal and Autoimmune Conditions
  Presenter: Jonathan Anzules
  All Authors:Jonathan Anzules, Kristen Valentine, Genevieve Mullins, Katrina Hoyer
  University of California Merced
   
 

Autoimmune disease is a phenomenon where an organism's immune system identifies parts of the body as foreign and targets it for destruction. There are several tolerance mechanisms that normally prevent self-reactive immune responses. However, this system sometimes fails and the progression of autoimmune disease is still largely unclear. It is theorized that autoimmune disease is initiated and perpetuated by a disruption to the homeostatic elements in the immune system. Understanding these homeostatic dynamics is crucial to understanding how autoimmune disease develops and is perpetuated. Cells are constantly interacting with each other, and the result of these conversations leads to increased survivability of a cell, stronger immune response, suppression, or change in activity. These conversations can be generalized and scaled up to a dynamic system of differing population interactions. Population dynamics can be represented by a system of differential equations. To further understand the homeostatic dynamics involved in autoimmunity, I propose to use mathematical modeling and experimental testing to define homeostatic constraints in healthy and autoimmune disease mice. The mathematical model will simulate the dynamics between naive CD4 T cells, activated CD4 T cells, T regulatory cells (Tregs), and the cytokine IL-2 in a developing system. I hypothesize that our model can predict whether autoimmune disease will develop when given the age and cellular population numbers.

   



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  Interferon gamma from acute Toxoplasma gondii infection during pregnancy impacts hematopoietic function in the fetus
  Presenter: April Apostol
  All Authors:April Apostol, Anna Beaudin, Kirk Jensen
  UC Merced
   
 

During fetal development, rapidly expanding populations of stem and progenitor cells generate the foundation of the mature immune system. In adulthood, infection can drive cytokine-mediated inflammation that directly influences hematopoietic stem cell (HSC) function and differentiation, but much less is known about the fetal immune response to maternal infection during pregnancy. Here, we investigated the fetal hematopoietic response to maternal Toxoplasma gondii (T. gondii) infection. T. gondii is an intracellular parasite that elicits Type II, IFN?-mediated maternal immunity to prevent vertical transmission and promote parasite clearance. The production of excessive IFN? from T. gondii infection has dire consequences for the developing fetus, such as lowered birth weights and premature abortion, but the effects to the developing immune system and the signals that mediate these interactions has not previously been studied. Our examination of fetal hematopoiesis in response to maternal T. gondii infection reveals that fetal HSCs proliferate in response to maternal infection. Additionally, we observe significant and robust proliferation and expansion of Flk2+ multipotent progenitors (MPPs), which are known to be lymphoid-biased in adults. The expansion of presumably lymphoid-biased progenitors in response to infection during the fetal period is in direct contrast to the pervasive expansion of myeloid progenitors in response to adult infection. Additionally, we found that maternal-derived IFN-y crosses the fetal-maternal interface and may play a significant role in determining fetal HSC response to maternal infection. Ongoing experiments explore the functional outcomes of fetal HSCs following aberrant maternal inflammation to define how changes to their steady-state lineage output can influence the trajectory of the immune system across the lifespan.

   



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  Maternal immune perturbation expands developmentally-restricted HSCs and progenitors in offspring
  Presenter: April Apostol
  All Authors:April Apostol, Anna Beaudin
  University of California Merced
   
 

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  Antigen-specific CD4+ T cell differentiation in murine malaria infection
  Presenter: Nicole Arroyo
  All Authors:Nicole Arroyo, Marion Pepper
  University of Washington
   
 

Malaria is a parasitic disease that kills approximately one million people a year. Although efforts to induce protection from malaria through vaccination have been ongoing for over sixty years, none have produced long-lasting immunity in malaria-endemic areas. This is likely due to a lack of knowledge of the immunological mechanisms required for protection. While it is clear that both CD4+ T cells and B cells are important contributors to immunity against the blood stage of infection, little is known about their specific functional contributions. We have used a murine model of Plasmodium infection, P. yoelii, to gain an understanding of the lymphocyte populations that form and are required for protection in both a primary and secondary infection. Using transgenic parasites and MHC class II tetramers to track CD4+ T cell responses during malaria, we have found that early in the response, the vast majority of CD4+ T cells that form are T follicular helper (Tfh)-like cells, despite the lack of a germinal center. We are currently using these tools to understand whether these cells are pathogenic or protective and how interactions with B cells drive their formation and expansion. Preliminary data further suggests that interactions between CD4+ T cells and B cells may be pathogenic during the early stage of the infection, but are required for parasite clearance. An effective vaccine may need to stimulate both the formation of antigen-specific T effector (Teff) and Tfh cells in order to provide effective immunity against rechallenge.

   



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  Antigen-specific CD4+ T cell differentiation in blood-stage malaria
  Presenter: Nicole Arroyo
  All Authors:Nicole Arroyo, Marion Pepper
  University of Washington
   
 

Malaria is a parasitic disease that kills approximately one million people a year. Although efforts to induce protection from malaria through vaccination have been ongoing for over sixty years, none have produced long-lasting immunity in malaria-endemic areas. This is likely due to a lack of knowledge of the immunological mechanisms required for protection. While it is clear that both CD4+ T cells and B cells are important components of the immune response to the blood stage of infection, little is known about their specific functional contributions. We have used a murine model of Plasmodium infection, P. yoelii, to gain an understanding of the lymphocyte populations that form and are required for protection. Using transgenic parasites and MHC class II tetramers to track CD4+ T cell responses during malaria, we have found that early in the response, the vast majority of CD4+ T cells that form are T follicular helper (Tfh)-like cells, despite the lack of a germinal center at this time point. Interestingly, in the absence of B cells, the CD4+ T cells alternatively develop a significant T effector (Teff) phenotype that appears to be primarily comprised of Th1 cells. Interestingly, while there are stark differences in the type of CD4+ T cells generated in the presence and absence of B cells, parasitemia remains comparable in both WT and uMT mice for approximately 1.5 weeks post-infection, at which point mice deficient in B cells are unable to control the parasite growth. We are currently investigating if CD4+ T cells are responsible for the parasite control at this early stage, how they may be contributing to this control, and which aspects of the Th1 and Tfh responses are important for maintaining low parasitemia. An effective vaccine may need to stimulate both the formation of antigen-specific Teff cells to manage parasite burden until Tfh cells have coordinated effective antibody responses by B cells in order to provide effective immunity against re-challenge.

   



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  Pathologic role of dual receptor T cells in chronic graft-versus-host disease.
  Presenter: Amritha Balakrishnan
  All Authors:Amritha Balakrishnan, Burhan Jama, Nicholas Gloude, Gerald Morris
  UCSD
   
 

Chronic graft versus host disease (cGVHD) is a complication resulting from HLA matched hematopoietic stem cell transplant (HSCT) that manifests as debilitating multi-organ immune pathology. Currently the mechanisms driving disease and the antigens recognized by the activated T cells remain unknown. We have previously shown that T cells naturally expressing two TCRs have disproportionate reactivity to auto and allo antigens and play a role in acute graft versus host disease (aGVHD). Based upon these findings we hypothesized that dual receptor T cells play an important role in the development of cGVHD and that these cells arise due to impaired thymic selection. To study this, we examined by flow cytometry T cells expressing two receptors in patients with cGVHD (n=5) 1.5 - 4 years post-transplant. Dual receptor T cells are increased in number (238.2 + 56.59 /103 T cells, mean+ SEM), and have an activated phenotype as compared to cells from healthy controls (n=5, 79 + 6.4 /103 T cells, P=0.028). To determine the origin of these cells we used a T cell depleted syngeneic HSCT mouse model and found a significant increase in the numbers of dual TCR T cells in post-selection thymocytes and in peripheral T cells of transplant mice. (P <0.05). The data indicates that defects in thymic selection result in the development of T cells with two receptors and these cells largely contribute to development of cGVHD. Using a novel single cell sequencing approach we will identify the cytokine and transcription profile of these cells. Furthermore, these results provide an opportunity to investigate the antigenic specificity of dual TCR T cells and mechanisms resulting in cGVHD by using single cell cloning techniques.

   



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  B7H6, a tumor biomarker, not only activates NK cells, but also demonstrates surprising inhibitory effects on tumor cell line proliferation, and migration
  Presenter: Nehla Banu
  All Authors:Nehla Banu, Riera-Leal A, Gutierrez-Silerio G, Ortiz-Lazareno PC, Bastidas-Ramirez BE, Bueno-Topete MR, Haramati J, Del Toro Arreola S
  Universidad de Guadalajara; Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Mexico
   
 

B7 homologue 6 (B7-H6) is a ligand for the NK cell activating receptor NKp30. Current research suggests that this ligand is selectively expressed on several cancer types including melanoma, prostate and breast cancer, thus making it an important molecule to be studied further. It is known that B7H6 binding to NKp30 results in NK cell mediated cytotoxic killing of B7H6–expressing tumor cells. However, it is also interesting to note that the intracytoplasmic domain of B7H6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) along with Src homology 2 (SH2) and src homology 3 (SH3) motifs, suggesting that this ligand may be involved in intracellular inhibitory signalling pathways. To investigate this idea, we stimulated B7H6 positive tumor cell lines with recombinant soluble NKp30 receptor, and evaluated possible inhibitory effects using WST-1 for cell viability, the impedance RTCA system for cell proliferation, the scratch method for cell migration, and cytometry for apoptosis. Notably, we observed that the sNKp30:B7H6 interaction significantly decreased tumor cell proliferation, cell viability and migration rate. We also showed an elevated level of soluble B7H6 in the cell supernatant, which may be a strategy of escaping NK cell mediated recognition and activation. In conclusion, we find that the ligand B7H6 may have a previously unknown inhibitory effect on tumor cells and may prove an interesting target for cancer therapy.

   



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  Translation inhibition by bacterial pathogens: Links to inflammation
  Presenter: Kevin C. Barry
  All Authors:Kevin C. Barry, Mary F. Fontana, Jonathan L. Portman, Aisling S. Dugan, Nicholas T. Ingolia and Russell E. Vance
  University of California, Berkeley
   
 

Legionella pneumophila is an intracellular bacterial pathogen that is the cause of a severe pneumonia in humans called Legionnaires’ Disease. L. pneumophila infects alveolar macrophages in the mammalian lung and requires a bacterial type IV secretion system (T4SS; dot-apparatus) that injects effector proteins into the host cytosol for virulence. Macrophages distinguish infection with T4SS+ (virulent) and T4SS- (avirulent) bacteria, and mount several distinct immune responses to virulent bacteria. Virulent L. pneumophila injects 5 bacterial effectors that block host translation. Host translation inhibition is increasingly being recognized as a pathogen-encoded activity that can lead to specific innate immune sensing of pathogens. Here we show that inflammatory signaling by the cytokine IL-1α, through the interleukin-1 receptor (IL-1R), protects the host during virulent L. pneumophila infection and we link the production of IL-1α to pathogen-induced blockade of host translation. We describe experiments aimed at understanding the molecular mechanism of host translation inhibition after infection with virulent L. pneumophila and undertake global analysis of translation in infected macrophages using ribosome profiling. These experiments define the role of translation regulation on production of inflammatory cytokines after pathogenic infection. Our data suggest that translation inhibition induced by pathogenic infection may be an important pathway that, somewhat paradoxically, induces inflammation and protects animals from multiple bacterial pathogens.

   



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  Elucidating IFN-[g]-induced human inflammasome responses to Legionella pneumophila
  Presenter: Antonia Bass
  All Authors:Antonia Bass, Sunny Shin
  University of Pennsylvania
   
 

Host recognition of intracellular bacterial pathogens results in the formation of a multiprotein complex termed the inflammasome, leading to the recruitment and activation of inflammatory caspases. These caspases promote IL-1 family cytokine secretion and pyroptosis, an inflammatory form of cell death, which are critical for anti-bacterial defense. In mice, interferon-gamma (IFN-γ) is a potent inducer of the canonical caspase-1-dependent and noncanonical caspase-1-independent inflammasomes. In mice, a family of interferon-inducible GTPases known as guanylate binding proteins (GBPs) promote inflammasome responses against a variety of bacteria. The functions of mouse GBPs include promoting rupture of pathogen-containing vacuoles and bacteriolysis of cytosolic bacteria in order to release LPS and other pathogen-derived products into the cytosol, resulting in host recognition and inflammasome activation. In contrast to mice, which possess 11 GBPs, humans have 7 GBPs. The role of these IFN-γ-inducible genes in inflammasome activation in human macrophages is poorly understood. Here, we use Legionella pneumophila, an intracellular gram-negative bacterium, to elucidate the functions of these IFN-γ-induced genes to inflammasome responses in human macrophages. Our findings elucidate aspects of human innate immune response to gram-negative bacterial pathogens and may provide insight into developing therapeutics to prevent gram-negative sepsis.

   



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  The role of IL-1[a] in the response to chronic Toxoplasma gondii infection
  Presenter: Samantha Batista
  All Authors:Samantha Batista, Katherine Still, Carleigh O'Brien, Tajie Harris
  University of Virginia
   
 

Toxoplasma gondii is an intracellular parasite that causes chronic infections, in which the parasite forms cysts inside neurons in the brain. Chronic infection is asymptomatic in healthy hosts, but immunocompromised individuals experience severe, even fatal, disease in which replicating parasites destroy brain tissue. Constant immune pressure is required to maintain control of the parasite throughout chronic infection, leading to the localized recruitment of immune cells. Thus, T. gondii is an excellent model to study neuroinflammation and immune cell recruitment into the CNS. Our lab observes cell death in focal areas of inflammation in the brain, suggesting the possibility of inflammatory molecule release from dying cells. The alarmin IL-1α is expressed in the brain, and is expressed predominantly by brain-resident microglia and infiltrating macrophages, implicating these cell types as potential sources of this cytokine. I have further evidence to suggest that microglia may be the relevant source in this model. Mice lacking IL-1α or its receptor (IL-1R1), but not IL-1β, have defects in monocyte/macrophage infiltration into the brain during chronic infection, as well as increased parasite burden in the brain. This IL-1α-dependent immune response appears to be dependent on pyroptosis, as caspase-1/11-/- mice also display decreased recruitment of myeloid cells into the brain and increased parasite burden. IL-1R1 is expressed largely on brain vasculature, and IL-1R1-/- mice display decreased mRNA expression of the adhesion molecules ICAM-1 and VCAM-1 in the brain. Blockade of the ICAM-1 ligand LFA-1 during chronic infection shows that macrophage recruitment into the brain is partly dependent on this molecule. Together, these data have led me to suggest a model in which during chronic T. gondii infection, IL-1α released from microglia in the brain through pyroptosis acts on the brain vasculature to modulate expression of adhesion molecules, facilitating the infiltration of immune cells to sites of infection.

   



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  IL-1[a] facilitates the entry of immune cells into the brain during chronic Toxoplasma gondii infection
  Presenter: Samantha Batista
  All Authors:Samantha Batista, Carleigh O'Brien, Tajie Harris
  University of Virginia
   
 

Toxoplasma gondii is an intracellular protozoan parasite that causes chronic infections in the brain. Chronic infection is asymptomatic in healthy hosts, but immunocompromised individuals experience severe, even fatal, disease in which quickly replicating parasites destroy brain tissue. Constant immune pressure is required to maintain control of the parasite throughout chronic infection, leading to the localized recruitment of immune cells, making T. gondii an excellent model to study neuroinflammation and immune cell recruitment into the CNS. During chronic T. gondii infection, cell death occurs in focal areas of inflammation in the brain, suggesting the possibility of the release of inflammatory molecules from dying cells. The alarmin IL-1α is expressed in the brain, and its expression is upregulated by chronic T. gondii infection. IL-1α is expressed predominantly by brain-resident microglia and infiltrating macrophages, implicating these cell types as potential sources of this cytokine. Mice lacking IL-1α or its receptor (IL-1R1), but not IL-1β, have defects specifically in monocyte/macrophage infiltration into the brain during chronic infection, as well as increased parasite burden in the brain. IL-1α has been the subject of much less investigation than IL-1β, but may still rely on inflammatory cell death for its release. Indeed, this IL-1α-dependent immune response appears to be dependent on pyroptosis, as caspase-1/11-/- mice phenocopy IL-1R1-/- mice displaying decreased recruitment of myeloid cells into the brain and increased parasite burden. IL-1R1 is expressed largely on endothelial and other stromal cells in the brain vasculature, and IL-1R1-/- mice display decreased mRNA expression of the adhesion molecules ICAM-1 and VCAM-1 in the brain. Together, these data suggest that during chronic T. gondii infection, IL-1α released from a macrophage population in the brain through pyroptosis acts on the brain vasculature to modulate expression of adhesion molecules, facilitating the infiltration of monocytes to sites of infection.

   



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  IL-31 limits allergen-induced lung inflammation
  Presenter: Renate Bauer
  All Authors:Renate Bauer, Theresa Neuper, Harald Schwarz, Muamera Sarajlic, Daniel Neureiter, Helen Strandt, Patrick Suchanek, Stacey R. Dillon, Angelika Stoecklinger, Peter Hammerl, Richard Weiss, Jutta Horejs-Hoeck
  Paris Lodron University of Salzburg
   
 

IL-31 is a Th2 cell-derived cytokine that is closely associated with pruritic (itching) skin inflammation. In fact, there are very promising therapeutic approaches inhibiting IL-31 signaling in people suffering from atopic dermatitis. Treatment significantly reduces pruritus or eczema area and allows patients to escape the vicious circle of itching and scratching. Recent work shows that increased IL-31 levels also correlate with asthma severity and might therefore play a role in allergic asthma or allergic rhinitis. However, the particular role of IL-31 in allergic lung inflammation is still unclear. Therefore, the main aim of this study was to investigate the contribution of IL-31 to allergen-induced lung inflammation. In order to mimic the human situation of enhanced IL-31 expression we used a murine model of IL-31 transgenic (IL-31tg) mice. We sensitized IL-31tg, IL-31 receptor knockout (IL-31RA-/-) and wildtype mice with the timothy grass (Phleum pretense) pollen allergen Phl p 5 and examined lung inflammation. Our data show that acute lung inflammation in terms of cell infiltration, cytokine secretion or mucin expression was clearly reduced in mice overexpressing IL-31 compared to wildtype or IL-31RA-/- animals. This suggests a regulatory or protective role of IL-31 in local lung inflammation.

   



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  Dietary regulation of tumor immune surveillance in the intestine
  Presenter: Semir Beyaz
  All Authors:Semir Beyaz, Khristian Bauer-Rowe, Michael E Xifaras, Stuart H Orkin, Omer H Yilmaz
  Harvard Medical School, Massachusetts Institute of Technology
   
 

Pro-obesity diets such as high fat diets (HFD) increase intestinal tumorigenesis in part by elevating the numbers and function of intestinal stem cell (ISC), which are the cells-of-origin for many intestinal tumors. Although the interaction between tumor and immune system plays a critical role in tumor progression, little is known about how a HFD regulates cancer cell intrinsic and extrinsic immune mechanisms during intestinal tumorigenesis. Here, we found that ISCs express high levels of MHC-II on their cell surface implicating a crosstalk between CD4+ T cells and ISCs in immune surveillance. A HFD led to significant downregulation of genes involved in MHC-II pathway, co-stimulation and inflammatory response in ISCs independent of obesity indicating less inflammatory cytokine milieu in the microenvironment of ISCs in mice fed with a HFD. Intestinal adaptation to a HFD consisted of reduction in bacterial diversity and immune infiltration of the crypt. Pharmacological inhibition of JAK/STAT pathway, antibiotics treatment and germ-free mice recapitulated the effects of a HFD on the intestine. Notably, adenomas that arise in a HFD have less MHC-II expression and loss of MHC-II expression in pre-malignant ISCs increased intestinal tumor incidence compared to MHC-II sufficient counterparts. These findings highlight how a HFD alters the immune recognition of ISCs and how such changes contribute to the development of intestinal tumors.

   



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  Tuft cell-derived acetylcholine regulates intestinal fluid and mucus secretion
  Presenter: Tyler Billipp
  All Authors:Tyler Billipp, Kathleen DelGiorno, Jakob von Moltke
  University of Washington
   
 

Tuft cells are rare chemosensory epithelial cells that initiate the intestinal Type 2 immune response to helminths and certain protists and commensal bacteria. Additional functions of tuft cells are not well understood. The intestinal epithelium maintains a barrier between the resident microbiota and the epithelium by the secretion of fluid and mucus. Secretion is regulated by acetylcholine (ACh) from enteric neurons; however, tuft cells also express the enzyme ChAT required for ACh synthesis and are uniquely positioned in the epithelium to coordinate localized fluid secretion in response to sensing of luminal signals. Here we show that the microbial metabolite succinate induces fluid secretion in the distal small intestine. The succinate-induced fluid secretion depends on tuft cell chemosensing and acetylcholine (ACh) synthesis and is independent of neuronal involvement. Succinate sensing by tuft cells may also regulate goblet cell compound exocytosis of mucus. Our findings suggest a model where intestinal tuft cells sense lumenal signals, such as the microbial metabolite succinate, and signal to neighboring enterocytes and goblet cells via ACh to induce rapid fluid and mucus secretion. Tuft cells might therefore replenish the mucus layer, coordinate the local removal of succinate-producing microbes, and regulate the microbial composition of the distal small intestine.

   



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  High Dimensional Analysis of Myeloid Cells Reveals Regulatory T Cell Control of Lymph Node Priming
  Presenter: Mikhail Binnewies
  All Authors:Mikhail Binnewies, Adriana Mujal, Joshua Pollack, Matthew Krummel
  UC San Francisco
   
 

CD4 T cells have been shown to play a critical role in supporting productive anti-tumor responses. Less clear is how CD4 T cell responses are initiated and organized in the tumor draining lymph node (tdLN). Here we utilize a combination of single-cell RNA sequencing and high dimensional flow cytometry to parse the full extent of heterogeneity present within the tdLN and functionally assess the ability of each antigen presenting cell to prime tumor-antigen specific naïve CD4 T cells. We identify two distinct IRF4-dependent CD11b+ dendritic cell (DC) populations that are uniquely able to prime naïve tumor-antigen-specific CD4 T cells in the tdLN. While the ability to initiate CD4 priming is intact, these CD11b+ DCs are functionally and spatially restrained by regulatory T cells (Treg), where upon Treg depletion, CD11b+ DCs display enhanced migration and improved function, resulting in superior CD4 T cell priming and CD4 Th1 differentiation. Data from human tumors confirms not only the presence of a human equivalent of these two CD11b+ DC populations (BDCA-1+ CD14-/+ DCs) but also a relationship between BDCA-1+ DCs and CD4 Th. These findings reveal the complexity of CD4 T cell activation in the tdLN and highlight potential therapeutic avenues in boosting CD4 T cell function in cancer.

   



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  HPRT impact on immune regulation effects the microenvironment
  Presenter: Eliza Bitter
  All Authors:Eliza Bitter , Michelle H. Townsend, Dallas Larsen, Claudia M. Tellez Freitas, Stephen R. Piccolo, K. Scott Weber, Richard A. Robison, and Kim L. O’Neill
  Brigham Young University
   
 

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a protein in the DNA salvage pathway that is responsible for converting inosine and guanine derivatives to their monophosphate forms. Previously, it has been shown that HPRT is upregulated within several cancer types. Due to significant elevation of HPRT within a large proportion of patient tumors, we evaluated if HPRT upregulation could impact immune infiltration in the tumor microenvironment. To first investigate the effects of HPRT levels on immune regulation, we evaluated the changes in immune gene expression in over 9000 lung squamous carcinoma patients. Patients were ranked according to HPRT high and low expression and analyzed for correlation against anti-inflammatory, anti-inflammatory and pro-inflammatory, and pro-inflammatory genes. Of the 194 total genes evaluated, 68% were negatively correlated with HPRT elevation (31 of 49 anti-inflammatory, 54 of 67 anti-inflammatory and pro-inflammatory, and 47 of 78 pro-inflammatory). Because HPRT upregulation appeared to have a significant impact on immune gene regulation, we evaluated the effects of elevated HPRT on patient survival to determine whether the change in immune regulation impacted overall patient health and longevity. Within the range of high and low HPRT expression, patient survival was compared between the top 30% and the bottom 30% in different cancer types. Among those in the higher 30% of HPRT expression there was a significant decrease in overall survival, with Uterine Corpus Endometrial Carcinoma and Renal Papillary Cell Carcinoma showing the greatest difference in survival (p = 0.001, p = 0.007 respectively). We then reasoned that HPRT is preferentially selected for in the tumor microenvironment for its impact on immune regulation and that due to this characteristic of HPRT, potentially there was a decrease in tumor infiltration of immune cell types. Immune cell infiltration was determined across several different cancer types for B cells, CD8+ T cells, CD4+, T cells, macrophages, neutrophils, and dendritic cells. Results from the analysis indicated an overall significant decrease in immune infiltration when HPRT expression is elevated—macrophages showed the least change in infiltration and CD4+ showed the most significant decrease (p = 0.001) across all cancer types tested. From our findings, we determined that HPRT significantly affects the ability of immune cell types to infiltrate the tumor. Our findings point to the tendency of tumors to select for HPRT in order to potentially promote tumor survival and its involvement in immune regulation.

   



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  Dendritic Cell Maturation Defines Immunological Responsiveness of Tumors to Radiation Therapy
  Presenter: Tiffany Blair
  All Authors:Tiffany Blair, Shelly Bambina, Alejandro Alice, Terry Medler, Jason Baird, Gwen Kramer, Victoria Troesch, Marka Crittenden, Michael Gough
  Oregon Health & Science University
   
 

Radiation therapy is capable of directing adaptive immune responses against tumors by stimulating the release of endogenous adjuvants and tumor-associated antigens. Within the tumor, conventional type 1 dendritic cells (cDC1s) are uniquely positioned to respond to these signals, uptake exogenous tumor antigens and migrate to the tumor draining lymph node (dLN) to initiate cross-priming of tumor reactive cytotoxic CD8+ T cells. Here we report that radiation therapy promotes the activation of intratumoral cDC1s in immunogenic tumors and this process fails to occur in poorly immunogenic tumors. In poorly immunogenic tumors, the adjuvant poly I:C overcomes this failure following radiation and successfully drives intratumoral cDC1 maturation, ultimately resulting in durable tumor cures. Depletion studies revealed that both cDC1s and CD8+ T cells are required for tumor regression following combination therapy. We further demonstrate that treatment with radiation and poly I:C significantly expands the proportion of proliferating CD8+ T cells in the tumor with enhanced cytolytic potential and requires T cell migration from LNs for therapeutic efficacy. Thus, we conclude that lack of endogenous adjuvant release or active suppression following radiation therapy may limit its efficacy in poorly immunogenic tumors, and co-administration of exogenous adjuvants that promote cDC1 maturation and migration can overcome this limitation to improve tumor control following radiation therapy.

   



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  Neutrophils Recruited through High Endothelial Venules of the Lymph Node via PNAd Intercept Disseminating Staphylococcus aureus
  Presenter: Ania Bogoslowski
  All Authors:Ania Bogoslowski, Eugene Butcher, Paul Kubes
  University of Calgary
   
 

There is growing evidence that the lymph node may function as not only an adaptive immune organ but also as a barrier to disseminating pathogens. However, the immune cells involved and the mechanisms by which this occurs has not been fully elucidated. We demonstrate that Staphylococcus aureus breach across skin leads to some migration of the pathogen to the draining lymph node but no further. Profound neutrophil recruitment to the lymph node begins within the first hour after infection and plateaus at 6 hours. How neutrophils reach the lymph node and their role upon arrival is only beginning to be understood. Using intravital multiphoton imaging and the LysM eGFP reporter mouse we have visualized the behavior of neutrophils in lymph node blood vessels and tissue. Almost no neutrophils enter via lymphatics. Neutrophils accumulate within lymph node High Endothelial Venules, and transmigrate into lymph node tissue. Peripheral Node Addressin (PNAd) together with its two ligands, L-selectin and platelet P-selectin, was absolutely critical for recruiting neutrophils via the HEV. Finally, blocking neutrophil recruitment to lymph nodes depleted of resident macrophages results in higher bacterial load in draining lymph node and liver, showing the important role of the lymph node neutrophils in barrier function. Neutrophils actively phagocytosed S. aureus and helped sterilize the lymph node and prevent dissemination to blood and other organs. This study establishes neutrophils as part of the innate defence system of the lymph node.

   



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  TGF[b] neutralization improves humoral immunity to vaccination in aged mice
  Presenter: Christine M. Bradshaw
  All Authors:Christine M. Bradshaw, Jennifer L. Uhrlaub, Natalie Iannuzo, Janko Nikolich-Žugich
  University of Arizona
   
 

Transforming growth factor β (TGFβ) is a pleiotropic cytokine involved in many cellular processes, including regulatory functions during anti-viral immune responses. Previously, we have shown in two models of age-related vulnerability, West Nile virus (WNV) and Chikungunya virus (CHIKV), that over-production of TGFβ correlates with decreased immune function. TGFβ neutralization during acute CHIKV infection decreases disease severity and improves neutralizing antibody titers. Ongoing experiments utilize RepliVAX-WN, a single cycle WNV vaccine, to elucidate the mechanism by which TGFβ blockade improves B cell responses in aged mice.

   



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  Dynamic regulation of LynA and c-Cbl impacts innate immunity
  Presenter: Ben F. Brian
  All Authors:Ben F. Brian, Whitney L. Swanson, Frances V. Sjaastad, Tanya S. Freedman
  University of Minnesota
   
 

Innate immune cells, like macrophages and mast cells, must be able to recognize and rapidly generate a response to diverse pathogenic stimuli. However, these cells must also be able to rapidly extinguish signaling to prevent the development of hyperinflammation that can produce tissue damage. Elucidating how signals are regulated downstream of these pathogen-sensing receptors is crucial to limiting harmful inflammation. We have discovered that the Src-family kinase LynA in macrophages is an important regulator of the antimicrobial response through immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors. We have shown previously that LynA is rapidly poly-ubiquitinated and degraded upon activation in macrophages, which prevents strong downstream signaling and the production of an immune response. Here we show that the ubiquitin ligase c-Cbl is responsible for poly-ubiquitinating LynA, and is a key negative regulator of ITAM signaling in macrophages. While macrophages express high amounts of c-Cbl and efficiently degrade LynA, we have shown that mast cells primarily express the closely related family member Cbl-b which is unable to poly-ubiquitinate LynA to the same extent. Differential expression of Cbl family members results in different signaling outcomes in mast cells and macrophages. We have also previously shown that macrophages stimulated with inflammatory cytokines like IFN-? upregulate LynA to amplify signaling downstream of ITAM-coupled receptors. Here we show that while LynA is upregulated in inflammatory conditions, c-Cbl is downregulated. Conversely, in anti-inflammatory conditions c-Cbl is upregulated and LynA downregulated. This dynamic regulation suggests that by altering the expression of membrane-proximal signaling components, environmental cues direct macrophage responsiveness, priming them for microbial elimination under inflammatory conditions and limiting aberrant inflammatory signaling in response to non-pathogenic stimuli.

   



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  A novel humanized mouse model incorporating non-fetal tissue
  Presenter: Matthew Brown
  All Authors:Matthew Brown, Ian G Norman, Ying Zhou, Wanying Lou, Jeremy A Sullivan and William J Burlingham.
  University of Wisconsin-Madison
   
 

Pre-clinical research into allogeneic tissue transplantation therapies, as well as patient-specific regenerative medicine studies, require animal models that closely mimic the typical patient’s immune response. Humanized mouse models offer a tractable in vivo system to experimentally test immunological hypotheses and therapeutic interventions in the context of a human immune system. In the work presented here, we explore the development of a novel humanized mouse model incorporating non-fetal tissue sources: cryopreserved human pediatric thymus tissue and umbilical cord blood hematopoietic stem cells. This model is equivalent to commonly used human fetal tissue models, such as the “BLT” mouse, with regard to frequency of human cell engraftment and distribution and function of various lymphocyte subsets relevant to transplant tolerance and rejection. Further, there are several benefits of this model over fetal tissue-based models. These benefits are associated with the sheer abundance of pediatric tissue available for mouse creation relative to fetal tissue, which allows for in-depth examination of a variety of parameters that are not feasible with limited quantities of fetal specimens. Additionally, and not insignificantly, this non-fetal tissue based model bypasses the ethical barriers associated with conducting fetal tissue research. Finally, the more developmentally mature tissues used in our model may afford a more accurate representation of clinical patient immune responses in terms of T cell gene expression patterns and function.

   



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  Transcriptional profiling with single-cell resolution reveals distinct fetal, newborn, and adult phenotypes of human naïve T cells and their hematopoietic stem cell progenitors
  Presenter: Daniel Bunis
  All Authors:Daniel Bunis, Rachel Lena Rutishauser, Ventura Mendoza, Chun Jimmie Ye, Trevor D. Burt
  University of California, San Francisco
   
 

During development in utero, many human fetal immune cells are predisposed towards tolerogenic responses. Compared to adult human counterparts, human fetal naïve CD4 T cells exhibit a higher propensity for differentiation into tolerogenic regulatory T cells (Tregs), and human fetal hematopoietic stem and progenitor cells (HSPCs) generate CD4 T cell progeny that are predisposed to Treg differentiation. After birth, naïve T cells must transition toward a more mature state that supports protection against pathogens and cancer. At the time of birth, it is unknown whether adult-associated protective programs are fully expressed, either universally within most naïve T cells, or heterogeneously within just a subset. Here, through single-cell transcriptional profiling of both naïve T cells and HSPCs, from fetal, full-term newborn umbilical cord blood, and adult human sources, we demonstrate that the fetal to adult transition is incomplete across most cord blood naïve T cells and their hematopoietic stem cell progenitors. Cells expressing a fully adult-like transcriptional signature are rare in cord blood, while most naïve T cells and HSPCs exhibit a distinct, intermediate transcriptional state. In complementary bulk transcriptional profiling of naïve CD4 T cells from the same samples, we also determined that the transcriptional state of naïve CD4 T cells in cord blood includes intermediate expression levels of Treg signature genes. Thus, cord blood naïve CD4 T cells likely retain a partial, fetal-like, Treg-predisposition. Our results may provide a novel mechanism that could explain why many vaccines, which were largely designed around elicitation of immunity in the protection-predisposed adult immune system, show reduced efficacy in neonates. Continued analyses of how expression of specific gene programs vary across ages will be carried out with the translational aim of informing specific ways that targeted, tolerogenic or protective, therapeutic responses might be elicited in both cord blood transplant and neonatal vaccination interventions.

   



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  Long-read Single Cell Sequencing reveals RNA Isoform diversity across single B cells
  Presenter: Ashley Byrne
  All Authors:Ashley Byrne, Roger Volden, Charles Cole, Theron Palmer, Anna Beaudin, Camilla Forsberg, Richard E Green, Christopher Vollmers
  University of California, Santa Cruz
   
 

Due to plummeting costs, short-read sequencing technology continues to dominate the field for interrogating whole RNA landscapes. However, due to fragmentation of full-length molecules, heavy computational tools are needed to estimate the transcriptome landscape. Despite this, there still remains limitations in evaluating RNA isoforms in short-read data due to long distant events. We recently employed a long-read cDNA sequencing approach using Oxford Nanopore Technology (ONT) to evaluate single B cells. Additionally, we have improved the Oxford Nanopore base accuracy using a new method called R2C2 (Rolling Circle Amplification to Concatameric Consensus). By combining both approaches we have been able to reconstruct accurate isoform-level transcriptomes across single B cells using our analysis pipeline MandalorION. We have applied our methods to evaluate single B cells and have discovered that much of the RNA isoform diversity observed can be found across B cell specific receptors. This could potentially have serious implications for immunotherapy design specifically for targeting B cell lymphomas.

   



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  Characterization of NK receptor expression on a subset of gamma delta T cells from a malaria endemic region
  Presenter: Perri Callaway
  All Authors:Perri Callaway, Perri Callaway, Lila Farrington, Emma Lutz, Felistas Nankya, Kate Naluwu, Kenneth Musinguzi, Margaret Feeney
  UCSF
   
 

V?9Vd2 T cells (Vd2s) are a subset of gamma delta T cells that recognize small non-peptidic molecules called phosphoantigens (pAgs) via their TCR. P. falciparum, the causative agent of malaria, produces pAgs in the plasmodial apicoplast. During in vivo P. falciparum infection, Vd2s can expand to up to 50% of the circulating T cells in the peripheral blood. However, the frequency of Vd2s has been shown to decrease after repeated malarial episodes and high exposure to P. falciparum. In contrast, expression of the Fc?R, CD16, on Vd2s increases. Previous lab data has indicated other natural killer receptors are also upregulated on Vd2s from individuals with high P. falciparum exposure. Among these is the killer immunoglobulin-like receptor (KIR) family. We found that KIR expression varies between NK cells and Vd2s from the same individual and KIRs are more highly expressed on CD16+ Vd2s. Further, Vd2s expressing KIR2DL4 were more likely to degranulate in response to P. falciparum antigens.

   



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  Non-typhoidal Salmonella Typhimurium ST313 isolates that cause bacteremia in humans stimulate less inflammasome activation than ST19 isolates associated with gastroenteritis.
  Presenter: Sarah Carden
  All Authors:Sarah Carden, Chinyere Okoro, Gordon Dougan, Denise Monack
  Stanford University School of Medicine, Wellcome Trust Sanger Institute
   
 

Salmonella is an enteric pathogen that causes a range of diseases in humans. Non-typhoidal Salmonella (NTS) serovars such as Salmonella enterica serovar Typhimurium generally cause a self-limiting gastroenteritis whereas typhoidal serovars cause a systemic disease, typhoid fever. However, S. Typhimurium isolates within the multi-locus sequence type ST313 that commonly cause bacteremia in humans have emerged in sub-Saharan Africa. S. Typhimurium ST313 are phylogenetically distinct from classical S. Typhimurium lineages such as ST19 that cause zoonotic gastroenteritis worldwide. Previous studies have shown that the ST313 lineage has undergone genome degradation when compared to the ST19 lineage, similar to that observed for typhoidal serovars. Currently, little is known about phenotypic differences between ST313 isolates and other NTS isolates. We find that ST313 isolates invade non-phagocytic cells less efficiently than the classical ST19 isolates that cause gastroenteritis. In addition, ST313 isolates induce less Caspase 1- dependent macrophage death and IL-1ß release than ST19 isolates. ST313 isolates also express lower levels of mRNA of the genes encoding the SPI-1 effector sopE2 and the flagellin, fliC, providing possible explanations for the decrease in invasion and inflammasome activation. The ST313 isolates have invasion and inflammatory phenotypes that are intermediate; more invasive and inflammatory than Salmonella enterica serovar Typhi and less than ST19 isolates associated with gastroenteritis. This suggests that both phenotypically and at the genomic level ST313 isolates are evolving to become more similar to typhoidal serovars and adapting to cause systemic disease in humans.

   



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  Trojan horses: Infected dendritic cells drive hyperdissemination of African Salmonella Typhimurium isolates
  Presenter: Sarah Carden
  All Authors:Sarah Carden, Gregory Walker, Jared Honeycutt Renee Tsolis, Juliana Idoyage, Denise MOnack
  Stanford University
   
 

Innate immune cells serve as one of the host's first lines of defense against invading pathogens. As the early responders to infection different innate immune cells can detect microbes, directly kill the pathogens, and help coordinate the activation of the adaptive immune response. In turn, microbes have evolved ways to exploit or subvert these actions to establish an infection. Salmonella Typhimurium classically causes a self-limiting gastroenteritis in humans. However, in Africa S. Typhimurium sequence type 313 (ST313) are routinely causing systemic disease such as bacteremia and meningitis. In thw mouse model of acute infection,both classical and African isolates have equal Salmonella burden in gut tissues, but the African isolates have a higher Salmonella load at systemic sites such as mesenteric lymph node, spleen and liver. This hyperdissemination phenotype mimics the disease we see in humans. The hyperdissemination is not due to differences in early bloodstream dissemination, cell death due to inflammasome activation, or differences in replication or survival at systemic sites. Analysis of infected cells in the mesenteric lymph node revealed that although macrophages, inflammatory monocytes and neutrophils were all infected, the largest reservoir of Salmonella are migratory dendritic cells (DC). The African Salmonella ST313 isolates infect a higher proportion and number of migratory dendritic cells. The Salmonella secreted effector SseI, inhibits the chemotaxis of dendritic cells towards CCL19. The African ST313 isolate lacks a functional SseI, making it likely that ST313-infected DC migrate better than those infected by a classical isolate expressing the wild-type protein. If we add back a functional SseI, the African isolates no longer disseminate better than classical Salmonella isolates. If we probe these interactions on the host side using CCR7 KO mice, the African isolates are no longer able to hyperdisseminate compared to classical isolates. While dendritic cells are vital to setting up an effective adaptive immune response, in the case of Salmonella they are trojan horses helping Salmonella, especially the African ST313 isolates, disseminate throughout the host.

   



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  Extracellular cGAMP is a cancer cell-produced immunotransmitter that promotes anti-cancer immunity
  Presenter: Jackie Carozza
  All Authors:Jackie Carozza, Volker Böhnert, Khanh Nguyen, Gemini Skariah, Kelsey Shaw, Jenifer Brown, Marjan Rafat, Rie von Eyben, Edward Graves, Jeffrey Glenn, Mark Smith, Lingyin Li
  Stanford University
   
 

Cancer immunotherapy targeting the adaptive immune system results in cures of previously considered terminally ill cancer patients. This remarkable achievement has excited basic researchers and clinicians alike to search for immunotherapies that can treat a higher percentage of patients and more cancers. We now know that an effective adaptive immune response to cancer depends on a robust innate immune response, and that Stimulator of Interferon Genes (STING) is the major innate immune pathway that sparks the anti-cancer immune cascade. The agonist of the STING pathway, 2’3’-cyclic GMP-AMP (cGAMP), is an intracellular second messenger synthesized in response to cytosolic DNA, which is prevalent in cancer cells. Here, we discover that cGAMP is also an extracellular signal that is regulated by its extracellular degradation enzyme ENPP1. After developing a potent ENPP1 inhibitor, we detected that cGAMP is continuously exported from cancer cell lines and can be increased by treatment with ionizing radiation. In mouse tumors, depletion of extracellular cGAMP abolished the curative effect of ionizing radiation. Together, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.

   



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  Identification of Interleukin 40, a novel B cell-associated cytokine
  Presenter: Jovani Catalan-Dibene
  All Authors:Jovani Catalan-Dibene, Monica I. Vazquez, Van Phi Luu1, Sean-Paul Nuccio, Alborz Karimzadeh, Jenna Kastenshmidt, S. Armando Villalta, Irina Ushach, Egest J. Pone, Amanda M. Burkhardt, Paolo Casali, Manuela Raffatellu, Marcela Hernandez, Gina Heller, Peter A. Hevezi and Albert Zlotnik.
  University of California, Irvine
   
 

We have identified a novel mouse and human cytokine. IL-40 is produced in the bone marrow, fetal liver and by activated B cells. Its sequence predicts a small (~27kDa) secreted protein unrelated to other cytokine gene families, that we have called Interleukin 40 (IL-40). IL40 is only present in mammalian genomes, suggesting a role in mammalian immune responses. Accordingly, IL-40 expression is induced in the mammary gland upon the onset of lactation, and an IL-40-/- mouse exhibits reduced levels of IgA in the serum, gut, feces, and milk. Furthermore, the IL-40-/- mouse has smaller and fewer Peyer’s patches and IgA secreting cells. The gut microbiome of IL-40-/- mice also exhibits altered composition, reflecting the reduced levels of IgA in the gut. Pre-B cells are altered in the bone marrow of the IL-40-/- mouse. Taken together, these observations indicate that IL-40 represents a novel B cell associated cytokine, that plays an important role in B cell development and humoral immune responses. IL-40 is also expressed by human activated B cells and by several human B cell lymphomas. The latter observation suggests that it may play a role in the pathogenesis of these diseases.

   



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  Crohn’s Disease-associated microbes produces metabolites to regulate Human T cell responses
  Presenter: Yu-Ling Chang
  All Authors:Yu-Ling Chang, Yu-Ling Chang, Maura Rossetti, Gemalene Sunga, David Casero, Jonathan Jacobs, Jonathan Braun
  University of California, Los Angeles
   
 

The unique microbial composition has been associated with Crohn’s Disease (CD) and the CD4+ T cell activation and plasticity are reported to regulate disease progression. However, it remains unclear how microbes crosstalk with host immune responses and regulate disease biology. This study hypothesized that microbial producing metabolites regulate T cell responses to control CD progression. We generated a list of predicted CD-associated microbial producing metabolites using metagenomic analyses and designed an in vitro screening system to look for metabolites regulating human T cell functions. Twelve out of 100 metabolites (12%) were found regulating human CD4+ T cell cytokine productions, including selectively inhibiting (enhancing) specific subsets of cytokines, or a pan-inhibition (enhancement) of all of tested cytokines. A further investigation suggested a novel mechanism of how ascorbate regulates T cell function. Ascorbate reduced the activated T cells, but not resting T cells, by causing apoptosis in the activated CD4+ T cells. A backward bioinformatics search demonstrates a group of bacteria that produces ascorbate and has been reported positively associated with CD disease progression. Together, this study presented that microbes producing ascorbate regulate host CD4 T cell responses and that is a novel mechanism to explain CD pathogenesis.

   



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  Novel chimeric antigen receptor converts soluble TGF-[b] into a potent T-cell stimulant
  Presenter: ZeNan Chang
  All Authors:ZeNan Chang, Michael H. Lorenzini, Eugenia Zah, Uyen Tran, Yvonne Y. Chen
  University of California, Los Angeles
   
 

A major challenge in adoptive T-cell therapy for solid tumors is the harsh immunosuppressive tumor microenvironment. Transforming growth factor beta (TGF-β) plays a key role in tumor immune evasion. Here, we report a novel TGF-β–binding chimeric antigen receptor that rewires the T-cell response to TGF-β, converting this potent immunosuppressive molecule into a stimulant. TGF-β CAR-T cells overcome TGF-β–mediated dysfunction and trigger TGF-β–specific Th1-type cytokine production as well as T-cell proliferation. We further elucidate mechanistic details of how CARs can be triggered by soluble ligands, as well as methods by which the signaling strength and ligand sensitivity of such CARs can be tuned. Due to the modular nature of the engineered receptor, this work informs future efforts to redirect T-cell responses to extracellular soluble molecules.

   



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  Impact of regulatory T cells on carcinogenesis of oral squamous cell carcinoma
  Presenter: Jaime L. Chao
  All Authors:Jaime L. Chao, Peter A. Savage
  University of Chicago
   
 

Although some head-and-neck squamous cell carcinomas (HNSCCs) are associated with human papillomavirus (HPV) infection, a large proportion of HNSCCs are HPV-negative and associated with heavy smoking, suggesting that HPV-negative HNSCCs are induced by carcinogen exposure. In many human cancers, including HNSCC, regulatory T (Treg) cells are found at elevated densities and are thought to be a major barrier to the generation of robust anti-tumor T cell responses. However, recent studies indicate that Treg cells can serve diverse functions in non-lymphoid sites. To study the functional impact of Treg cells on the development and progression of HPV-negative HNSCC, we utilized an autochthonous mouse model of carcinogen-induced oral squamous cell carcinoma (SCC), in which mice are exposed long-term to the carcinogen 4-nitroquinoline-1-oxide (4-NQO) in the drinking water. Immunohistochemistry and flow cytometric analysis revealed elevated densities of CD3+ T cells and Foxp3+ Treg cells in pre-malignant dysplastic lesions and SCC lesions, suggesting that many lesions exhibit a T cell-inflamed phenotype. Single-cell TCR sequencing demonstrated the presence of recurrent Treg cell clones within carcinogen-induced lesions, suggesting clonal expansion of Treg cells in the tumor microenvironment. Surprisingly, systemic depletion of Treg cells at the later stages of carcinogen exposure did not induce tumor regression, but instead induced the opposite effect, driving increased incidence and burden of SCC. These findings suggest that Treg cells may play an unexpected role in restricting the progression of carcinogen-induced SCC in the oral cavity, prompting a re-examination of the common paradigm that Treg cells promote tumorigenesis by suppressing anti-tumor immunity.

   



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  High-throughput interrogation of the HSV-1 genome under type-I IFN pressure
  Presenter: Maxime Chapon
  All Authors:Maxime Chapon, Jeffrey S Zhao, Genhong Cheng
  UCLA
   
 

Herpes Simplex Virus type 1 (HSV-1) is the highly contagious causative agent for cold sores. While the virus has been extensively studied, the infection still has no cure. Most of previous works tested viral mutants individually to determine the target gene’s functions. This approach has been very successful but can prove laborious to test the effect of different selective pressures on viral fitness. High-throughput sequencing combined with transposon mutagenesis has been used to screen the complete genomes of several bacteria species for major fitness factors. With the maturation of this technique and its associated computational analysis tools, we created a transposon insertion library in the HSV-1 genome. This allowed us to test the fitness of viral mutants with and without type-I interferon pressure, and identify novel anti-interferon viral protein candidates. Furthermore, this unique approach could be used to interrogate the viral genome under different selection pressures, improving our understanding of the host-viral relationship, and provide novel targets for antiviral therapy. Recently, HSV-1 is utilized as a platform for innovative cancer immunotherapies, by taking advantage of its large genome and cytopathic effects. Our tool could help further develop this technology by identifying novel viral immunomodulatory proteins.

   



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  A Novel Role for the 4E-BP/eIF4E Axis in B Cell Antibody Class Switching
  Presenter: Honyin Chiu
  All Authors:Honyin Chiu, Leandra Jackson, Anni Mai, and David A. Fruman
  University of California, Irvine
   
 

During an adaptive immune response, activated mature B cells give rise to antibody secreting plasma cells to fight infection. B cells undergo a process known as antibody class switch recombination (CSR) to produce different classes of antibodies with varying effector functions. The mammalian target of Rapamycin (mTOR) signaling pathway is activated during this process and B cells deficient in mTOR have impaired survival, proliferation and differentiation into antibody secreting cells. It has been shown that antibody class switch recombination requires B cell division, however it is unclear if there is another regulatory mechanism through mTOR that is independent of proliferation. We and others have found that the mTORC1 allosteric inhibitor rapamycin, at non-immunosuppressive concentrations in vivo, can significantly decrease the amounts of germinal center B cells while still maintaining IgM secreting B cells. We have also found that rapamycin, when added after a B cell has committed to divide, can suppress class switching and cap-dependent translation while preserving proliferation. Additionally, restricting Glutamine availability phenocopies low concentrations of Rapamycin, in vitro. Mechanistic investigation of the mTORC1 substrate 4E-BP, an inhibitor of eIF4E and cap-dependent translation, showed that blocking the 4E-BP/eIF4E axis can decrease antibody class switching independently of proliferation. We have found that the mechanism involves decreased translation of the Aicda mRNA encoding activation-induced cytidine deaminase (AID) protein. These results uncover a novel role for mTORC1 and the 4E-BP/eIF4E axis in AID protein levels and B cell antibody class switching, suggesting that cap-dependent translation regulates key steps in B cell differentiation. Further study of this pathway may provide mechanistic insight into antibody-mediated autoimmune diseases such as lupus and arthritis as well as the formation of protective antibody responses following vaccination.

   



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  BCAP promotes TLR-induced IFN[a] production in pDC
  Presenter: Talyn Chu
  All Authors:Talyn Chu, Hayley Waterman, Griffin Gessay, and Jessica A. Hamerman
  University of Washington
   
 

Systemic lupus erythematosus (SLE) severity is correlated with elevated serum levels of type I interferons (IFN), specifically IFNα. pDC are important in the pathogenesis and etiology of SLE due to their ability to produce large amounts of IFNα in response to endocytosed nucleic acids. TLR7 and TLR9-induced IRF7 translocation to the nucleus and subsequent IFNα production by pDC is dependent on phosphatidylinositol-3 kinase (PI3K), but how PI3K is activated and regulates this process remains undefined. We showed that the cytosolic signaling adapter B cell adaptor for PI3K (BCAP) links TLRs to PI3K activation in macrophages, thus we asked whether BCAP plays a role in pDC IFNα production and SLE pathogenesis. Here, we show BCAP promoted many aspects of TLR7-driven lupus-like disease including interferon-stimulated gene expression in the blood. Consistent with these findings, we found BCAP promoted TLR7 and TLR9-induced IFNα production in pDC. Strikingly, BCAP-/- mice produced significantly less serum IFNα after injection of TLR9 agonist than WT mice, consistent with a pDC IFNα defect. TLR-induced IFNα production in pDC involves dual signaling pathways that run in parallel. There is a TLR-independent pathway that is initiated by ligand recognition at the plasma membrane and involves Dock2-mediated activation of Rac1, required for the phosphorylation of IKKα, and a TLR-dependent pathway that occurs upon RNA or DNA recognition by endosomal TLR7 or 9 respectively and leads to MyD88 activation. The pathways converge upon the phosphorylation and activation of IKKα leading to the phosphorylation and nuclear translocation of the transcription factor IRF7 and IFNα gene transcription. BCAP regulated IFNα production independently of the TLR-MyD88 pathway and both BCAP and PI3K activation were required for CpG DNA-induced early actin remodeling, a readout of Rac1 activation, and IKKα phosphorylation in pDC. These data suggest BCAP and PI3K specifically regulate the TLR-parallel pathway in pDC. Overall, we show a novel role for BCAP in regulating IFNα production in pDC and lupus pathogenesis.

   



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  Effect of TGF-[b] on the CD8 T cell response to rectal LCMV infection
  Presenter: Benjamin Cohn (1,2)
  All Authors:Benjamin Cohn (1,2), Timothy Borbet (3), Julie Luong (1), Erik Woodruff (1), Shahzada Khan (1), Martin Trapecar (1), Shomyseh Sanjabi (1,4)
  1. Gladstone Institute of Virology and Immunology, San Francisco, California, USA. 2. Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, California, USA. 3. Department of Environmental Medicine, School of Medicine, New York University, New York, New York, USA. 4. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA
   
 

Transforming growth factor (TGF)-β is a pleiotropic cytokine involved in both suppressive and inflammatory immune responses. In the colon, TGF-β plays a pivotal role in maintaining tolerance against innocuous food and commensal bacteria, but little is known about how TGF-β affects the anti-viral immune response against rectally transmitted pathogens. We have previously shown that TGF-β signaling selectively causes apoptosis in CD127-KLRG1+ short-lived effector cells, leading us to predict that this effect may be amplified in the TGF-β-rich environment of the gut. To address the role of TGF-β in colon immunity, we have established a model for rectal lymphocytic choriomeningitis virus (LCMV) infection in mice, allowing us to compare the rectal immune response to the systemic response elicited against LCMV. We demonstrate that LCMV is transmitted rectally without causing any intestinal damage, and passes from the colon to the iliac (iLN) and mesenteric draining lymph nodes. Compared to the systemic route, CD8 T cell priming and activation is delayed in rectal infection: mucosal priming occurs in the iLN 3 days post rectal inoculation, followed by a systemic activation 5 days post infection. The kinetics of CD8 T cell activation correlate with the kinetics of viral transmission and the amount of time it takes the virus to spread from the rectum to the draining lymph nodes. Using mice carrying a CD8 T Cell-specific deletion of TGF-β Receptor II (RII), we determined the effect of TGF-β signaling on three distinct phases of the CTL response: priming within regional lymph nodes, clonal expansion and memory formation. Determining the effect of TGF-β on the mucosal anti-viral CD8 T cell response will help us understand the requirements for viral control in the colon and other mucosal tissues. A better understanding of how immunological tolerance is overcome in mucosal tissues such as the colon will pave the way for effective therapies against HIV and other sexually transmitted viral infections.

   



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  SLC19A1 is an importer of the immune second messenger 2’3’-cGAMP
  Presenter: Anthony Cordova
  All Authors:Anthony Cordova, Christopher Ritchie, Gaelen Hess, Michael Bassik, Lingyin Li
  Stanford University School of Medicine
   
 

Analogs of the innate immune second messenger 2’3’-cGAMP have potent antitumoral effects in mice, and several of these analogs are now in clinical trials for metastatic solid tumors. 2’3’-cGAMP and other cyclic dinucleotides (CDNs) bind to the ER-membrane protein STING, leading to transcription of type I interferons and downstream immune activation. However, it is unknown how extracellular 2’3’-cGAMP and its analogs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMP's role as an immunotransmitter, and will aid in the development of better therapeutic STING agonists with greater target cell selectivity, fewer off-target effects, and better patient stratification.

   



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  Characterization of long non-coding RNA GAPLINC in mouse macrophages
  Presenter: Apple Cortez Vollmers
  All Authors:Apple Cortez Vollmers, Author Names separated by commas
  UC Santa Cruz
   
 

The innate immune system is the first line of defense against microbial and common bacterial infections. The first cells to respond to infection are macrophages, which are differentiated from cells called monocytes. Activated macrophages, in response to infection, trigger an inflammatory response through toll-like receptors (TLRs) that initiate the release cytokines and chemokines. Dysregulation of the inflammatory response following activation can lead to detrimental effects in the host such as septic shock, auto-immune disease, and cancer. More recently, long non-coding RNAs (lncRNAs) have been implicated to have functional roles in immunity. LncRNAs comprise the largest group of RNA transcripts produced by the cell, are greater than 200 nucleotides in length, and lack protein-coding ability. RNA-seq data acquired from primary human monocytes and macrophages identified a lncRNA called gastric adenocarcinoma predictive long intergenic noncoding (GAPLINC) to be highly upregulated (>1000-fold) during the monocyte to macrophage differentiation process. We observed that siRNA knockdown of GAPLINC in primary cells activated inflammatory response genes. Alternatively, increased GAPLINC during differentiation showed a decrease in inflammatory gene expression levels, but upon TLR4 stimulation, became highly attenuated. Here, we identify a mouse homolog of GAPLINC. Our findings recapitulate preliminary results found in human, as well as show that we can effectively target GAPLINC using CRISPRi technology.

   



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  IL-10 deficiency promotes microbiota-driven IL-22 mediated defenses against acute C. difficile infection
  Presenter: Emily Cribas
  All Authors:Emily Cribas, Emily S. Cribas, Jeffrey Maslanka, Joshua Denny, Michael C. Abt
  University of Pennsylvania
   
 

Clostridium difficile infection is the most common hospital-acquired infection in the United States. Pathogenesis is initiated by toxin-mediated damage to the intestinal epithelium and is exacerbated by non-C.difficile bacterial translocation. While the contributions of commensal bacteria and host immune responses in shaping C.difficile pathogenesis have been well-studied, the indirect role of commensal bacteria-mediated immunoregulation on disease progression remains poorly understood. Given the importance of the anti-inflammatory cytokine IL-10 in microbial tolerance, we use Il10-/- mice as a model to study dysregulated proinflammatory responses to commensal bacteria in the context of C. difficile infection. We show that specific pathogen-free Il10-/- mice have reduced morbidity and mortality to acute C. difficile infection compared to littermate Il10 heterozygous (Il10HET) mice. Improved survival was independent of C.difficile burden and toxin levels at peak infection, suggesting a critical role for immune defenses against non-C.difficile bacteria. Although the immune response at the peak of infection did not differ significantly between Il10-/- and Il10HET mice, there was elevated baseline expression of il22 and ifng in Il10-/- mice; both cytokines are critical in mediating innate immune defense against C.difficile. Genetic ablation of T-bet, the master transcriptional regulator of IFN-? production, in Il10-/- mice, did not diminish protection against severe C. difficile infection. In contrast, il10.il22 double knockout mice were significantly more susceptible compared to il10-/- mice. We conclude that absence of IL-10 improves host defense against C.difficile pathogenesis in an IL-22 dependent manner. Future work will involve defining the mechanism of IL-22 induction and identifying downstream effectors required for protection upon loss of IL-10 and commensal tolerance.

   



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  SUMO-2/3 prevents the activation of a spontaneous Type I Interferon response
  Presenter: Ty Crowl
  All Authors:Ty Crowl, Daniel Stetson
  University of Washington
   
 

Detection of intracellular nucleic acids by innate immune sensors triggers the production of Type I Interferons (IFNs), a family of cytokines that activate an antiviral response. While IFNs are critical for host defense against viral infection, dysregulated production underlies multiple autoinflammatory diseases in human patients. We have found that disruption of any essential ligase of the sumoylation pathway results in a potent and spontaneous IFN response. Vertebrates possess three small ubiquitin-like modifiers (SUMOs) that can be conjugated onto target proteins and alter protein function in diverse, but still poorly characterized ways. We have discovered that regulation of IFN by sumoylation is redundantly mediated by both SUMO-2 and SUMO-3, but not SUMO-1, revealing a previously unknown and specific function of SUMO-2/3. Remarkably, this IFN response is not dependent on any known nucleic acid sensing pathway and does not require either of the canonical, IFN-inducing transcription factors IRF3 or IRF7. Taken together, these findings demonstrate that SUMO-2 and SUMO-3 are essential regulators of Type I IFN responses that prevent the activation of a non-canonical and potentially novel mechanism of IFN induction.

   



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  Evolutionary Tradeoffs Between RNA Interference and Rig-I Like Receptors
  Presenter: John Crowl
  All Authors:John Crowl, Hannah Volkman, Dan Stetson
  University of Washington
   
 

RNA Interference is a post-transcriptional regulatory mechanism through which genes can be silenced by the down-regulation of template matched RNAs. Most invertebrates possess an amplified RNAi response mediated by RNA Dependent RNA Polymerases (RdRPs), which generate a pool of secondary small RNAs. This allows organisms that possess RdRPs, such as the nematode C. elegans, to undergo robust, heritable silencing and to use RNAi as a potent antiviral defense mechanism. Despite the apparent benefits of RdRPs, these enzymes were abruptly lost early in the chordate lineage. However, most of these organisms that lack RdRPs possess an alternative means of antiviral defense in the Rig-I like receptors (RLRs), which detect the RNA products of viral RdRPs and activate a type I interferon-mediated antiviral response. We propose that sensitive detection of viral RdRP products by RLRs necessitated the abrupt loss of endogenous RdRPs during chordate evolution, along with an accompanying loss of potency in RNAi. To test this, we have introduced C. elegans RdRPs into mammalian cells, and have found that one of them activates a potent type I Interferon response dependent on RLR signaling and the RdRP’s catalytic activity.

   



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  Role of miR-27 in controlling T cell immunity
  Presenter: Leilani Cruz
  All Authors:Leilani Cruz, Sunglim Cho, Chen-Jang Wu, Tomoharu Yasuda, Ling-Li Lin, Klaus Rajewsky, Li-Fan Lu
  UCSD
   
 

miR-23~27~24 clusters are highly enriched in regulatory cells while expressed at low levels in conventional T cells. However, despite a selective expression pattern of miR-23~27~24 clusters in T cells, to date, studies of this miRNA family have primarily focused on their role in tumorigenesis. Here, we show miR-23~27~24 clusters play a pivotal role in regulating T cell immunity. Particularly, our results have demonstrated that the majority of miR-23~27~24 cluster-dependent phenotypes could be attributed to a single member of this miRNA family, miR-27. Mice with T cell-specific overexpression of miR-27 spontaneously developed autoimmune phenotypes. On the other hand, in vitro polarization studies revealed that T cells with miR-27 overexpression exhibited impaired differentiation and effector function of multiple T helper cell lineages. Finally, our mixed bone marrow chimeras studies further demonstrated that miR-27 could control different T cell responses through either a T cell-intrinsic or -extrinsic manner. Collectively, Our results identify a new

   



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  Immunoregulatory Dendritic Cells During Persistent Viral Infection
  Presenter: Cameron R. Cunningham
  All Authors:Cameron R. Cunningham, Elizabeth B. Wilson, David G. Brooks
  UCLA
   
 

Immunosuppressive factors drive the immune dysfunctions that prevent virus clearance thereby enabling viral persistence. Ablation of these suppressive pathways restores immune activity facilitating enhanced control of the persistent virus. Understanding the sources, developmental cues, and mechanisms of induction of the suppressive factors will enable directed approaches to specifically block these pathways to augment an immune response crippled by suppression. Recently, we identified a specific immunoregulatory antigen presenting cell (iregAPC) population during persistent lymphocytic choriomeningitis virus (LCMV) infection in mice that specifically produces potent immunoregulatory factors, including IL-10, PD-L1, and indoleamine 2,3-dioxygenase (IDO), that suppress antiviral T cell responses. Interestingly, we recently demonstrated that in addition to its critical antiviral role throughout viral persistence, sustained type I interferon (IFN-I) signaling led to many of the suppressive mechanisms (including IL-10 and PD-L1 expression) and immune dysfunctions associated with persistent viral infections. Yet, how the iregAPC are generated, acquire suppressive activity (and if these events are separable), and the role of IFN-I in this process were all unclear. To interrogate iregAPC, we identified protein expression unique to these cells and used these to demonstrate that IFN-I suppresses the expansion of stimulatory DCs during persistent LCMV infection, while simultaneously endowing suppressive activity to iregAPC. However, direct IFN-I signaling was not required for the development of iregAPC, but instead generation of this population was dependent on MyD88 and IFN? signaling. We are currently investigating the molecular mechanisms and cellular interactions through which MyD88/IFNγ drive the emergence of this population and why these cells are susceptible to IFN-I mediated induction of an immunosuppressive function while other APC are not. Thus, our data indicate that immunosuppression during viral persistence is a multi-step process wherein MyD88 and IFNγ signaling lead to the development of a population of APC with suppressive potential and that ongoing IFN-I signaling then secondarily endows this suppressive function. Ultimately, we anticipate that these studies and future exploration will provide a greater understanding of the sources and dynamics of regulatory factors produced during a persistent infection and potentially identify novel therapeutic targets to boost an otherwise suppressed immune response.

   



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  Transcriptional profiling defines unique gene expression signatures in CD8 T follicular cells
  Presenter: Oscar Davalos
  All Authors:Oscar Davalos, Genevieve Mullins, Kristen M. Valentine, Katrina K. Hoyer
  Department of Molecular and Cell Biology, University of California Merced
   
 

Cytotoxic CD8 T cells have been largely overlooked when defining autoimmune pathology. A population of CXCR5+ CD8 T cells arises during autoimmune disease. This research aims to identify the similarities and differences in CXCR5+ CD8 T cells relative to CD4 T follicular helpers and bulk activated CD8 T cells using the IL-2-KO autoimmune model. RNA-seq was performed on CXCR5+PD-1+ CD8 T cells and CD4 T follicular helper cells from IL-2 deficient mice at 15 days of age. For transcriptional profiling, CXCR5+ CD8 T cells were compared to the average of CD4 T follicular helper and bulk CD8 T cell expression. We identified 2030 differentially expressed genes, with 1157 upregulated and 873 downregulated. CXCR5+ CD8 T cells retained cytotoxic gene expression and expressed a subset of genes specific to this population. Top 15 significant gene ontology categories indicated immune responses and cellular movement. We are currently evaluating the functionality of the genes found specifically in CXCR5+ CD8 T cells, and exploring the transcriptional differences in CXCR5+ T cells based on autoimmune disease pathology.

   



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  Identifying factors that inhibit host protein synthesis during Legionella pneumophila infection
  Presenter: Justin De Leon
  All Authors:Justin De Leon, Kevin C. Barry, Jonathan L. Portman, Mary F. Fontana, and Russell E. Vance
  UC Berkeley
   
 

Legionella pneumophila (Lp) is a Gram-negative facultative intracellular pathogen and the etiological agent of Legionnaire’s disease. Our laboratory uses Lp as a model to understand the mechanisms of pathogenesis by intracellular pathogens and the innate immune responses they elicit. Recent work in our lab has shown that Lp infection promotes a potent immune response dependent on interleukin-1 alpha (IL-1a). We have shown in vitro that translation inhibition and Toll-like receptor (TLR) signaling are sufficient to induce an IL-1a response. However it has proven difficult to test whether these factors account for IL-1a induction by Lp. In order to formally test this idea, an Lp strain deficient in translation inhibition is required. However, such a strain has not been generated despite the deletion of seven putative effectors that inhibit translation (?7). This leads to two hypotheses: (1) there are additional Lp-derived factors that inhibit host translation, or (2) Lp infection activates a novel host pathway that inhibits translation. To address the possibility of additional Lp-derived factors we have devised a genetic screen in Lp to identify mutants that fail to inhibit host translation. To search for possible host pathway candidates, we have utilized a host proteomic approach to identify novel signaling pathways that may modulate host translation during infection.

   



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  The Notch signalling pathway controls the differentiation of effector CD8 T cells during chronic infection
  Presenter: Dave De Sousa
  All Authors:Dave De Sousa, Frédéric Duval, Jean-François Daudelin, Marie-Ève Lebel, Alain Lamarre, Salix Boulet, Nathalie Labrecque
  Université de Montréal
   
 

During chronic infection, the persistence of antigen and inflammation leads to the differentiation of CD8 T cells into an exhausted state characterised by increased expression of inhibitory receptors (IRs), such as PD1, and the progressive loss of T cell functionality. In addition, it has been shown that different subsets of exhausted CD8 T cells (Tex) are generated following chronic infection. Among these subsets, Tex progenitors expressing intermediate level of PD1 (PD1int) will give rise to terminally differentiated Tex cells (PD1hi). PD1/PDL1 blockade will only reinvigorate PD1int cells. The molecular events controlling Tex cell differentiation are still poorly defined. As Notch signalling will be sustained during chronic infection by persistent TCR stimulation and inflammation, we postulated that Notch signalling influences Tex cell differentiation. To test that, we infected mice, lacking (N1N2Δ/Δ) or not (N1N2fl/fl) expression of Notch1/2 in mature CD8 T cells, with LCMV clone 13. Following infection, N1N2?/? Tex cells express higher levels of IRs and are less functional when compared to N1N2fl/fl CD8 T cells. Moreover, N1N2Δ/Δ effectors preferentially differentiate into the PD1hi subset at the expense of the PD1int and, as a consequence, Notch-deficient CD8 Tex cells cannot control viral load following PD-1/PDL-1 blockade. Analysis of the transcriptome of N1N2Δ/Δ Tex cells suggests that Notch signalling influences Tex differentiation by regulating response to cytokines and CD4 help, which are two signals controlling T cell exhaustion. Manipulation of Notch signalling may provide a unique opportunity to improve CD8 T cell response during chronic infection and cancer.

   



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  Investigating a role for the NAIP/NLRC4 inflammasome in generation of an adaptive T cell response
  Presenter: Katie Deets
  All Authors:Katie Deets, Russell Vance
  UC Berkeley
   
 

The NAIP/NLRC4 inflammasome is a powerful tool of innate immunity that aids in the elimination of cytosolic bacteria. Several groups have looked into the role of inflammasome activation on adaptive T cell responses, but that work has largely been done in the context of bacterial infections, which make isolating the role of NAIP/NLRC4 difficult. The Vance lab has developed a novel in vivo genetic system where we can specifically induce expression of flagellin (Fla), a potent activator of NAIP/NLRC4, along with the model antigen ovalbumin (Ova) as a single fusion protein (OvaFla) in the cytosol of cells under the control of a tamoxifen-inducible Cre recombinase. We will use this tool to determine if NAIP/NLRC4 activation is sufficient to generate an adaptive T cell response by addressing the following questions: (1) Are antigens released from pyroptotic cells taken up and displayed by antigen presenting cells? (2) What type of adaptive T cell response is generated? (3) Is this adaptive T cell response protective against challenge with flagellin-expressing pathogens?

   



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  Investigating a role for the NAIP/NLCR4 inflammasome in adaptive T cell responses
  Presenter: Katie Deets
  All Authors:Katie Deets, Jakob Von Moltke, Russell Vance
  UC Berkeley
   
 

Activation of the NAIP/NLCR4 inflammasome aids in the elimination of certain cytosolic bacteria from the intestinal epithelium. NAIP/NLCR4 activation in these cells results in pyroptosis and expulsion of the infected cell into the lumen of the gut. However, it remains unclear what effect this rapid cell expulsion has on pathogen-specific CD8 T cell responses. To address the role of the NAIP/NLCR4 inflammasome activation and cell expulsion on a subsequent CD8 T cell response, we are using a novel in vivo genetic system that uses a tamoxifen-inducible Cre recombinase to express flagellin (Fla), a potent activator of NAIP/NLRC4, along with the model antigen ovalbumin (Ova) as a single fusion protein (OvaFla) in the cytosol of intestinal epithelial cells (IECs). This tool allows us to address several key questions: (1) Are antigens from pyroptotic IECs released into the underlying intestinal tissue? (2) Are these antigens taken up and cross-presented to CD8 T cells? (3) Is this cross-priming response sufficient to generate protection against future infections? We will share our ongoing results addressing these questions.

   



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  Investigating a role for the NAIP/NLRC4 inflammasome in T cell immunity
  Presenter: Katie Deets
  All Authors:Katie Deets, Russell Vance
  UC Berkeley
   
 

The work that has been done thus far to investigate the role of the NAIP/NLRC4 inflammasome in generating an adaptive immune response has been carried out in the context of bacterial infections, which introduce a variety of PAMPs and make isolating specific response pathways difficult. To address this problem, the Vance lab has developed a novel in vivo genetic system in which we can specifically induce expression of flagellin (Fla), a potent activator of NAIP/NLRC4, along with ovalbumin (Ova) as a single fusion protein (OvaFla) in the cytosol of cells under the control of a tamoxifen-inducible Cre recombinase. With this tool, we will address whether NAIP/NLRC4 is sufficient to generate an adaptive T cell response through the following questions: (1) Are antigens released by pyroptotic cells taken up and displayed by antigen presenting cells? (2) What type of adaptive T cell response is generated? (3) Is NAIP/NLRC4 activation sufficient to generate a successful memory T cell response?

   



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  Alveolar and monocyte-derived macrophages differentially engage antibacterial programs during adaptive immunity to Mycobacterium tuberculosis.
  Presenter: Jared Delahaye
  All Authors:Jared Delahaye, Courtney Plumlee, Christopher Plaisier, Sara Cohen, Eliza Peterson, Nitin Baliga, Kevin Urdahl
  University of Washington
   
 

Cognate interactions between antigen-specific CD4 T cells and Mycobacterium tuberculosis (Mtb)-infected cells are critical for mucosal immunity to tuberculosis (TB). However, Mtb resides intracellularly within a variety of myeloid cell populations, and the relative impact of CD4 T cells on distinct infected cell types remains unknown. Here we report that intratracheally-transferred, Mtb-specific T cells are unable to reduce the bacterial burden in the first week after aerosol infection, when Mtb resides almost exclusively within alveolar macrophages (AMs). However, transferred T cells do mediate protection by day 14, at which point Mtb has disseminated to monocyte-derived macrophages (MDMs). Using microscopy in combination with a fluorescent reporter strain of Mtb, we observe that AM harbor more bacteria than MDM on a per cell basis even after T cells traffic to the lung. RNA-Seq analysis of these two macrophage populations reveals early and sustained enrichment for proinflammatory pathways (i.e. NFkB signaling) in Mtb-infected MDMs compared to AMs. In parallel, we analyzed the bacterial transcriptome and found that Mtb from AM engage distinct response programs compared to Mtb from MDM. Together, our results indicate that AMs may serve as a privileged niche for Mtb, despite the presence of Mtb-specific T cells. As many current TB vaccine candidates seek to induce lung-resident T cells that can recognize and control Mtb early after aerosol infection, the relative resistance of Mtb-infected AMs to T cell-mediated immunity could represent a previously unappreciated barrier to this strategy.

   



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  Novel recruitment of NEMO to TCR microclusters during T cell activation
  Presenter: Elizabeth DeRiso
  All Authors:Elizabeth DeRiso, Andrea Workman, Lawrence P. Kane, and Stephen C. Bunnell
  Tufts University Sackler School of Graduate Biomedical Science
   
 

Recent advancements in the world of immune therapy for cancer treatment take advantage of T cell signaling controls, highlighting the importance of fully understanding basic signaling mechanisms for therapeutic targeted use. The NFκB essential modulator protein (NEMO), also known as IKKγ, regulates canonical activation of the cell survival and growth transcription factor NF?B, downstream of the T cell receptor (TCR). Patients with mutated or deficient NEMO protein generally have some form of immune deficiency. Despite this important role, however, little is known about the specific mechanisms regulating the sub-cellular localization of NEMO during immunoreceptor signaling. Through high-resolution, dynamic imaging analysis in primary human PBMCs, and Jurkat T cells, we demonstrate that NEMO is recruited into functionally relevant, ubiquitin-dependent microclusters that coincide with TCR-associated kinase Zap70. Additionally, NEMO is recruited into mobile vesicles and larger, slow moving, membrane-bound macroclusters. Our studies indicate that entrance of NEMO into microclusters also depends on Zap70 kinase activity, yet is independent of other proximal signal molecules such as SLP76 and LAT. Finally, recruitment of NEMO into Zap70 clusters occurs within five minutes of TCR engagement, indicating rapid recruitment of NEMO to the TCR. Shifting from the assumed cytosolic localization of NEMO, our results indicate a novel recruitment pathway for NEMO into functionally relevant, TCR-associated microclusters required for NFκB activation.

   



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  Living on the Edge: Evaluating the Constitutive Innate Immunity of Anna’s Hummingbirds (Calypte anna)
  Presenter: Andrea M. DeRogatis
  All Authors:Andrea M. DeRogatis, Kirk C. Klasing, Lisa A. Tell
  University of California, Davis
   
 

Hummingbirds are indispensable pollinators in many ecosystems, making their conservation essential. As is the case with many species, hummingbirds are now faced with new challenges resulting from anthropogenic changes. Unfortunately, there is currently a very limited understanding of what factors may influence the success of hummingbird conservation including changing disease dynamics and new diseases. There has been little research done investigating the pathogenic threats that hummingbirds face. Hummingbirds are the smallest vertebrates and have the highest metabolic rates, indicating that their immunological defense may differ from more commonly studied taxa. The objective of this study is to gain a basic understanding of the constitutive innate immunity of hummingbirds. This was evaluated for Anna’s hummingbirds (Calypte anna) by examining the in vitro microbicidal capacity of whole blood. Age and sex were found to have no effect on the microbicidal killing ability of whole blood when exposed to E. coli. When multiple bacterial species were tested, there was a significant difference in the killing ability based on species. This basic knowledge improves conservation efforts by providing important insight into how different diseases may impact hummingbird populations based on their innate immunity.

   



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  Modification of a Novel Immune Assay for use with Japanese Quail (Coturnix japonica)
  Presenter: Andrea DeRogatis
  All Authors:Andrea DeRogatis, Kirk C. Klasing
  University of California, Davis
   
 

Knowledge about an organism’s immune system is important to a variety of biological sciences, not just the specific field of immunology. Although there are a variety of techniques available to study the avian immune system, there are also currently numerous limitations. Thus, there is a need for research that builds on existing methods and develops new techniques that can be used to study all components of the avian immune system in both laboratory and field environments. The main objective of this study was to determine if a novel immunological assay, designed for chicken samples, could be modified using quantitative polymerase chain reaction (qPCR; aka real-time PCR or RT-PCR) analysis to make it applicable for samples from Japanese quail (Coturnix japonica). Secondary to the main aim, was to determine whether lipopolysaccharide (LPS) dose, time of feather removal, or injection site significantly influenced the innate immune response as reflected by measurable changes in interleukin-1-beta expression (IL1β). Two experiments were performed to address these aims. In the first experiment, blood feathers on both the left and right wings of Japanese quail were injected with either 1X concentration endotoxin free phosphate buffered saline (PBS), a medium (0.1 μg) or high dose (1 μg) of LPS. The same experimental design was used for the second experiment, however only feathers from the right wing were injected. Using quantitative RT-PCR, we were able to successfully track changes in IL1β expression level in the feather tissue (pulp) in response to LPS injection. In both experiments, there were significant effects of time (P < 0.001), treatment (P <0.001) and the interaction of both time and treatment (P < 0.001) on IL1β expression. These results demonstrate that qPCR can be used to successfully measure an important modulator of an innate immune response using tissue from the growing feathers of Japanese quail. This technique will provide researchers with a valuable tool that can be used to help address avian based research questions important to numerous fields ranging from ecology to immunology.

   



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  Affinity for self drives the preferential accumulation of promiscuous CD4+ T cells over the lifespan.
  Presenter: Neha Deshpande (1)
  All Authors:Neha Deshpande (1), Sing Sing Way (2) and Michael S. Kuhns (1)
  1 The University of Arizona, 2 Cincinnati Children's Hospital Medical Center.
   
 

T cells discriminate self from foreign peptides presented in the context of self-major histocompatibility complex (pMHC) molecules via clonotypic T cell receptors (TCRs). CD8+ T cell recognition and responsiveness to foreign pMHC is known to diminish over the lifespan, which is consistent with gradual thymic involution over time. It is further supported by experimental evidence that restricting the diversity of class I MHC peptides during positive and negative selection results in selection of fewer CD8+ T cells that are highly specific for pMHC. How the affinity of the CD4+ T cell compartment for self-pMHC, and its capacity to bind foreign-pMHC change over the lifespan are fundamental aspect of T cell biology that remain largely unexplored. Experimentally restricting thymic selection is known to allow degenerate CD4+ T cells to develop. This suggests that they might accumulate in the CD4+ T cell compartment over time. We report that, while old mice (18-22 months) contain fewer CD4+ T cells than adults (8-12 weeks), those that remain have a higher intrinsic affinity for self-pMHC. Old mice also have more cells that bind distinct foreign-pMHCs, either alone or in combination. The numerical increase of these subsets with age directly correlates with their affinity for self-pMHC. However, no relationship was observed between affinity for self-pMHC and responsiveness to foreign-pMHC. These data demonstrate that the CD4+ T cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T cell receptor interactions with self-pMHC. These results have important implications for the design of immunotherapeutics targeting CD4+ T cells for longterm efficacy.

   



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  Innate immune cell influence on adaptive immune cell function during Coccidioides infection
  Presenter: Anh Diep
  All Authors:Anh Diep, Kelly Otsuka, Katrina H. Hoyer
  University of California, Merced
   
 

Coccidioidomycosis is a fungal, respiratory disease caused by Coccidioides immitis and Coccidioides posadasii. This emerging infectious disease ranges from asymptomatic to pulmonary disease with disseminated infection. There is little understanding as to what differentiates hosts that resolve the disease and hosts that development chronic infection. Intervention at early disease stages is difficult as symptoms presented are broad and generic. There remains a critical need for understanding coccidioidomycosis and identifying biomarkers of disease progression for more effective diagnosis and treatment. Coccidioides infection occurs when arthroconidia is inhaled into the lung and undergoes morphological changes from soil (arthroconidia) to host form (spherule). The initial immune response is mediated by local cells within the lung epithelium tissue and recruited immune cells. Effective Coccidioides clearance requires monocyte migration into the site of infection and subsequent differentiation. Macrophages and neutrophils mediate fungal clearance via phagocytosis and effector cytokine secretion. Long lasting immunity to Coccidioides requires dendritic cell activation of effector helper cells. Little is known about the dynamics and interactions between immune cells and Coccidioides at the start of infection and how these interactions influence later adaptive immune cell recruitment and function. Our lab observed a striking difference between chronic disease patients and patients who cleared. These differences in immune cell profiles and cytokines in serum lead us to investigate innate immune cell response to Coccidioides and their influences on adaptive immunity in vitro and in vivo.

   



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  Immunological method for measuring LDL-Cholesterol Using the Streptolysin O and Apolipoprotein B-100 anitibody.
  Presenter: Kim Dowon
  All Authors:Kim Dowon, Author Names separated by commas
  Hallym University
   
 

Cholesterol is one of the important components for necessary to maintain the body. It is related with function as component of cell membranes and it is also important raw material of sex hormone and cortical hormone. For function of cholesterol, it needs movement through blood. Because cholesterol does not dissolve in water, it moves through lipoprotein in blood. The lipoproteins can be divided into LDL(Low Density Lipoprotein) and HDL(High Density Lipoprotein) depending on the density. When LDL-cholesterol is too much present in blood, wall of blood vessels will be covered with lipid component. Therefore, it is important to measure value of LDL-cholesterol and diagnose diseases such as cardiovascular disease (CVD), obesity, diabetes, atherosclerosis, hypertension and stroke. In this paper, it shows that measuring the LDL-cholesterol in blood through an immunological method. Paper suggest double bio marker using two types of immunological measuring methods. One is streptolysin O (SLO) recombinant protein that it can specific combine with cholesterol because carboxyl-terminal of SLO has tryptophan-rich. The other is monoclonal antibody about Apolipoproetin B-100 existing in surface of LDL particle. The experimental results show that measuring LDL-cholesterol is dependent concentration and LDL- cholesterol of 2?/ml recognize the value of O.D.630nm <0.77. Also, the method recognized concentration of cholesterol until 125 to 250ng/ml. For those reason, this method can consider measuring LDL- cholesterol in serum. Keywords: LDL-Cholesterol, Apolipoprotein B-100, Streptolysin O, Immunoassay.

   



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  BCAP is a negative regulator of myeloid cell development
  Presenter: Jeffrey Duggan
  All Authors:Jeffrey Duggan, Griffin Gessay, Akshay Krishnamurty, Tobias Hohl, Jessica Hamerman
  Dept. of Immunology, Univ. of Washington; Dept. of Immunology, Benaroya Research Institute at Virginia Mason
   
 

B cell adaptor for PI3-kinase (BCAP) is a signaling adaptor protein expressed in cells of the hematopoietic lineage, including the myeloid cell compartment. BCAP has a variety of functions within immune cells, including participating in B cell signaling and homeostasis, NK cell development and function, and macrophage TLR signaling. Here we examined the role of BCAP in myeloid cell homeostasis and development. When examining myeloid cell populations within wild-type (WT) and BCAP-/- mice, we found that, BCAP-/- bone marrow had an increased number of inflammatory monocytes compared to WT bone marrow, whereas splenic populations of inflammatory monocytes, neutrophils, and dendritic cells were similar in number between WT and BCAP-/- mice. Additionally, we observed that mixed bone marrow chimeras generated with a 1:1 ratio of WT and BCAP-/- bone marrow exhibited a skewing towards BCAP-/- monocytes and neutrophils in the bone marrow, blood and spleen, showing a competitive advantage of BCAP-/- myeloid cells. We therefore hypothesized that BCAP regulates myeloid cell development. WT and BCAP-/- bone marrow had similar numbers of myeloid progenitor cells, including the LSK (Lineage-Sca-1+cKit+), CMP (Common Myeloid Progenitor) and GMP (Granulocyte-Macrophage Progenitor) cells. However, in mixed bone marrow chimeras the LSK, CMP, and GMP populations were skewed toward BCAP-/- cells similar to the mature myeloid cells, showing that BCAP-/- progenitors can out-compete their WT counterparts. In an in vitro myeloid colony forming unit assay, sorted BCAP-/- LSK, CMP, and GMP produced more total cells than WT progenitor cells, supporting a cell-intrinsic role of BCAP in regulating myeloid differentiation. To examine the development of inflammatory monocytes from WT and BCAP-/- progenitors in vivo, we used WT and BCAP-/- mice expressing the Ccr2-DTR transgene. After Diphtheria Toxin-mediated depletion of inflammatory monocytes, BCAP-/- mice replenished their peripheral monocyte numbers more rapidly than WT mice. Additionally, when infecting WT and BCAP-/- mice with Listeria monocytogenes, we observed markedly enhanced bacterial clearance in the spleen of BCAP-/- mice compared to WT mice, which correlated with increased numbers of inflammatory monocytes and neutrophils by day 2 post-infection. Together, these data show that BCAP acts as a cell-intrinsic negative regulator of myeloid cell development during both homeostatic and inflammatory settings.

   



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  BCAP inhibits proliferation and differentiation of myeloid progenitors at the steady state and during demand situations
  Presenter: Jeffrey Duggan
  All Authors:Jeffrey Duggan, Matthew B. Buechler, Rebecca M. Olson, Tobias M. Hohl, Jessica A. Hamerman
  Benaroya Research Institute, University of Washington
   
 

B cell adaptor for PI3-kinase (BCAP) is a signaling adaptor expressed in mature hematopoietic cells including monocytes and neutrophils. Here we investigated the role of BCAP in the homeostasis and development of these myeloid lineages. BCAP-/- mice had more bone marrow (BM) monocytes than WT mice, and in mixed WT:BCAP-/- BM chimeras, monocytes and neutrophils skewed towards BCAP-/- origin, showing a competitive advantage for BCAP-/- myeloid cells. BCAP was expressed in bone marrow hematopoietic progenitors, including LSK (Lineage-Sca1+cKit+), CMP (Common Myeloid Progenitor) and GMP (Granulocyte/Macrophage Progenitor) cells. At the steady state, BCAP-/- GMP expressed more IRF8 and less CEBPa than WT GMP, which correlated with an increase in monocyte progenitors and a decrease in granulocyte progenitors amongst GMP. Strikingly, BCAP-/- progenitors proliferated and produced more myeloid cells of both neutrophil and monocyte/macrophage lineages than WT progenitors in myeloid colony forming unit (CFU) assays, supporting a cell-intrinsic role of BCAP in inhibiting myeloid proliferation and differentiation. Consistent with these findings, during cyclophosphamide-induced myeloablation or specific monocyte depletion, BCAP-/- mice replenished circulating monocytes and neutrophils earlier than WT mice. During myeloid replenishment after cyclophosphamide-induced myeloablation, BCAP-/- mice had increased LSK proliferation, and increased numbers of LSK and GMP cells, compared to WT mice. Furthermore, BCAP-/- mice accumulated more monocytes and neutrophils in the spleen than WT mice during Listeria monocytogenes infection. Together, these data identify BCAP as a novel inhibitor of myelopoiesis in the steady state and of emergency myelopoiesis during demand conditions.

   



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  STAT4 Directs Protective Innate Lymphoid Cell Responses to Gastrointestinal Infection
  Presenter: Sarah Dulson
  All Authors:Sarah Dulson, Emily Watkins, and Laurie E. Harrington, Ph.D.
  University of Alabama at Birmingham
   
 

Foodborne infection with Listeria monocytogenes (Lm) carries a mortality rate of 20-30% in high-risk individuals and leads to serious complications such as meningitis and miscarriage. Therefore, studies evaluating gastrointestinal responses to Lm are needed to better enhance therapeutic interventions. Our data show that adaptive immune responses are dispensable in the first 5 days of infection since bacterial burden is similar between Rag1-deficient and wildtype mice. Innate Lymphoid Cells (ILCs) are tissue-resident lymphocytes that are enriched at barrier surfaces, and we hypothesize that intestinal ILCs are central mediators of the early response to Lm infection. Using cytokine reporter mice, we demonstrate a robust and early IFNγ response to Lm by Group 1 ILCs (ILC1s) in the large intestine lamina propria. In addition, the systemic IFNγ response is significantly reduced in the absence of ILCs. Consistent with this, mice devoid of ILCs mice suffer higher mortality and increased bacterial dissemination compared with ILC-sufficient littermates. Mechanistically, ILC1s lacking the transcription factor STAT4 are unable to produce IFNγ, and STAT4-deficient mice readily succumb to bacterial dissemination and mortality following Lm infection. Interestingly, STAT4-deficient animals with intact ILC populations were no longer protected against infection compared with ILC-depleted littermates, indicating that STAT4 signaling is critical for ILC-mediated protection. Furthermore, inhibition of STAT4 activity immediately prior to cytokine stimulation demonstrates that STAT4 directly promotes IFNγ production in ILC1s. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with Lm and identify STAT4 as a central modulator of ILC-mediated protection.

   



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  Maintenance of the unlicensed NK cell subpopulation in F1 MHC hybrid mice
  Presenter: Cordelia Dunai
  All Authors:Cordelia Dunai, Ethan G. Aguilar, Lam T. Khuat, William J. Murphy
  UC Davis
   
 

The activation of NK cells is controlled by the integration of activating and inhibitory signals. NK cells can be divided into subsets termed licensed and unlicensed based on the expression of inhibitory receptors with varying affinities for MHC class I molecules. Our group has shown that licensing delineates helper and effector NK cell subsets during viral infection. Unlicensed NK cells localized to lymph nodes and produced proportionately higher levels of GM-CSF which correlated with maturation of dendritic cells, which in turn fostered antigen-specific T cells. This division of labor occurs in mice and humans, but is mediated by different families of receptors. We studied two different strains of mice (C57BL/6 and BALB/c), as well as their F1 hybrids. These two strains have different MHC haplotypes and the F1 offspring express both, leading to different dynamics of licensed and unlicensed NK cells. NK cells expressing Ly49C/I, Ly49G2, and Ly49A are licensed in F1 offspring as these inhibitory receptors bind MHC class I molecules H-2Kb, H-2Db and H-2Dd, respectively. We have observed that to compensate for the increased frequency of licensed NK cells, there is a compensatory increase in the frequency of Ly49C/I-Ly49G2-Ly49A- NK cells, indicating the maintenance of the unlicensed subpopulation in F1 hybrids. Understanding more about the subsets of NK cells has relevance to clinical applications of de novo generated or adoptively-transferred NK cells for the treatment of cancer.

   



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  Opposing roles of natural killer cell subsets in a mouse model of acute myeloid leukemia and hematopoietic stem cell transplant
  Presenter: Cordelia Dunai
  All Authors:Cordelia Dunai, Ethan G. Aguilar, Lam T. Khuat, and William J. Murphy, Ph.D.
  UC Davis
   
 

Natural killer (NK) cells are lymphocytes that bridge innate and adaptive immune responses. The activity of NK cells is controlled by the integration of activating and inhibitory signals. NK cells can be divided into subsets termed licensed and unlicensed based on the expression of inhibitory receptors with varying affinity for MHC class I molecules. In general, licensed NK cells have higher cytotoxic potential than unlicensed NK cells. Hematopoietic stem cell transplant (HSCT) is a treatment for a number of hematological malignancies, including acute myeloid leukemia (AML). Patients who are not eligible to receive intensive cytoreductive therapy, are at risk for primary refractory disease, or are experiencing relapse are all candidates for HSCT. NK cells are the first lymphocytes to recover post-HSCT and have been shown to be critical for an anti-leukemia response. Here we investigate the contribution of NK cell subsets to anti-leukemia responses in the context of a mouse model of HSCT.

   



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  Interaction between HIV-1 integrase and viral RNA drives proper virion morphogenesis and is necessary for successful infection
  Presenter: Jennifer Elliott
  All Authors:Jennifer Elliott, Jenna E. Eschbach, Pratibha C. Koneru, Wen Li, Maritza Puray-Chavez, Dana Townsend, Dana Lawson, Alan N. Engelman, Mamuka Kvaratskhelia, Sebla B. Kutluay
  Washington University School of Medicine
   
 

Recognition of non-self RNA or DNA is a critical step in the immune response to many viral pathogens. Viruses in turn have evolved multiple means of evading or subverting sensing mechanisms, with a common strategy being limiting the accessibility of viral nucleic acids. In the case of HIV-1, the viral genomic RNA is enclosed inside a conical capsid lattice, which is released into the target cell upon viral entry. In addition to its structural role, the HIV-1 capsid is thought to be essential for shielding the viral genomic RNA from cytosolic pattern recognition receptors. Recent findings indicate a key role for the HIV-1 integrase (IN) enzyme in proper virion morphogenesis, and mutations within IN can lead to the generation of morphologically aberrant viral particles with the viral genomic RNA mislocalized outside the capsid core. Despite containing all the components necessary for replication, such particles cannot successfully infect target cells. To determine how multiple mutations within IN can lead to similar defects in virion morphology and viral replication, we characterized a panel of IN mutant viruses for defects in the viral lifecycle. Human primary CD4+ T cells or cultured cells were infected with IN mutant viruses and viral RNA, DNA, and proteins were analyzed by either immunoblot, qPCR, or confocal microscopy. All of the mutant viruses screened were severely attenuated in infectivity, and encountered a block early after entering target cells. Viral RNA and IN itself were prematurely lost from target cells, and reverse-transcribed viral DNA did not accumulate. Similar defects were seen in viruses in which the capsid protein (CA) was mutated to destabilize the capsid core. These findings support a model in which the HIV-1 capsid core is critical for protecting entering viral RNA and replicative enzymes from the host environment.

   



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  Characterizing the immune response to AAV9-CRISPR/Cas9 in a humanized mouse model of Duchenne muscular dystrophy using single cell RNA-sequencing
  Presenter: Michael Emami
  All Authors:Michael Emami, Michael R Emami, Courtney Young, Feiyang Ma, Matteo Pellegrini, Melissa Spencer
  University of California, Los Angeles (UCLA)
   
 

Duchenne muscular dystrophy (DMD) is an x-linked disease caused by out-of-frame mutations in the DMD gene, which encodes dystrophin. Due to the nature of these mutations, DMD is amenable to gene-editing and gene replacement therapies. Ongoing phase I/II clinical trials for DMD rely on a gene replacement strategy using adeno-associated virus (AAV) to achieve systemic delivery. However, immune responses against AAV vectors have resulted in serious adverse events (SAEs) in current and past studies. Yet, it is poorly understood why certain patients develop SAEs following AAV gene therapy and what specific immune responses arise. To study immune responses in vivo following AAV administration, we dosed a humanized dystrophic mouse model (hDMD del45) with AAV serotype 9 carrying CRISPR/Cas9 and performed 10x Genomics single cell RNA-sequencing (scRNA-seq). We tested three different AAV9-CRISPR/Cas9 doses: 1.2E12vg, 6E12vg, and 1E13vg and isolated PBMCs pre-AAV administration and 4 weeks post-AAV administration. Four main immune cell populations were observed: monocytes, NK cells, B cells, and T cells, all of which demonstrated a clear shift in phenotype between pre- and post-AAV treatment. Within the T cell population, we detected shifts in the phenotypes of all subpopulations including CD4+, CD8+, regulatory T cells (Tregs), and gamma delta (?d)T cells between pre- and post-AAV treatment, regardless of dose. Assessment of gene expression within T cell subpopulations revealed upregulation of Dusp1 and Dusp2 in CD8+, CD4+ and ?dT cells. These two genes are downstream of Toll-Like Receptor signaling and may represent a potential target to suppress SAEs with AAV administration. Additional analysis and validation studies are needed identify critical immune cell populations and genes that elicit responses to AAV, and to separate capsid vs transgene-specific immune reactions. These studies will enable the identification of new target genes involved in immune responses to AAV.

   



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  Interrogating the Molecular Mechanisms of STING Signaling
  Presenter: Sabrina L. Ergun
  All Authors:Sabrina L. Ergun, Daniel Fernandez, Thomas M. Weiss, and Lingyin Li
  Stanford University
   
 

The STING (STimulator of INterferon Genes) pathway is an innate immune signaling cascade which promotes essential anti-cancer, anti-viral, and anti-bacterial responses. Conversely, STING overactivation is linked to several autoimmune and inflammatory diseases such as Lupus, Multiple Sclerosis, heart attack, and Parkinson’s disease. Despite its significance in the disease context, the precise molecular mechanism of STING activation and attenuation remains unclear. Using structural biology and biochemistry, we report that the metazoan second messenger 2’3’-cGAMP induces the release of the auto-inhibiting STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Autoimmune disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the putative C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2’3’-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a novel therapeutic hypothesis for autoimmune syndrome treatment.

   



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  Exploring the potential role of NLRC3 as a negative regulator of T cell function
  Presenter: Nichole Escalante
  All Authors:Nichole Escalante, Vinicius Motta, Jerry Zhou, Dave Prescott, Stephen Girardin, Dana Philpott
  Department of Immunology, University of Toronto
   
 

NLRC3 is an understudied member of the Nod-like receptor family whose function in maintaining immunity is poorly characterized. Previously published work has suggested that NLRC3 may act as a negative regulator of T cell activation however the specific mechanisms through which it acts in T cells remains to be discovered. We have found high mRNA expression of NLRC3 in Natural Killer cells and T cells, with slightly lower expression in B cells and little to undetectable expression in macrophages. In T cells, NLRC3 protein expression was found in the cytoplasm and nucleus. After in vitro T cell receptor (TCR) stimulation, both mRNA and protein NLRC3 expression decreased. NLRC3 knockout CD4+ T cells, however, did not have altered proliferation, activation or IL-2 secretion after in vitro stimulation and analysis of T cells in NLRC3 knockout mice showed normal frequencies of CD4+ αβ TCR+, CD8+ αβTCR+ and γδTCR+ T cells in the thymus, spleen, mesenteric lymph nodes and intestines. Interestingly, NLRC3 knockout mice have decreased colon pathology after Citrobacter Rodentium infection compared to wild type mice. This recent finding suggests that NLRC3 is an important regulator of mucosal immunity in vivo.

   



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  Enter a Title for your Abstract
  Presenter: Katrina Evans
  All Authors:Katrina Evans, Author Names separated by commas
  University of California Irvine
   
 

Breast cancer brain metastasis affects some 10-20% of patients, and there are very few effective treatments. The native immune effectors in the brain are macrophage-like cells called microglia, and astrocytes. These cell types represent close to 50% of cells in the brain and function to survey the blood brain barrier (BBB) for disruptions, protect the brain from invading pathogens and respond to injury and inflammation. Activated infiltrating microglia and astrocytes are a major component of metastatic breast tumors in the brain, but it is not known whether these cells are involved in the promotion or rejection of breast cancer cells. To address this gap in knowledge we isolated microglia, macrophages and astrocytes from metastatic and control mouse brains and generated single cell RNA libraries to interrogate their gene expression. Using the Seurat analysis pipeline we have identified metastasis associated microglia gene signatures are similar to microglia in other neuroinflammatory and neurodegenerative diseases.

   



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  Beyond innate immunity: the role of IRF3 in CD4+ T cell function
  Presenter: Nancy Fares-Frederickson
  All Authors:Nancy Fares-Frederickson, Dennis Otero, Monica Macal, Elina Zuniga, Michael David
  UC San Diego
   
 

Interferon regulatory factor 3 (IRF3) is a transcription factor found in the cytoplasm of most nucleated cells. Upon pathogen recognition, IRF3 becomes activated and translocates to the nucleus where it mediates the production of type I interferon (IFN). Whereas IRF3 has been studied extensively in the context of type I IFN production and innate immunity, its role in adaptive immunity has remained largely unexplored. The objective of our research is to elucidate the role of IRF3 in CD4+ T cell function. Our findings demonstrate that IRF3-deficient CD4+ T cells show robust proliferation in response to TCR engagement, but are compromised in their ability to differentiate into specialized T helper cell subsets—most notably IL-17-producing Th17 cells. As such, CD4+ T cells lacking IRF3 expression fail to induce disease in the T cell transfer model of colitis. These studies reveal a novel, T cell-intrinsic function of IRF3 and extend its role beyond the innate immune response to pathogens.

   



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  A Gut Commensal Bacteria Influences Resistance to Pneumonia.
  Presenter: Krysta Felix
  All Authors:Krysta Felix, (Kevin) Fei Teng, C. Pierce Bradley, Christina Klinger, Nhan Tran, and H-J. Joyce Wu
  University of Arizona
   
 

The gut microbiota can have profound effects on an individual’s health. The immune system reaction to intestinal colonization mediates a large portion of these effects. Segmented Filamentous Bacteria (SFB) is a commensal bacterium that exerts a powerful pro-inflammatory influence over the immune system, resulting, among other things, in increased antibody production. Immune responses initiated in the intestines can spread to other mucosal sites, such as the respiratory system. We hypothesized that SFB colonization would boost the T-independent II antibody response to Pneumovax-23, an anti-pneumococcal vaccine, to increase resistance to pneumonia induced by Streptococcus pneumoniae. To test this, we used a mouse model of pneumonia in mice colonized by SFB. We monitored the mice for symptoms of illness, bacterial burden, and immune response. In an immunization setting, we found that SFB+ Pneumovax-23 immunized mice are more resistant to pneumococcal pneumonia than are SFB- mice given the same vaccine. Understanding the mechanisms of protection against S. pneumoniae will contribute to devising better ways to prevent infection and design vaccine to this pathogen.

   



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  Segmented Filamentous Bacteria Confer Protection Against a Lung Infection Through Innate Mechanisms.
  Presenter: Krysta Felix
  All Authors:Krysta Felix, Nhan Tran, Debdut Naskar, Walid Raslan, and Hsin-Jung Joyce Wu
  University of Arizona
   
 

Segmented Filamentous Bacteria (SFB), commensal bacteria that colonize the small intestine under normal, healthy conditions, profoundly impact the host immune system. In particular, they induce protection against intestinal pathogens such as Citrobacter rodentium and Entamoeba histolytica, potentially in an IL-17-dependent manner. Immune responses initiated in the intestines can extend to other mucosal sites, such as the respiratory system. Because of this, we hypothesized that SFB colonization would increase resistance to a lung infection caused by Streptococcus pneumoniae. We found that SFB colonization does not make a difference in disease susceptibility in WT mice. We further identified B cells as key players in protecting mice against S. pneumoniae infection. In the absence of B cells, mice that lack SFB develop more severe disease, while those that are colonized by SFB maintain resistance, as demonstrated by protection against body temperature decrease and weight loss, as well as decreased lung bacterial load and lower neutrophil infiltration. These data suggest that SFB protect the host against S. pneumoniae infection in a manner dependent on the innate immune system. In a B cell transfer model, B cell-mediated protection requires B cell-intrinsic MyD88, as in contrast to mice receiving WT B cells, mice receiving MyD88-/- B cells are unable to maintain normal temperature after infection, a phenotype comparable to total lack of lymphocytes. Our data demonstrate the significant impact of the gut microbiota on infections even in gut-distal locations such as the lungs.

   



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  MODULATING MITOCHONDRIAL RESPIRATION ALTERS IMMUNE HOMEOSTASIS AT STEADY STATE
  Presenter: Adrien Fois
  All Authors:Adrien Fois, Claudine Beauchamp, Geneviève Chabot-Roy, Guy Charron, Yan Burelle, Christine Des Rosiers, John D. Rioux, Sylvie Lesage
  Hôpital Maisonneuve Rosemont
   
 

Leigh syndrome French Canadian type (LSFC) is an inheritable disease characterized by the occurrence of acute crises of fulminant metabolic acidosis, which considerably increase disease severity and mortality. This syndrome is caused by a mutation in the LRPPRC gene. LRPPRC regulates mRNA stability of many mitochondrial proteins necessary for the assembly of the respiratory chain. As such, mutations in LRPPRC modulate mitochondrial respiration, known to polarize immune responses. Interestingly, metabolic crises in LFSC patients often follow infections. These results highlight the possible contribution of the immune system in the onset of metabolic acidosis crises. To investigate the role of LRPPRC in immune function, we generated various mouse models. Due to the embryonic lethal phenotype of LRPPRC-/- mice, we generated LRPPRCfl/fl x Mx1-Cre mice. Deletion of LRPPRC in adult mice results in a depletion of many immune cells, including NK cells and plasmacytoid dendritic cells. In an attempt to better understand the impact of the LSFC causal mutation, we also generated LRPPRC-354V-KI mice. Mice homozygous for the A354V mutation die in utero. However, we were able to analyze LRPPRC-354V-KI+/- and observed an increase in T cells when compared to WT mice. Altogether, our preliminary data suggest that a deficiency in LRPPRC, which modulates mitochondrial respiration, influences immune homeostasis. We are currently investigating its role in ongoing immune responses. It remains to be seen whether these observations will be generalizable to other, less severe, metabolic syndromes.

   



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  Immuno-PET of murine T helper lymphocytes with an anti-CD4 cys-diabody
  Presenter: Amanda Freise
  All Authors:Amanda Freise, Richard Tavaré, Kirstin Zettlitz, Felix B. Salazar, Anna M. Wu
  Crump Institute for Molecular Imaging
   
 

OBJECTIVE: Investigating the immune system presents a unique challenge because immune cells traffic between and localize at multiple sites throughout the entire body. Standard diagnostic methods for assessing the dynamic immune system include biopsies, which are site-specific, and blood draws, the results of which can be variable and represent only a small fraction of the total lymphoid population. In contrast, noninvasive imaging of the presence of lymphocytes, specifically CD4+ T cells, throughout the entire body would be an improvement upon current limited methods of analysis. Selective imaging of CD4+ T cells can be accomplished with immuno-positron emission tomography (PET), which utilizes antibody-based probes to detect and quantify cell surface markers. Engineering antibodies to produce fragments allows for customization of pharmacokinetics, clearance route, and conjugation to suit in vivo imaging applications. Based on the GK1.5 rat anti-mouse CD4 antibody, we have engineered a bivalent cys-diabody (cDb) fragment for detecting CD4+ T cells in vivo. The present study aims to characterize the functional properties and imaging capability of GK1.5 cDb. METHODS: GK1.5 cDb is comprised of two single-chain variable fragments connected by a linker. A C-terminal cysteine was introduced to enable site-specific conjugation to maleimide-Alexa488, maleimide-biotin, or maleimide-desferrioxamine (malDFO), a radiometal chelator. The cDb lacks the Fc region of the parental antibody, resulting in increased clearance compared to intact antibodies and removal of Fc-mediated functionality. Flow cytometry was used to confirm GK1.5 cDb binding specificity in vitro and ELISA binding curves were performed to assess affinity. The effect of GK1.5 cDb on surface expression of CD4 on T cells in vivo was investigated by injecting GK1.5 cDb and measuring expression in various lymphoid organs over time. Functional effects of GK1.5 cDb at several doses were assessed using in vivo and in vitro T cell proliferation assays. For imaging studies, GK1.5 cDb-malDFO was radiolabeled with 89Zr and 10-16 µg (25-35 μCi) was injected into untreated, CD4-blocked (bolus co-injection of cold cDb), or CD4-depleted mice. Mice were PET scanned at 20 hours post-injection, followed by biodistribution studies. Additionally, a dose escalation study using 2, 6, 12, or 40 μg GK1.5 cDb was performed to test the effect of protein dose on imaging and biodistribution. RESULTS: Flow cytometry on murine primary lymphocytes demonstrated that GK1.5 cDb retains its specificity for murine CD4. ELISA binding curves showed that the dissociation constant of the cDb (~2.6 nM) was similar to that of the parental antibody (~0.99 nM). Administration of GK1.5 cDb in vivo resulted in temporarily decreased T cell expression of CD4, which recovered within three days. Analysis of CD3+ and CD4+ cell populations showed that GK1.5 cDb does not deplete T cells. Both in vitro and in vivo studies of CD4+ T cell proliferation showed that higher doses of cDb had an inhibitory effect on proliferation, and that lower doses did not alter proliferative capacity. In imaging and biodistribution studies, axillary LNs and spleen showed high uptake of 89Zr-radiolabeled cDb in untreated mice (45, 30% ID/g respectively), in contrast to CD4-depleted mice (3.9, 4.1% ID/g) and CD4-blocked mice (7.1, 6.1% ID/g). The dose escalation study showed that the lowest protein/radiation dose (2 μg/7 μCi) gave better target:background ratios and better targeting of spleen and LN compared to higher doses (6-40 μg/20-136 μCi). CONCLUSION: Characterization at several doses demonstrated that low-dose GK1.5 cDb did not perturb CD4+ T cell numbers or proliferative capacity, and was an effective imaging agent for CD4 expressed in lymphoid organs in wild type mice. GK1.5 cDb is a promising agent for further noninvasive investigation of CD4+ T lymphocytes in vivo.

   



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  Intra-spike crosslinking overcomes antibody evasion by HIV-1
  Presenter: Rachel Galimidi
  All Authors:Rachel Galimidi, Joshua S. Klein, Maria S. Politzer, Shiyu Bai, Michael Seaman, Michel Nussenzweig, Pamela J. Bjorkman
  California Institute of Technology
   
 

Antibodies developed during HIV-1 infection lose efficacy as the virus mutates its envelope spike. We postulated that HIV-1 uses its small spike number to impede bivalent binding of IgGs through inter-spike cross-linking, thus hindering avidity, potent neutralization, and expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody based molecules capable of avid binding through intra-spike cross-linking. We used DNA as a “molecular ruler” to measure distances between epitopes on virion-bound envelope and to construct intra-spike cross-linking molecules. Bivalent binding resulted in synergy (>100-fold average increased potency) and shed light on dynamic states of the HIV-1 envelope protein. These results support the hypothesis that low spike densities facilitate antibody evasion and demonstrate that intra-spike cross-linking lowers the concentration of antibodies required for neutralization by up to 2.5 orders of magnitude.

   



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  Overcoming HIV-1 evasion of antibody avidity by intra-spike crosslinking
  Presenter: Rachel Galimidi
  All Authors:Rachel Galimidi, Maria S. Politzer, Joshua S. Klein, Shiyu Bai, Michael S. Seaman, Michel C. Nussenzweig, Anthony P. West, Jr., Pamela J. Bjorkman
  California Institute of Technology
   
 

Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 spike antibodies primarily bind monovalently because HIV’s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike trimer structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and neutralization potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a “molecular ruler” to measure intra-epitope distances on virion-bound spikes and to construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude of increased potency (>100-fold average increases across a virus panel) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection.

   



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  Hypothalamic Cooperative Defenses during Sepsis
  Presenter: Robert Gallant
  All Authors:Robert Gallant, Karina Sanchez, Jose Puerta, Janelle Ayres
  Salk Institute for Biological Studies
   
 

Host defense against infections utilizes two distinct strategies. Resistance mechanisms function to promote health by decreasing pathogen burden while cooperative defenses promote health with a neutral to positive impact on pathogen burden. We sought to identify novel cooperative defenses during sepsis by utilizing the concept of lethal dose 50 (LD50). We developed an LD50 polymicrobial sepsis model in which mice are infected with a 1:1 mixture of E. coli O21:H+ and S. aureus by intraperitoneal injection. Within 8-10 hours, half of the infected mice succumb to infection while half become asymptomatic carriers with no difference in pathogen burden. The hypothalamus likely plays a critical role in this cooperative defense-driven sepsis survival since it regulates many homeostatic and endocrine pathways important during infection. To identify hypothalamic cooperative defense mechanisms promoting this survival, we conducted RNA-seq on the hypothalami of infected healthy, infected morbid, and uninfected mice then looked for genes elevated in infected healthy mice compared to both infected morbid and uninfected mice. We are currently investigating how these transcriptomic differences reveal hypothalamic cooperative defenses during sepsis.

   



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  Discover T cell fate in a dish: generation and profiling of pathogen-specific CD4 T cells
  Presenter: Yajing Gao
  All Authors:Yajing Gao, Edward K Wakeland, Chandrashekhar Pasare
  University of Texas Southwestern Medical Center
   
 

CD4 T cells are vital for optimal functioning of the adaptive immune system. In addition to secreting effectors that have direct effects, they are critical for CD8 T cell and B cell responses. Naïve CD4 T cells are activated by antigen-presenting cells (APCs, especially dendritic cells) and differentiate into distinct effector subtypes, distinguished by their profiles of cytokine production. CD4 T cell effector cytokines such as IFNγ, IL-17A and IL-13 are specialized and critical for elimination of specific spectrum of pathogens. Therefore, CD4 T cell response, depending on the nature of the invading enemy, is rather evolved to generate an optimal proportion of each subsets in order to provide effective host protection. The pathogen-specificity of effector CD4 T cells hence is a combination of TCR recognition of pathogen epitopes and the customized wiring of effector functions. So far little is known about the exact mechanisms by which the later specificity is determined. Here we describe an in vitro method to generate and trace pathogen-specific CD4 T cells, allowing us to unbiasedly assess pathogen-directed clonal expansion and differentiation of naïve CD4 T cells. Utilizing this system, we have been able to prime pathogen-specific CD4 T cells in vitro, which as a result exhibit cytokine profiles consistent with the established in vivo response for a given pathogen. Adoptive-transfer of these CD4 T cells to a naïve mouse, followed by pathogen challenge, showed specificity towards the priming pathogen. Using fate-mapping and transcriptional profiling, we found evidence of heterogeneity and specificity between Th17 subsets generated by different pathogenic stimuli. Collectively, our results suggest an effective way to study pathogen specificity of T cell differentiation and we are rigorously using this tool to define early programming that imprints terminal effector function on primed CD4 T cells.

   



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  Antigen-specific transfer of CD40L (CD154) from helper T cells to B cells
  Presenter: Jennifer Gardell
  All Authors:Jennifer Gardell, David Parker
  Oregon Health and Science University
   
 

It has been known for decades that the delivery of T cell help for B cells is antigen-specific, MHC-restricted, and depends on CD40L. However the mechanisms by which CD40L, a transmembrane cytokine, is delivered to the T cell surface and engages CD40 on antigen-presenting B cells remains to be determined. Huse et al. (2006 Nat Immunol 7: 247-55) showed that a subset of cytokines is delivered directionally to the point of contact between the T cell and the antigen-presenting cell, also known as the immunological synapse, but CD40L was not among the cytokines investigated in that study. Although CD40L, like other cytokines, is made de novo following T cell activation, Koguchi et al. (2007 Blood 110(7): 2520-7, 2011 J Immunol 187(2): 626-34, 2012 PLoS One 7(2): e31296) showed that CD40L protein is stored in effector and memory CD4 T cells in intracellular vesicles that come to the cell surface rapidly following antigen recognition in sufficient amounts to activate antigen-presenting B cells. It has been thought that when a T cell recognizes an antigen-presenting B cell, CD40L expressed on the T cell surface engages with CD40 on the surface of B cells for a period long enough to lead to productive signaling. Here we show for the first time that CD40L does not remain on the surface of the T cell, but is actually transferred to the B cells. This transfer is absent from bystander B cells that are not presenting antigen, and is only partially dependent upon the presence of CD40 on the antigen-presenting B cells. Choudhuri et al. (2014 Nature 507(7490): 118-23) have recently shown that TCRs are deposited in microvesicles on the antigen-presenting cell at the immunological synapse. Our data suggests that CD40L might be deposited in a similar manner in some type of membrane vesicle on antigen-presenting B cells. This transfer would allow for sustained CD40L-CD40 signaling after a very brief (Allen et al. 2007 Science 315(5811): 528-31, Victora et al. 2010 Cell 143(4): 592-605, Shulman et al. Science 345(6200): 1058-62, Liu et al. 2014 Nature) interaction with helper T cells in the germinal center. If delivery of CD40L is proportional to the amount of antigen on the B cell, transfer of CD40L could be the mechanism by which B cells with higher affinity for antigen following somatic hypermutation are selected in the germinal center reaction, as recently proposed by Dustin (2014 Mol Cell 54(2): 255-62).

   



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  Investigating the role of intratumoral dendritic cells in anti-tumor immunity
  Presenter: Alycia Gardner
  All Authors:Alycia Gardner, Álvaro de Mingo Pulido, Brian Ruffell
  H. Lee Moffitt Cancer Center
   
 

CD8α+ and CD103+ type 1 classical dendritic cells (cDC1s) have been shown to be the dendritic cell (DC) subset critical for inducing a cytotoxic T cell response against tumors. However, while the role for DCs in educating T cells within the lymph nodes is well established, relatively little is known about the impact that DCs have within the tumor. In this study, we made use of orthotopically-implanted tumors derived from C57BL/6 MMTV-PyMT transgenic mice. Bone marrow dendritic cells (BMDC) were generated by ex vivo culture in media containing Fms-like tyrosine kinase 3 ligand (Flt3L). Using these model systems, we show that orthotopically-implanted PyMT tumors are responsive to single-agent checkpoint blockade therapy, indicating the presence of a baseline immune response. Similar decreases in the rate of tumor growth were achieved by systemic administration of Flt3L, and by intratumoral administration of polyinosinic-polycytidylic acid (poly(I:C)), which expand and activate DCs, respectively. Intratumoral administration of BMDCs also significantly slowed tumor growth in C57BL/6 mice, an effect which was further enhanced by co-injection with poly(I:C). In order to examine whether DC trafficking to the lymph node is required for the BMDC-mediated reduction in tumor growth, we used CCR7-/- BMDCs, which do not migrate to the lymph node. Mice treated with these BMDCs exhibited therapeutic response, albeit not to the extent observed with WT BMDC treated mice. These findings suggest a role for DCs locally within the tumor microenvironment, and provide the basis for further investigation of local DC activity in the tumor.

   



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  An atypical splenic progenitor population supports antibody production during Plasmodium infection in mice.
  Presenter: Debopam Ghosh
  All Authors:Debopam Ghosh, Brian Kennedy, Johnasha Stuart, Jason S. Stumhofer
  UAMS
   
 

Hematopoietic stem and progenitor cells (HSPCs) function to maintain the immune cell repertoire in the steady state, as well as during emergency situations including infection or stress. Although the bone marrow serves as the primary site of hematopoiesis, extramedullary mobilization and differentiation of HSPCs occurs in the spleen – a critical step in the host immune response during acute Plasmodium infection. Here, we have identified an atypical HSPC population in the spleen of C57BL/6 mice with a Lineage-Sca-1+c-kit- (LSK-) phenotype. These LSK- cells were found to expand in response to acute P. yoelii 17X infection and upon transfer into naïve congenic mice they differentiated predominantly into mature follicular B cells. However, when transferred into infection-matched hosts, LSK- cells matured into B cells capable of responding to Plasmodium by differentiating into germinal center and memory B cells, as well as plasma cells that secreted parasite-specific antibodies. Incubation with parasitized RBC lysate, enhanced the ability of splenic LSK- cells to differentiate into B cells in vitro, suggesting that a component of the parasite directly stimulates expansion and differentiation of LSK- cells. However, differentiation of LSK- cells into B cells after infection was independent of MyD88 signaling. Collectively, we have identified a population of atypical lymphoid progenitor cells in the spleen that is capable of differentiating into B cells in response to Plasmodium infection and ultimately contributes to the overall humoral response by producing antigen-specific antibodies.

   



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  An IL-2 receptor partial agonist expands FoxP3+ regulatory T cells in vivo
  Presenter: Caleb Glassman
  All Authors:Caleb Glassman, Leon Su, Sonia Majri, K. Christopher Garcia
  Stanford University
   
 

IL-2 is critical for the development, survival and effector function of multiple lymphoid cell populations. Using mutations previously identified by our group that impair interactions with the common gamma chain, we generated a series of IL-2 receptor partial agonists and assessed their activity in vivo. One mutein, REH, showed specific activity on FoxP3+ regulatory T cells with reduced activity on CD8s. This mutein mirrored Treg specific difference in IL-2 signaling with increased dependence on the high-affinity IL-2 receptor, CD25, and heightened sensitivity to negative regulation by suppressor of cytokine signaling 1 (SOCS1). Consistent with its effect on Tregs, REH administration delayed disease onset and decreased disease severity in the context of experimental autoimmune encephalomyelitis. Together, these results demonstrate that intrinsic differences in IL-2 sensitivity can be exploited to generate variants with cell-type specific activity.

   



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  Exploring the mechanisms that establish the chromatin state of T cells
  Presenter: Naomi Goldman
  All Authors:Naomi Goldman, Aditi Chandra, Maria Fasolino, and Golnaz Vahedi
  University of Pennsylvania
   
 

Cell fate-specific gene expression programs are established in part by alterations in chromatin accessibility via the action of lineage-determining transcription factors (TFs). Work in our lab has recently identified that the transcription factor TCF-1—integral for normal thymic development—targets and is essential for the opening of repressed chromatin in T cells1. However, the mechanism through which TCF-1 acts at enhancers in order to regulate genes during T cell development is unknown. To characterize the role of TCF-1 in the establishment of the T cell epigenome, I immunoprecipitated TCF-1 in thymocytes followed by a mass spectrometry (MS) analysis to identify interacting proteins. Previous work has shown that exogenous expression of TCF-1 in NIH3T3 fibroblasts leads to a gain in chromatin accessibility at T cell regulatory regions that are normally silent in fibroblasts. Given this observation, I have developed a system to validate candidate interacting proteins from my MS results. I have utilized CRISPR/cas9 to knock down candidates in fibroblasts prior to TCF-1 transduction followed by RNAseq and ATAC-seq to assess gene expression and chromatin state. Analysis of these results will allow me to determine if other factors are required to enable TCF-1 to modulate the chromatin state. These studies aim to provide mechanistic insight into how transcription factors work to establish cell identity in addition to adding to our understanding of how cell fate might be reprogramed at will for therapeutic purposes.

   



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  Microbial experience influences tumor infiltrating CD8+ T cells
  Presenter: Hanna Groeber
  All Authors:Hanna Groeber, Cody Morrison, Kristin Renkema
  Grand Valley State University
   
 

Immune cells have been harnessed for anti-cancer therapy with varying degrees of success. One potential reason for immunotherapy failures in clinical trials may be that typical specific pathogen free (SPF) mice do not model human microbial experience. Indeed, previous studies have shown that SPF mice immunity closely resembles newborn human immunity, whereas immune systems from mice exposed to diverse microbes more closely resemble adult human immunity. We have adopted a model of microbial experience by co-housing SPF mice with mice purchased from local pet stores, therefore exposing the SPF mice to various viral, bacterial, fungal, and parasitic pathogens. Pathogen testing confirmed that the co-housed (CoH) mice are exposed to pathogens and that the SPF controls remain pathogen-free; CoH mice also gain KLRG1+ CD44+ CD8+ T cells in the blood and spleen. We injected B16 melanoma cells subcutaneously into SPF and CoH mice and monitored tumor development and T cell activation ex vivo and in vitro. CoH tumors had increased frequencies of KLRG1+ CD44+ CD8+ T cells compared to SPF tumors, and CoH tumor-infiltrating CD8+ T cells exhibited increased activation upon in vitro stimulation. Ultimately these findings will contribute to our understanding of how microbial experiences shape anti-tumor immunity and have significant implications for future immunotherapy research.

   



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  Development of NY-ESO-1 TCR bearing T cells from HSCs is dependent on HLA-A2.1
  Presenter: Eric Gschweng
  All Authors:Eric Gschweng, Eric H Gschweng, MacLean Sellars, Michael L Kaufman, Antoni Ribas, Donald B Kohn
  University of California, Los Angeles
   
 

Engineering hematopoietic stem cells (HSC) for cancer immunotherapy has the potential to provide a life time supply of engineered T cells, overcoming current limitations associated with the use of a differentiated T cell based infusion product. However, the development of T cells from an engineered HSC transplant may be negatively affected when using T cell receptors (TCR) with supraphysiologic affinity. The transplant of human CD34 enriched peripheral blood HSCs transduced with a lentiviral vector encoding an affinity enhanced NY-ESO-1 TCR to immunocompromised mice (NSG) was used to evaluate the development of engineered T cells from HSCs in vivo. We observed an HLA-A2.1 expression dependent effect on the efficiency of development of NY-ESO-1 TCR bearing T cells from HSCs tied to signaling during thymopoiesis. In non A2.1 matched donor and recipient tissue, the majority of NY-ESO-1 bearing cells were halted in the double positive stage of thymopoiesis, with reduced numbers of cells in CD4 and CD8 single positive stages, and few CD8 SP cells in the periphery. Examination of markers of activation in the thymus between the TCR positive and negative fractions revealed a failure of positive selection in this model. Using HLA-A2.1 positive NSG mouse recipients and HLA-A2.1+ donor tissue allowed for positive selection during thymopoiesis, and a greater proportion of peripheral engraftment of CD8 SP NY-ESO-1 TCR bearing T cells. The supraphysiologic affinity of the TCR did not result in negative selection during development, providing support for the use of affinity enhanced TCRs for HSC based engineered immunity.

   



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  A subset of CD73 expressing IgM+ antigen-experienced cells is dependent on T-B interaction.
  Presenter: Sneh Lata Gupta
  All Authors:Sneh Lata Gupta, Lucas D'Souza, Vineeta Bal, Satyajit Rath, Anna George
  National Institute of Immunology
   
 

Memory B cells in the murine system are composed of Switched memory and IgM memory. The maturation markers CD73, CD80 and CD273 have previously been used to characterize multiple phenotypically and functionally distinct populations within this compartment. The factors involved in the formation of these subsets, however, remain unclear. We report that a subset of CD73 expressing IgM+ antigen-experienced cells with memory characteristics is diminished in T-cell deficient mice and provision of T cells by adoptive transfer or in mixed bone marrow chimeras rescued this deficiency. We also identify a role for CD40-CD154 signaling in this phenomenon, as CD73+ subsets were reduced in CD40-/- mice and also in the CD40-/- compartment of mixed-marrow chimeras. In the absence of CD40-CD154 signaling, we find CD73 expression to be reduced in splenic T cells and Age-associated B cells (ABCs), suggesting the importance of T-B interactions in the induction of this marker.

   



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  B cell-intrinsic and extrinsic IL-10 signaling are critical for the generation of germinal center B cells and protective anti-Plasmodium humoral immunity
  Presenter: Jenna Guthmiller
  All Authors:Jenna Guthmiller, Amy Graham, Ryan Zander, Rosemary Pope, Noah Butler
  University of Oklahoma Health Sciences Center
   
 

Plasmodium parasites cause over 200 million cases of malaria and nearly half a million deaths each year. Clinical and experimental studies show that humoral immunity is essential for Plasmodium parasite control and clearance. However, naturally acquired humoral immunity is short-lived and non-sterilizing, leaving individuals susceptible to repeated episodes of malarial disease. The mechanisms that skew B cell activity towards short-lived, inefficient humoral immune reactions are not fully understood. Recent reports highlight that excessive IFN-γ exacerbates malarial disease by limiting germinal center (GC) responses and that IL-10 can limit severe malarial disease by suppressing the activity of IFN-γ. Here we test the hypothesis that Plasmodium infection-induced IL-10 and IFN-γ directly counter-regulate B cell function and anti-Plasmodium humoral immunity. We found that direct IL-10 signaling in B cells is essential for GC B cell responses, parasite-specific IgG secretion, parasite clearance, and host survival. By contrast, IFN-γ signaling in B cells directly triggers non-protective, short-lived plasmablast responses and limits GC B cell responses resulting in reduced secreted parasite-specific IgG. Thus, IL-10 both directly and indirectly, by antagonizing IFN-γ, promotes GC B cell development and anti-Plasmodium humoral immunity. We further identified that both B cell-intrinsic IL-10 signaling and IL-10-mediated suppression of IFN-γ limit T-bet expression in B cells. Genetic deletion of T-bet in B cells abrogates short-lived plasmablast responses, promotes robust GC B cell responses, and markedly enhances parasite-specific IgG secretion. Collectively, our data identify that B cell-intrinsic IL-10 and IFN-γ signaling counter-regulate expression of T-bet, which acts as a molecular switch to promote either short-lived plasmablast responses or limit GC B cell responses. Our findings encourage reconsideration of IFN-γ and IL-10 as correlates of protection and pathology during clinical malaria.

   



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  Manipulating cell death pathways to promote anti-tumor immune responses
  Presenter: Cassidy Hagan
  All Authors:Cassidy Hagan, Annelise G. Snyder, Nicholas Hubbard, Michelle Messmer, Sigal Kofman, Brian Daniels, Andrew Oberst
  University of Washington
   
 

Engaging immune responses as a strategy to treat cancer has become a major focus of modern cancer therapeutic regimens. Recently, it has been established that various mechanisms of cell death drive distinct immune responses to tumor antigens and can drastically alter the outcome of tumor progression. Inflammatory cell death modalities such as necroptosis have been shown to induce robust immune activation and tumor control while apoptosis, an immunologically silent form of cell death, can be tolerogenic. We have created systems and reagents which directly trigger immunogenic necroptosis in tumor cells in order to exploit the immunogenicity of programmed cell death. Using these, we have defined induction of necroptosis as an immunogenic event that synergizes with immune checkpoint blockade to promote durable tumor clearance. Because cell death releases cancer antigens in combination with distinct immunomodulatory signals, future treatment strategies modulating cell death pathways may benefit the immunotherapeutic goals of modern cancer therapy.

   



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  A systematic approach to characterizing CD4 T cell function in Borrelia burgdorferi infection
  Presenter: Elizabeth Hammond
  All Authors:Elizabeth Hammond, Kimberly Olsen, Nicole Baumgarth
  University of California Davis
   
 

Borrelia burgdorferi is a spirochete bacteria that is the causative agent of Lyme disease. It is widely accepted that B. burgdorferi has evolved a wide variety of mechanisms to subvert host immune responses, which leads to persistent infection. Our lab has previously demonstrated that T-dependent B cell responses are impaired in a murine model of B. burgdorferi infection. Specifically, we observed structural defects in germinal centers (GCs), early collapse of GCs, and impaired formation of long lived plasma cells and memory B cells. In light of other studies that demonstrated the ineffectiveness of T cells in providing protection against B. burgdorferi, we have developed the hypothesis that the CD4 T cell response is impaired during B. burgdorferi infection, thus contributing to spirochete persistence. In order to systematically determine which aspects of the CD4 T cell response are impaired during B. burgdorferi infection, we first identified an immunodominant T-dependent antigen, arthritis related protein (Arp). Furthermore, we determined a highly immunogenic Arp epitope in C57BL/6 mice. Using the identified Arp epitope, we developed an I-Ab restricted tetramer in order to track an antigen-specific CD4 T cell response using flow cytometry. Furthermore, we developed a FACS staining protocol to identify follicular T helper cells (TFH) which are localized in the GC. Using these reagents and techniques, we tracked the CD4 T cell response over the course of acute infection with B. burgdorferi.

   



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  A role for T-bet in coordinating T cell activation
  Presenter: Gretchen Harms Pritchard
  All Authors:Gretchen Harms Pritchard, David Christian, Nathan Roy, Janis Burkhardt, Christopher Hunter
  University of Pennsylvania
   
 

The T-box transcription factor T-bet is perhaps most prominently known as a master regulator of Th1 differentiation and IFN? production. Recent work from our laboratory has identified a novel role for this transcription factor in coordinating the effector T cell responses necessary to control the intracellular parasite Toxoplasma gondii in peripheral tissues, and suggests that T-bet may function in a diverse array of immunological processes. The integrin CD11a is involved in cell:cell interactions during T cell priming and activation. Here, we show that T-bet is required for optimal upregulation of CD11a and ICAM-binding, an observation that suggests that T-bet is required for optimal cell:cell interactions during T cell activation. Support for this idea is provided by studies which showed that in mice challenged with T. gondii, the absence of T-bet or CD11a blockade results in a similar reduction in T cell responses. Thus, these findings highlight the importance of these proteins for in vivo T cell responses and suggest a role for T-bet in the coordination of the very early events in T cell activation that are necessary for optimal CD8+ T cell responses.

   



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  Determining the effect of diverse host-associated microorganisms on tolerance induction
  Presenter: Erika Hayes
  All Authors:Erika Hayes, Minh Pham, Marc Gavin, Daniel Campbell
  Benaroya Research Institute
   
 

Regulatory T cells (TR) are required for maintaining immune homeostasis and preventing loss of tolerance to self-antigens. Therapeutic manipulation of TR for induction or restoration of tolerance has been pursued for treatment of autoimmunity, but while it has been relatively easy to induce tolerance in specific pathogen free (SPF) mice, this has not translated to human patients. It was recently reported that diverse host-associated microorganisms are acquired by traditional SPF mice through co-housing with pet store mice. This cohabitation dramatically altered the SPF mouse immune phenotype and response to antigenic challenge, ultimately inducing an immune transcriptomic signature akin to that of an adult human. We have found that co-housed mice have an enriched inflammatory dendritic cell (DC) population in the spleen, as well as a ratio of effector to regulatory T cells that is skewed heavily towards effector lymphocytes. We hypothesize that the increased lymphocyte activation and effector/memory phenotypes in CH mice are a product of increased DC activation. Based on evidence that antigen-experienced T cells can reciprocally affect DC responses, we further predict that the Teff-skewed CD4+ T cell compartment will perpetuate DC activation, leading to a less tolerant immune state. As this co-housing model is able to better recapitulate the heightened basal activation status of lymphocytes observed in humans, utilization of this model to evaluate key factors for tolerance induction will be informative on why efforts to translate therapies from SPF mice to humans have failed. For example, when antigen-specific immunotherapy (ASI) is administered in SPF mice, it results in efficient induction of TR, but this is abrogated in the presence of an inflammatory stimulus. These studies will give evidence for use of more realistic pre-clinical animal models to provide better predictive power for results of immune targeted therapies in humans, and reveal important parameters for induction or restoration of tolerance that will aid in designing therapeutics.

   



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  Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation
  Presenter: Samuel J. Hobbs
  All Authors:Samuel J. Hobbs, Jeffrey C. Nolz
  Oregon Health and Science University
   
 

Tissue-resident memory (TRM) CD8+ T cells permanently reside in non-lymphoid tissues where they are important mediators of both host defense and inflammatory disease. TRM cells are derived from effector cells after they have been recruited into non-lymphoid tissues, but the molecular and transcriptional mechanisms that control TRM differentiation are largely undefined. We have recently used a model of localized epicutaneous infection with Vaccinia viruses (VacV) expressing individual model antigens to demonstrate that local antigen recognition in the skin microenvironment enhances TRM formation by ~50-100 fold. To understand how T cell receptor (TCR) engagement may promote TRM differentiation, we sought to identify effector CD8+ T cells that were actively recognizing cognate peptide-MHC in vivo. Using an endogenous interferon-gamma (IFNγ)-YFP reporter, we found that only ~20% of the antigen-specific CD8+ T cells in the skin microenvironment produce IFNγ during the course of a VacV skin infection. However, genome-wide transcriptional profiling revealed that the T cells producing IFNγ in the skin are also undergoing dramatic changes in gene expression that resemble mature TRM cells. In fact, effector CD8+ T cells producing IFNγ also expressed the transcription factor Blimp1, which has previously been shown to be critical for the acquisition of the core TRM transcriptional profile. We further show that TCR stimulation is sufficient to induce Blimp1 expression in effector, but not naïve CD8+ T cells, suggesting that antigen recognition by effector T cells results in unique transcriptional consequences not observed during the initial activation of naïve T cells. Together, these studies demonstrate that antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor impacting gene expression, which ultimately promotes the differentiation of TRM cells during viral skin infection.

   



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  Tissue-resident memory CD8+ T cells promote local antigen specific inflammation
  Presenter: Sam Hobbs
  All Authors:Sam Hobbs, Jeffrey C. Nolz
  Oregon Health and Science University
   
 

Tissue-resident memory (Trm) CD8+ T cells permanently reside in non-lymphoid tissues where they are important mediators of both host defense and inflammatory disease. In particular, Trm cells have been implicated in multiple skin inflammatory diseases including psoriasis and allergic contact dermatitis. Here, we sought to model contact-dependent inflammation of the skin by topical application of antigen to the site of Trm formation. We show that stimulation of resting Trm cells with antigen results in a local inflammatory response that is characterized by swelling and recruitment of leukocytes into the skin. Using IFNγ-YFP reporter TCR transgenic CD8+ T cells, we are able to identify Trm cells exhibiting local effector function within hours of stimulation with antigen. CD8+ T cells can produce inflammatory cytokines including IFNγ and TNFα, which could increase the expression of adhesion molecules on the skin-associated vascular endothelium. In fact, administration of P- and E- selectin blocking antibodies reduced both swelling and leukocyte recruitment into the skin following peptide challenge. Importantly, inflammatory responses did not occur in skin lacking Trm cells, was not affected by the depletion of circulating memory CD8+ T cells, and was antigen specific. Finally, we show that repeated challenges of the skin with antigen resulted in substantially increased numbers of CD69+ antigen-specific Trm cells. These results demonstrate that Trm CD8+ T cells mediate antigen-specific inflammation and expand locally during repeated exposures to cognate antigen, which could have implications for the treatment or prevention of inflammatory disorders of the skin.

   



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  Targeted expansion of tissue-resident CD8+ T cells to boost cellular immunity in the skin
  Presenter: Samuel Hobbs
  All Authors:Samuel Hobbs, Jeffrey Nolz
  Oregon Health & Science University
   
 

Effective cellular immunity against intracellular pathogens requires direct recognition of peptide-MHC by CD4+ and CD8+ T cells. Therefore, protective memory T cells must either be already positioned at the site of pathogen entry or be able to rapidly localize to inflamed tissue microenvironments following re-infection. Traditionally, the goal of vaccination strategies targeting the durable formation of cellular immunity has focused on generating large populations of circulating antigen-specific memory T cells using booster immunizations and/or strong adjuvants. However, in many human vaccination trials, the numbers of circulating memory T cells do not correlate with protection. This lack of protection by circulating memory T cells has generated a strong interest in developing vaccines that seed tissue-resident memory (TRM) T cells at sites of pathogen entry. Although their protective capacity is well established, whether pre-existing TRM populations can be specifically boosted to increase tissue-specific protective immunity is largely unknown. Here, we demonstrate that repeated activation of rare, endogenous TRM CD8+ T cells using only topical application of antigenic peptide caused delayed-type hypersensitivity and increased the number of antigen-specific TRM CD8+ T cells specifically in the challenged skin by ~15-fold. Expanded TRM CD8+ T cells in the skin were derived from memory T cells recruited out of the circulation that became CD69+ tissue-residents following local antigen encounter. Notably, recruited circulating memory CD8+ T cells of a different antigen-specificity could be coerced to become tissue-resident using a dual peptide challenge strategy. This ‘recruit and capture’ technique significantly increased anti-viral protection in the skin, suggesting this procedure could be used to rapidly boost tissue-specific cellular immunity. Overall, our study demonstrates that established TRM T cells can be amplified in a tissue-specific manner to rapidly enhance protective cellular immunity without relying on booster immunizations or adjuvants, but also suggests a mechanism of how pathogenic T cells specific for commensal or environmental antigens could continually accumulate in the skin following repeated exposures.

   



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  Anti-viral type I interferons induce naive-like T cells with reprogrammed metabolic signatures
  Presenter: Namit Holay
  All Authors:Namit Holay, Barry E. Kennedy, J. Patrick Murphy, Prathyusha Konda, Michael Giaccomontonio, Joao A. Paulo, Mariam Elaghil, Gary Sisson, Youra Kim, Derek Clements, Christopher Richardson, Steven P. Gygi and Shashi Gujar
  Dalhousie University
   
 

Early events during the acute phase of an immune response to virus exposure, specifically in the context of T cells, remain poorly understood. In this study, we describe the early induction of naïve-like T cells in vivo after reovirus exposure and dissect the molecular mechanisms that drive these cells. Using a combination of mass spectrometry approaches for multiplex proteomics and metabolomics, we first identified a crucial regulatory role for type I interferon signaling in the induction of these cells after virus exposure. We further demonstrated that naïve-like T cells had a completely rewired metabolic signature when compared to naïve T cells. Elucidating the molecular mechanisms of early T cell phenomena after virus exposure will improve our understanding of anti-viral immunology and guide the development of viral vaccines and virus-based treatments for cancer.

   



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  Monocytes promote the generation of effector T cells through localized IL-12 production in draining lymph nodes
  Presenter: Jessica Huang
  All Authors:Jessica Huang, Joseph Leal, Karan Kohli, Michael Gerner
  University of Washington
   
 

Cells of the innate immune system are integrally involved in the generation of adaptive immunity. Particularly, conventional dendritic cells (cDCs) are known to mediate T cell activation and differentiation in lymph nodes (LNs) during inflammation. While other innate cell subsets can contribute, their roles remain less well-defined. Here, we utilize immunization models and West Nile virus (WNV) infection, coupled with advanced microscopy approaches to study the responses of different innate cell populations in draining LNs during inflammation. We found that within hours following immunization with various type-1 inflammatory adjuvants and after WNV infection, monocytes (MOs) were rapidly recruited to draining LNs in large numbers. MOs infiltrated the draining LNs predominantly through high endothelial venules, and once in the LN, differentiated into monocyte-derived DCs (MoDCs). These MoDCs further infiltrated the deep T cell zone, and physically interacted with T cells undergoing activation by cDCs. While MoDCs did not play a major role in antigen presentation, they did constitute a significant source of IL-12 production within the T cell zone, suggesting a likely role in T cell differentiation. Indeed, blockade of monocyte trafficking into LNs with a CCR2 blocking antibody resulted in decreased IL-12 levels in the T cell zone, and in a significant reduction in fully differentiated effector CD4+ and CD8+ T cells. Interestingly, some adjuvants induced highly polarized infiltration of MOs across the lymph node, generating regions dominantly enriched in MOs and MODCs. Spatial analysis of T cell responses revealed that early-effector Tbet+TCF1- T cells were preferentially located in these MO-rich regions, while less differentiated Tbet-TCF1+ T cells were generally observed in regions relatively devoid of MOs, suggesting localized T cell differentiation. Together, these data suggest that during type-1 inflammation, monocytes infiltrate the LNs to create inflammatory microenvironments, and by cooperating with cDCs, they promote the generation of optimally differentiated effector T cells.

   



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  Paradoxical pro-tumor functions of dendritic cells on colorectal cancer
  Presenter: Hsin-I Huang
  All Authors:Hsin-I Huang, Gianna Hammer
  Department of Immunology, Duke University
   
 

The immune system’s ability to eradicate cancer requires coordination between innate and adaptive immune cells. No other cell type bridges innate and adaptive immunity better than the dendritic cell (DC). For this reason, DCs, specifically the Batf3-dependent DC subset, are thought to be key to anti-tumor immunity. Although the anti-tumor functions of Batf3-dependent DCs have been well described in skin cancer, whether these DCs have similar anti-tumor functions in other cancers is unknown. Here we investigated the roles of Batf3-dependent DCs in colon cancer and paradoxically found that these DCs actually promote colon tumorigenesis. Thus, in a spontaneous colorectal cancer mouse model, loss of Batf3-dependent DCs resulted in decreased colon tumor number. Although Batf3-dependent DCs are best known for expanding IFNγ-producing T cells, in colon tumors, we surprisingly found that Batf3-dependent DCs were required to expand tumor-infiltrating γδT cells producing IL-17. IL-17 is known to promote colon tumorigenesis and our findings pinpoint IL-17-producing γδT cells as a key source of IL-17 for tumor growth, and surprisingly describe a requirement for Batf3-dependent DCs to expand an IL-17-producing T cell population. In contrast, we found that Batf3-dependent DCs were not required for expanding tumor-infiltrating IFNγ-producing T cells suggesting that anti-tumor cytokine IFNγ was not compromised after losing Batf3-dependent DCs. Furthermore, we identified Batf3-dependent DCs were required for γδT cells survival in colon tumor, as a potential mechanism that Batf3-depednent DCs regulated γδT cells. In addition to this function, we identified PD-L1 expression as a second mode of action by which Batf3-dependent DCs promote colon cancer. While we found that all DC subsets enter colon tumors, tumor-infiltrating Batf3-dependent DCs uniquely upregulate high levels of PD-L1. PD-L1 upregulation and high PD-L1 expression on Batf3-dependent DCs was specific within the tumor microenvironment. Importantly, PD-L1 was not expressed on colon tumor cells themselves, suggesting that PD-L1 expression on Batf3-dependent DCs may be dominant for PD-L1-mediated immunosuppression in colorectal cancer. Collectively, these paradoxical pro-tumorigenic functions and phenotypes of Batf3-dependent DCs suggest that these DCs are not universally anti-tumor and that targeted therapies that modulate Batf3-dependent DCs could benefit colorectal cancer patients.

   



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  Monocytes promote the generation of effector T cells through localized IL-12 production in draining lymph nodes
  Presenter: Jessica Huang
  All Authors:Jessica Huang, Karan Kohli, Joseph Leal, Michael Gerner
  University of Washington
   
 

Cells of the innate immune system are integrally involved in the generation of adaptive immunity. Particularly, conventional dendritic cells (cDCs) are known to mediate T cell activation and differentiation in lymph nodes (LNs) during inflammation. While other innate cell subsets can contribute, their roles remain less well-defined. Here, we utilize immunization models and Type-1 inflammatory adjuvants to study the responses of different innate cell populations in the draining LNs. We found that within hours of immunization with distinct TLR agonists, monocytes rapidly migrated to the draining LNs in large numbers and differentiated into monocyte-derived DCs (MoDCs). These MoDCs further infiltrated the deep T cell zone where they physically interacted with T cells undergoing activation by cDCs. While MoDCs did not capture large quantities of antigen, they did constitute a major source of IL-12 production in the T cell zone, suggesting their likely role in T cell differentiation. Indeed, early-effector T-bet high T cells were preferentially enriched in regions heavily infiltrated by MoDCs, suggesting localized T cell polarization. Blockade of monocyte trafficking into LNs with a CCR2 blocking antibody resulted in decreased IL-12 levels in the T cell zone, and in a significant reduction in effector CD4+ and CD8+ T cells 4 days post-immunization. Together, these data suggest that during the generation of adaptive immune responses, monocytes in draining LNs create a localized spatial niche and cooperate with cDCs to promote the production of optimally differentiated effector T cells.

   



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  The role of CCR5 in the maintenance of intestinal immune homeostasis
  Presenter: Xin Huang
  All Authors:Xin Huang, Daniel T. Utzschneider, Arnaud Delpoux, Chenyen Lai, Steve M. Hedrick
  1Molecular Biology Section, Division of Biological Sciences, UC San Diego, La Jolla, CA 92093 2Cellular and Molecular Medicine, UC San Diego, La Jolla, CA 92093
   
 

The intestinal epithelium provides a crucial barrier against external pathogens. Disruption of this intestinal barrier leads to microbial translocation, compromised immune surveillance and thus can result in systemic infections. Specialized immune cells play an important role in maintaining intestinal integrity and microbial homeostasis. In order to analyze the impact of individual cell populations on sustaining the intestinal integrity, we created a novel mouse model, which allows us to identify, sort, or specifically delete individual cell subsets based on the expression of the chemokine receptor type 5 (CCR5). In this mouse model, we replaced the coding region of CCR5 with a cDNA coding primate diphtheria toxin receptor (pDTR) and eGFP fusion, so that the altered allele constitutes a CCR5 null and renders the cells carrying this allele sensitive to non-inflammatory apoptosis via injected diphtheria toxin(DT). Here, we showed that administration of DT lead to a complete loss of NK cells as well as a partial loss of NKT cells, CD44+CD4 cells, CD44+CD8 cells, gdT cells and CD11c+ dendritic cells. Interestingly, sustained DT-treatment induced dramatic weight loss, hypothermia and ultimately mortality, which we found was due to the loss of hematopoietic cells. Finally, the administration of a broad-spectrum antibiotic cocktail prevented the occurrence of illness, indicating its dependency of gut integrity. All together, we generated a novel and flexible mouse model in which CCR5+ cells can be identified, sorted, or killed with the administration of DT. Our findings characterized the key importance of CCR5 on different immune cell subsets and suggest that CCR5+ immune cells play an essential role in intestinal immune homeostasis on several levels.

   



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  Cell death without the mess: Understanding specific contributions of cell death programs in shaping immunity.
  Presenter: Nicholas Hubbard
  All Authors:Nicholas Hubbard, Andrew Oberst, Annelise Snyder, Michelle Messmer
  University of Washington
   
 

Inflammatory and non-inflammatory forms of programmed cell death (PCD) such as necroptosis, pyroptosis and apoptosis has been increasingly indicated a key player in the development of pathogenic and protective immune responses, particularly in privileged environments such as the gut, lung and skin. Notably, each of these cell death programs is hypothesized to yield distinct immune consequences due unique features intrinsic to each process resulting in unique cytokine, chemokine and DAMP profiles. Despite these implications, programmed cell death in disease settings occurs downstream of complex, pleiotropic signals such as viral, bacterial or fungal infection, complicating our understanding of the specific effects of cell death on the immune response. To address this question directly, we have developed a novel system, for rapid and specific induction of ‘pure death’ via necroptosis, pyroptosis or apoptosis using constitutively active forms of the cell death effectors Caspase-9 (apoptosis), RIP3K (necroptosis) or Caspase-1 (pyroptosis). When delivered to target tissue using recombinant adeno-associated virus (AAV), these proteins induce rapid, specific, controlled in vivo cell death. Further, we extend this approach to selectively activate the individual molecular events contained within each cell death program, allowing us to further dissect specifically how each process contributes to the downstream immune response, focusing initially on the lung as a target organ. Programmed cell death in the lungs represent a challenge due to their extensive contact with the outside environment, variety of immune challenges and intolerance to damage. Highlighting this, damage the lung epithelium by a variety of allergens, environmental pollutants and respiratory infections has been linked to the development of asthma and allergy, leading us to predict that multiple forms of programmed cell death mechanisms are relevant in this in this tissue. We hypothesize that activation of different cellular death pathways in the lung epithelium will differentially impact innate and adaptive immunity, and that this contributes to both appropriate and inappropriate host responses.

   



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  Activation of ZBP1 dependent necroptosis during homeostasis and development.
  Presenter: Nicholas Hubbard
  All Authors:Nicholas Hubbard, Megan Maurano, Daniel Stetson, Andrew Oberst
  University of Washington, Department of Immunology
   
 

Necroptosis is a form of inflammatory programmed cell death mediated by Receptor-Interacting-Protein-Kinase 3 (RIPK3) that likely evolved as an antiviral defense mechanism. In the context of infection, the critical upstream initiator of necroptosis is the sensor Z-DNA binding protein 1 (ZBP1). However, the cognate ligand and activation mechanism for ZBP1 remains poorly understood, complicated by the observation that a diverse array of both RNA and DNA viruses, including Influenza, Cytomegalovirus and West Nile Virus, trigger ZBP1-dependent cell death. Recent reports of sterile ZBP1 activation during development point to the possibility that ZBP1 can also be activated in response to endogenous cellular ligands. This suggests that ZBP1 signaling relies on a combination of tightly controlled expression, regulated activation, and cellular state with respect to infection or stress. Our studies indicate, that under homeostatic conditions, ZBP1 activation of necroptosis is controlled by ADAR1, an RNA-modifying enzyme that suppresses aberrant immune responses to self double-stranded RNA (dsRNA). Notably, ZBP1 and ADAR1 are the only mammalian proteins to contain a Z-DNA Binding Domain. These preliminary findings implicate dsRNA as a ligand for ZBP1, and identify ZBP1 as potential mediator of pathology in settings of ADAR1 deficiency. Using a mouse model of ADAR1 deficiency, we demonstrate that deletion of ZBP1 rescues the associated growth and survival defects. These combined findings indicate a previously unknown and physiologically relevant role for ZBP1 during the homeostatic response to dsRNA. Future work will focus on further defining this unexpected mechanism for the activation of necroptosis by an endogenous ligand, and explore the pathways required for negative regulation during the maintenance of cellular homeostasis and viral infection. These findings will identify and characterize a previously unknown potential source of inflammation and pathology which may contribute during a range of human autoimmune diseases.

   



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  Role of maternal mucosal immunity on progeny susceptibility to type 1 diabetes
  Presenter: Baweleta Isho
  All Authors:Baweleta Isho, Leili Marandi and Philippe Poussier
  University of Toronto and Sunnybrook Research Institute
   
 

Type 1 Diabetes (T1D) is a disorder of glucose homeostasis resulting from the destruction of insulin-producing pancreatic β-cells by T lymphocytes. Multiple genetic and environmental risk factors contribute to disease susceptibility. While environmental determinants remain elusive in humans, studies in the spontaneously diabetic NOD mouse have demonstrated that the differential susceptibility of males and females to disease is associated with the composition of the intestinal microbiome and its impact on systemic testosterone levels. Here, using the progeny of crosses between NOD and immunocompromised NOD.scid mice, we tested the hypothesis that maternal mucosal immunity plays a role in the pathogenesis of diabetes through its impact on microbiome composition. We observed that in the prediabetic stage, the number of animals with anti-insulin autoantibodies was lower in the progeny of NOD.scid mothers than in that of NOD mothers (13.3%, n = 15 vs 58.8%, n = 17, p = 0.008). Later, female mice born to NOD.scid mothers had a lower T1D incidence when compared to those born to NOD mothers (61.1%, n = 20 vs 95.0%, n = 20, p<0.05) while the maternal immune status did not influence disease incidence in male progeny born to NOD.scid or NOD mothers (43.4%, n = 25 vs 61.5%, n = 13, p>0.05). Strikingly, disease protection conferred by NOD.scid mothers was associated with changes in sex hormone levels in the plasma. Specifically, testosterone levels in male progeny of NOD.scid mothers were significantly lower than in males born to NOD mothers (1.0±1.5 ng/ml, n = 8 vs 10.7±11.3 ng/ml, n = 8, p<0.05). Similarly, plasma levels of the active estrogen, estradiol, were lower in female progeny of NOD.scid mothers than in that of NOD mothers (30.2±21.7 pg/ml vs 113.1±33.5 pg/ml, p<0.05). Ongoing studies are investigating the contribution of the intestinal microbiome and maternal secretory IgA responses to these hormonal changes and the modulation of islet inflammation.

   



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  Enterohemorrhagic E. coli type III-secreted immunomodulatory effectors suppress acute inflammation in vivo and promote lethal disease.
  Presenter: Laurice Jackson
  All Authors:Laurice Jackson, Ilan Rosenshine, John Leong
  Tufts University, The Hebrew University of Jerusalem
   
 

Enterohemorrhagic Escherichia coli (EHEC) is an important enteric pathogen. It belongs to the family of attaching and effacing pathogens (AE pathogens), which include Enteropathogenic E. coli (EPEC) and the natural murine pathogen Citrobacter rodentium (CR). Upon binding host cells, these organisms efface microvilli and trigger localized assembly of host actin, forming pedestal-like structures beneath the attached bacterium. EHEC is distinguished from EPEC and CR by its ability to cause systemic disease through the production of the EHEC phage-encoded Shiga toxin (Stx), which can cause hemolytic uremic syndrome (HUS), involving the destruction of red blood cells, consumption of platelets, and kidney failure. Antibiotic use, which can result in phage induction and high-level production of Stx, is contraindicated and there is no therapy to protect individuals against this infection. To colonize and cause disease EHEC, EPEC, and CR utilize a type three system (TTSS) to inject dozens of protein ‘effectors’ (i.e. toxins) into host cells. Amongst these effectors is a subset that modulates innate inflammatory responses. In vitro characterization of these immunomodulatory effectors using HeLa cells has revealed several modes of action. Of note are the effectors NleE, NleH, and NleB, which act to inhibit NF-kB signaling, apoptosis, and TNF-alpha signaling, respectively. CR ( ΦStx2dact), an Stx-producing version C. rodentium (CR), was recently shown, upon infection of mice, to mimic many of the features of human EHEC disease, including renal dysfunction. We show here that CR ( ΦStx2dact) ?BEH, which lacks NleB, NleE, and NleH, was able to colonize the intestines of mice and triggered a much greater acute intestinal inflammatory response than wild type CR ( ΦStx2dact). However, this strain was unable to cause lethal infection. Moreover, in an initial experiment, infection with CR ( ΦStx2dact) ?BEH elicited an immune response that prevented subsequent colonization or disease after an otherwise lethal challenge with CR ( ΦStx2dact). Thus, NleB, NleE and/or NleH play essential roles in suppressing the acute intestinal inflammatory response and the development of a protective immune response.

   



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  Innate control of memory CD4 T cell effector function
  Presenter: Aakanksha Jain
  All Authors:Aakanksha Jain, Aakanksha Jain, Chandrashekhar Pasare
  University of Texas Southwestern Medical Center
   
 

Pathogen recognition by DCs via activation of pattern recognition receptors results in the up-regulation of MHC and co-stimulatory molecules and secretion of pro-inflammatory cytokines. While naive CD4 T cells require all three of these signals from DCs for successful activation and differentiation, MHC-TCR interaction is thought to be sufficient for memory CD4 T cell reactivation. Surprisingly, we find that reactivation of memory CD4 T cells via TCR alone induces significantly less effector cytokine production as compared to DC mediated reactivation suggesting that additional innate signals still play a role. Further investigation revealed a requirement for IL-1β dependent signaling in addition to TCR activation for optimal cytokine production by reactivated Th17 cells. This is consistent with previous studies showing that lack of IL-1R on T cells leads to defective Th17 responses. Interestingly, here we find that IL-1R deficient mice harbored similar proportions of T cells committed to the Th17 lineage as measured by Rorγt expression and IL-17A production. Instead, T cell intrinsic IL-1R signaling was required for Th17 cytokine secretion upon reactivation of polyclonal or antigen specific memory CD4 T cells from secondary lymphoid organs. We obtained similar results from in vivo reactivation of lamina propria resident Th17 cells highlighting the global requirement for IL-1R signaling for Th17 effector function irrespective of their tissue of residence. Collectively, our data point to a novel innate controlled regulation of memory Th17 cell function and warrants revisiting the currently established paradigm for memory CD4 T cell reactivation.

   



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  Genomic Analysis Of Bone Marrow Progenitors During Viral Infection Reveals Novel Dendritic Cell Regulators
  Presenter: Yeara Jo
  All Authors:Yeara Jo, Kai Zhang, Wei Wang, Elina Zuniga
  University of California, San Diego
   
 

Dendritic cells (DC) play a central role in immune responses and can be broadly subdivided into conventional (c) as well as plasmacytoid (p) DCs. Notably, we and others have described several adaptations of DCs and their progenitors during acute and chronic infections, including impaired DC development, maturation and altered cytokine production. To understand the mechanisms underlying such adaptations we determined the transcriptional and chromatin landscapes of bone marrow (BM) DC progenitors from lymphocytic choriomeningitis virus (LCMV) infected mice, via RNA-Seq and ATAC-Seq, respectively. Initial analysis indicated that infection induced multiple alterations in gene signatures, including type-I-interferon signaling and metabolic pathways, as well as changes in chromatin accessibility, which were more enriched within intergenic regions. We next used the Taiji algorithm, which integrates chromatin accessibility and gene expression to assess the global importance of transcription factors (TFs) at the systems level. This analysis identified 11 known DC regulators and 24 TFs with no previous connection to DC biology. Follow-up knock-down experiments revealed that Glucocorticoid Modulatory Element Binding Protein 1 (Gmeb1), which was predicted to exhibit increased activity in progenitors from LCMV-infected mice, suppressed DC development and maturation. On the other hand, Zinc Finger Protein 524 (Zfp524), whose activity was predicted to be reduced in progenitors from LCMV-infected mice, promoted pDC cytokine production while inhibiting the same function in cDCs. These results highlight two novel TFs that regulate DC development and/or function, significantly deepening our understanding of DC biology and providing potential new targets for DC-based immunotherapies in infectious and non-infectious diseases.

   



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  Determining how the Helicobacter pylori chemoreceptor TlpA modulates host inflammation
  Presenter: Kevin Johnson
  All Authors:Kevin Johnson, Christina Yang, J. Elliot Carter, Karen M. Ottemann
  UC Santa Cruz
   
 

Helicobacter pylori is Gram-negative pathogen that colonizes the stomach of half of the world’s population. Colonization results in chronic inflammation, and a subset of individuals develop ulcers, and gastric cancer. H. pylori utilizes its chemotaxis system to promote chronic stomach colonization and to modulate host inflammation. Chemotaxis enables the bacteria to regulate its motility in response to environmental signals that are sensed by chemoreceptors. The chemoreceptor TlpA was previously shown to suppresses the severity of host inflammation, as ?tlpA H. pylori caused hyperinflammation in a murine model but colonized to wild-type (WT) levels. Given that the severity of inflammation drives the progression of more serious disease outcomes, such as peptic ulcers and gastric cancer, we want to understand how TlpA modulates host inflammation. Since this work, it has become appreciated that chemotaxis regulates the fine scale localization of H. pylori between the gastric glands and mucosa of the stomach corpus and antrum. Accordingly, we revisited this phenotype, preforming an 8-month time course examining the localization of WT and ?tlpA H. pylori SS1 within the gastric glands and mucus of the stomach corpus and antrum. Gland localization was assessed using a method our lab developed, termed Bacterial Localization in Isolated Glands (BLIG), which utilizes H. pylori strains expressing GFP under the control of the urease promoter from pTM115. Additionally, inflammation was assessed by determining the density and distribution of lymphocytes recruited to the stomach corpus and antrum via histology. From this study, we discovered ?tlpA H. pylori becomes trapped within corpus glands, reaching high numbers per gland, and induces more inflammation, compared to a WT infection. Future work will aim to determine whether gland localization alone drives the progression of hyperinflammation by ?tlpA H. pylori and will characterize this hyperinflammatory response.

   



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  Repertoire Analysis of influenza-specific Tbet+ and Tbet- memory B cell pools
  Presenter: John Johnson
  All Authors:John Johnson, James J. Knox, Arpita Myles, Rebecca L. Rosenthal, Mariya Kostiv, Shannon R. Christensen, Jonathan Yewdell, David H. Canaday, Jinfang Zhu, Adrian B. McDermott, Yoav Dori, Max Itkin, Wenzhao Meng, Aaron Rosenfeld, Shannon Barbour, Scott E. Hensley, E. John Wherry, Norbert Pardi, Drew Weissman, Ali Naji, Michael R. Betts, Eline Luning Prak, and Michael P. Cancro
  University of Pennsylvania
   
 

Antibodies that bind the conserved stalk region of influenza hemagglutinin can be broadly-neutralizing, but the B cell response that produces stalk-specific antibody is poorly understood. We have recently identified a subset of hemagglutinin-specific B cells that co-expresses the transcription factor T-bet and the myeloid lineage marker CD11c during influenza challenge that establish a long-lived memory population after infection. Using a B-lineage knockout of T-bet we have also shown that this B cell subset is required for the hemagglutinin stalk antibody response, but the relationship between T-bet+ and T-bet- hemagglutinin-specific B cells is poorly understood. To address the question of whether these memory B cell subsets are stable and separate pools we sequenced the BCRs of both subsets within individual mice after immunization. Examination of heavy-chain CDR3s shows a similar distribution of CDR3 length for both T-bet+ and T-bet- hemagglutinin-specific B cells. Moreover, both memory pools show a similar degree of somatic hypermutation which is consistent with our observations that both these subsets transit through germinal centers during the primary response. Analysis of the clonal overlap between the T-bet+ and T-bet- subsets reveals some clonal sharing, but also indicates that many clones are unique to each subset. Lineage tree analysis of the shared clones demonstrates that T-bet+ can bifurcate from T-bet- cells after somatic hypermutation and that these two subsets largely remain as separate pools after their bifurcation. We are following up on these observations using genetic fate-mapping tools to better understand the fate of T-bet+ hemagglutinin-specific B cells.

   



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  Engineering High Affinity T-Cell Receptors Specific for Listeria monocytogenes.
  Presenter: Deborah Johnson
  All Authors:Deborah Johnson, Bryce E. Anderson, Kemais Ehlers, Stephen P, Persuad, and K. Scott Weber
  Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT; Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO
   
 

CD4+ T cells are central players in immunity to infection and help coordinate both the adaptive and the innate responses to infection. CD4+ T cell activation is dependent on the binding interactions between the T cell receptor (TCR) and peptide-MHC (pepMHC). TCRs have very low affinity for pepMHC; thus we determined to generate pathogen specific high affinity T cell receptors to test if T cell activation and memory cell formation is improved with increased pepMHC affinity. To do this we used two CD4+ T cells, called LLO118 and LLO56, which are specific for the same naturally occurring Listeria monocytogenes epitope. These two T cells have similar affinity for pepMHC and differ by 15 amino acids found mainly in the CDR3????region. Despite these similarities, LLO118 has a stronger primary response while LLO56 has a stronger secondary response. We generated single chain LLO118 and LLO56 TCRs (Vβ2-linker-Vα2) and reasoned that we could use directed evolution to generate stabilized and then high affinity mutants. Single chain LLO118 and LLO56 were fused to the yeast surface protein Aga-2 and error prone PCR was used to generate mutagenic libraries. Stabilized single chain TCRs were selected for using biotinylated Vβ2 and Vα2 antibodies and anti-biotin beads. LLO118 and LLO56 stabilized clones were then used as templates to generate affinity libraries using site directed mutagenesis of the CDR3? region. We have sorted the LLO118 library with pepMHC tetramers (LLO190-201) and have isolated clones with improved binding affinity. Generation of high affinity pathogen specific TCRs should increase our understanding of the relationship between TCR:pepMHC avidity and T cell activation. These TCRs will also aid in attempts to identify the characteristics of CD4+ T cells needed for effective memory cell generation and should provide useful insights for improving vaccine design and immunotherapies.

   



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  Determining how the Helicobacter pylori chemoreceptor TlpA modulates host inflammation
  Presenter: Kevin Johnson
  All Authors:Kevin Johnson, Christina Yang, J. Elliot Carter, Karen M. Ottemann
  UC Santa Cruz
   
 

Helicobacter pylori is Gram-negative pathogen that colonizes the stomach of half of the world’s population. Colonization results in chronic inflammation, and a subset of individuals develop ulcers, and gastric cancer. H. pylori utilizes its chemotaxis system to promote chronic stomach colonization and to modulate host inflammation. Chemotaxis enables the bacteria to regulate its motility in response to environmental signals that are sensed by chemoreceptors. The chemoreceptor TlpA was previously shown to suppresses the severity of host inflammation, as ?tlpA H. pylori caused hyperinflammation in a murine model but colonized to wild-type (WT) levels. Given that the severity of inflammation drives the progression of more serious disease outcomes, such as peptic ulcers and gastric cancer, we want to understand how TlpA modulates host inflammation. Since this work, it has become appreciated that chemotaxis regulates the fine scale localization of H. pylori between the gastric glands and mucosa of the stomach corpus and antrum. Accordingly, we revisited this phenotype, preforming an 8-month time course examining the localization of WT and ?tlpA H. pylori SS1 within the gastric glands and mucus of the stomach corpus and antrum. Gland localization was assessed using a method our lab developed, termed Bacterial Localization in Isolated Glands (BLIG), which utilizes H. pylori strains expressing GFP under the control of the urease promoter from pTM115. Additionally, inflammation was assessed by determining the density and distribution of lymphocytes recruited to the stomach corpus and antrum via histology. From this study, we discovered ?tlpA H. pylori becomes trapped within corpus glands, reaching high numbers per gland, and induces more inflammation, compared to a WT infection. Future work will aim to determine whether gland localization alone drives the progression of hyperinflammation by ?tlpA H. pylori and will characterize this hyperinflammatory response.

   



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  CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors
  Presenter: Lokesh A. Kalekar
  All Authors:Lokesh A. Kalekar, Shirdi E. Schmiel, Na Zhang, Gretta L. Stritesky, Deepali Malhotra, Kristin A. Hogquist, Marc K. Jenkins, Daniel L. Mueller
  Center for Immunology, University of Minnesota - Twin Cities
   
 

Selective suppression of effector T cell functions in the periphery is required for preventing immune cell-mediated damage to healthy tissues. This is especially true during many health conditions such as pregnancy and organ transplants. Anergy, an acquired state of T cell functional unresponsiveness, is one way in which this suppression can be achieved. However, anergy as a peripheral tolerance mechanism remains poorly understood. In this study, we demonstrate that anergy is selectively induced in fetal antigen-specific maternal CD4+ T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4+ T cells, enriched in self antigen-specific T cell receptors, is also observed in healthy hosts. Neuropilin-1 expression in anergic conventional CD4+ T cells is associated with thymic regulatory T cell (Treg cell)-related gene hypomethylation, and this correlates with their capacity to differentiate into Foxp3+ Treg cells that suppress immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity, but it also generates precursors for peripheral Treg differentiation.

   



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  Autophagy protects monocytes from Wolbachia heat shock protein 60 (rWmhsp60) induced apoptosis and senescence
  Presenter: Vijayan Kamalakannan
  All Authors:Vijayan Kamalakannan, Shiny C, Babu S, Narayanan RB
  Center for Biotechnology, Anna University
   
 

Monocyte dysfunction by filarial antigens have been postulated as major mechanism underlying immune evasion following hypo responsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis in monocytes, lymphocyte anergy, etc. Here we showed that rWmhsp60 (recombinant Wolbachia heat shock protein 60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal, but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced following TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-?B p65 nuclear translocation which lead to inflammation and apoptosis via TNF-receptor pathway following the increase in IL-6 and TNF-a levels. Here for the first time, we report that in addition to apoptosis, rWmhsp60 a possible antigen in filarial pathogenesis also induce molecular senescence in monocytes. Targeting TLR-4 therefore presents a promising candidate for treating rWmhsp60 induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin, detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction thus protecting TLR-4 mediated apoptosis and senescence. Also, rapamycin induced monocytes were unresponsive to rWmhsp60 and triggered lymphocyte activation and proliferation following PHA stimulation. With the perspective of the evidences put forward, targeting inherent autophagic degradation pathway may provide perfect catalyst to herald a potential area of research for the development of inherent methods to treat filariasis and other inflammatory conditions.

   



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  Tie2 Signaling Enhances Mast Cell Progenitor Adhesion to Vascular Cell Adhesion Molecule-1 (VCAM-1) through [a]4[b]1 Integrin
  Presenter: Kazumasa Kanemaru
  All Authors:Kazumasa Kanemaru, Emiko Noguchi, Takahiro Tokunaga, Kei Nagai, Takashi Hiroyama, Yukio Nakamura, Satoko Tahara-Hanaoka, Akira Shibuya
  Department of Immunology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
   
 

Mast cell (MC) activation contributes considerably to immune responses, such as host protection and allergy. Cell surface immunoreceptors expressed on MCs play an important role in MC activation. Although various immunoreceptors on MCs have been identified, the regulatory mechanism of MC activation is not fully understood. To understand the regulatory mechanisms of MC activation, we used gene expression analyses of human and mouse MCs to identify a novel immunoreceptor expressed on MCs. We found that Tek, which encodes Tie2, was preferentially expressed in the MCs of both humans and mice. However, Tie2 was not detected on the cell surface of the mouse MCs of the peritoneal cavity, ear skin, or colon lamina propria. In contrast, it was expressed on mouse bone marrow–derived MCs and bone marrow MC progenitors (BM-MCps). Tie2 is a receptor tyrosine kinase containing two tyrosine kinase domains in the cytoplasmic portion. Upon binding with its ligand Angiopoietin-1 (Ang1), Tie2 mediates an activating signal. Since Ang1 is expressed by peri-endothelial mural cells, we hypothesized that Tie2 would be involved in the transmigration of MCps across the vascular endothelium. Indeed, in vitro assay demonstrated that Tie2 signaling enhanced MCp adhesion to VCAM-1 via α4β1 integrin. These results suggest that Tie2 play an important role in MCp adhesion to VCAM-1 on vascular endothelial cells and transmigration to inflammatory sites.

   



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  B cell responses to vaccines in the aged
  Presenter: Senthil Kannan
  All Authors:Senthil Kannan, Hildegund C.J. Ertl, Raj Kurupati, Larissa Haut, Ken E. Schmader, Susan Doyle,
  University of Pennsylvania , The Wistar Institute
   
 

Vaccines have prevented millions of deaths ranging from primarily pediatric diseases like Rubella to more diverse ones like Influenza, Measles and Shingles. However, the efficiency of vaccines drops sharply with age, and thus leaves our aging population at a disadvantage. This decrease in immune function – immunosenescence – is thus a vital area of interest. My research addresses immunosenescence in mice and humans, specifically looking at B cells and Antibody Secreting Cells and their responses to the inactivated Influenza vaccine. This has resulted in a number of interesting findings. A number of differences were observed by multicolor flow cytometry between young and aged mice including differences in expression of immune regulators like Programmed Death -1 (PD-1) and its ligand Programmed Death Ligand-1 (PD-L1). Metabolic differences were also observed, including higher levels of basal mitochondrial respiration in the young mice. We conducted similar studies in young and aged humans receiving the inactivated influenza vaccine, and observed a role of the B and T Lymphocyte Attenuator (BTLA) - Herpes Virus Entry Mediator (HVEM) pathway in generating antibody responses, and ASC numbers.

   



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  A role for group 2 innate lymphoid cells in muscular dystrophy
  Presenter: Jenna Kastenschmidt
  All Authors:Jenna Kastenschmidt, Ileen Avetyan, Rayan Yahia, Rachel Ayer, S. Armando Villalta
  University of California, Irvine
   
 

A recently identified innate lymphocyte population - group 2 innate lymphoid cells (ILC2s) - were shown to be critical regulators of type II immune responses and tissue repair. Given that type II immune responses are required for efficient skeletal muscle regeneration, we sought to test the hypothesis that ILC2s promote muscle regeneration by regulating type II inflammatory responses in mdx mice. The mdx mouse is a model of Duchenne muscular dystrophy (DMD) that is characterized by cyclic bouts of muscle degeneration and regeneration. We show that ILC2s are activated, reflected by increased numbers in dystrophic muscle and elevated expression of IL-5. IL-2/anti-IL-2 complex (IL-2c) and IL-33 effectively increased the number of ILC2s and expression of type 2 cytokines (IL-5 and IL-13), indicating that ILC2s are capable of promoting type II immune responses in dystrophic muscle. Additionally, we found IL-2c/IL-33 treatment increased myofiber cross sectional area, suggesting enhanced muscle regeneration. Collectively our data support a working model in which ILC2s promote type II immune responses in dystrophic skeletal muscle to promote regeneration.

   



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  The role of group 2 innate lymphoid cells in the pathogenesis of mdx skeletal muscle
  Presenter: Jenna M. Kastenschmidt
  All Authors:Jenna M. Kastenschmidt, Ileen Avetyan, S. Armando Villalta
  University of California, Irvine
   
 

Group 2 innate lymphoid cells (ILC2s) are critical regulators of type 2 inflammatory responses, and function to promote tissue repair and restoration of homeostasis. However, whether these repair processes include regenerative responses that encompass ILC2 and tissue stem cell interactions is largely unknown. To address this question, we examined the role of ILC2s in regulating muscle regeneration, a tissue with a high regenerative capacity that is dependent on a well-defined muscle stem cell population (i.e. satellite cells). We show that ILC2s are activated and numbers are elevated in skeletal muscle of mdx mice, a model of Duchenne muscular dystrophy (DMD) in which the regulation of disease pathogenesis is controlled in part by the balance between type I and type II immune responses. Gain-of-function studies using IL-2/anti-IL2 complex (IL-2c) showed that this treatment effectively increased the number of ILC2s in mdx skeletal muscle and increased myofiber cross sectional area, suggesting enhanced muscle regeneration. In addition, ablation of IL-13-producing cells, including ILC2s, inhibited the recruitment of muscle eosinophils, an innate immune cell population previously implicated in muscle regeneration. Collectively our data support a working model in which ILC2s ameliorate the pathogenesis of muscular dystrophy by promoting muscle regeneration, likely through the regulation of satellite cell function and type II inflammatory responses that promote skeletal muscle regeneration.

   



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  Increased recruitment of inflammatory monocytes contributes to age related lung pathology during respiratory syncytial virus infection
  Presenter: Gerald Kellar
  All Authors:Gerald Kellar, Kathryn L. Pothoven PhD, Sabine Spath PhD, Steven F. Ziegler PhD
  Benaroya Research Institute; University of Washington Immunology Department; U.S. Army Medical Department
   
 

With decreasing air quality worldwide due to manmade and natural circumstances chronic pulmonary disorders are becoming an ever-increasing healthcare burden. Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma; with no vaccine available and only one catastrophic trial attempted in the late 1960’s, a better understanding of the infectious progression of this pathogen is critical to better understanding how to combat it. The immune profile of RSV infected 3-week versus 8-week old C57BL/6 mice were examined to differentiate a juvenile vice adult profile, respectively; 3-week old mice displayed increased recruitment of C-C chemokine receptor-2 (CCR2) positive inflammatory monocytes (MO) with increased production of C-C chemokine ligand-2 when compared to 8-week old mice. CCR2+ MO have been linked to the exacerbation of pulmonary pathology during influenza virus infection where these cells are thought to contribute to tissue destruction while minimally contributing to viral clearance; therefore, we hypothesize that the increased recruitment of CCR2+ MO in the 3-week old RSV infected mice contribute to tissue remodeling that is characteristic of the progression to asthma. Histological staining of lungs from the age-related mouse groups demonstrate that the 3-week old mice display increased accumulation of extracellular matrix (ECM) components, particularly hyaluronan and versican, both of which are traditionally increased in the asthmatic lung and can be produced and/or degraded by the MO. Additionally, the 3-week old mice have increased production of interleukin-17 and elevated lung neutrophils at 72-hours post infection, both of which have been linked to complications of RSV infection while possibly further contributing to tissue destruction. This data suggests that the immune profile of the juvenile lung could result in unproductive tissue & ECM destruction that could facilitate the conditions which support the development of asthma.

   



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  Increased recruitment of inflammatory monocytes contributes to age related lung pathology during respiratory syncytial virus infection.
  Presenter: Gerald Kellar
  All Authors:Gerald Kellar, Katie Barrow, Kathryn L. Pothoven, Steven Reeves, Sabine Spath, and Steven F. Ziegler
  University of Washington-Benaroya Research Institute
   
 

With decreasing air quality worldwide chronic pulmonary disorders are becoming an ever-increasing healthcare burden. Early life respiratory syncytial virus (RSV) infection has been linked to the onset of asthma; with no vaccine available and only one catastrophic trial attempted in the late 1960’s, a better understanding of the infectious progression of this pathogen is critical to better understanding its nature. The initial immune profile of RSV infected 3-week versus 8-week old C57BL/6 mice were examined to differentiate a juvenile vice adult profile, respectively; 3-week old mice displayed an enduring myeloid presence (including monocytes (MO), alveolar macrophages (AM), and neutrophils) with increased production of C-C chemokine ligand-2 (CCL2), CCL3, and CCL4 when compared to 8-week old mice at 72-hours post infection. Histological staining demonstrates that the 3-week old mice display increased accumulation of the extracellular matrix (ECM) component hyaluronan (HA) in alveolar spaces, which is traditionally increased in the asthmatic lung and can be generated and/or degraded by the MO. MO have also been linked to the exacerbation of pulmonary pathology during influenza virus infection where these cells are thought to contribute to tissue destruction while minimally contributing to viral clearance; therefore, we hypothesize that the increased recruitment of MO in the 3-week old RSV infected mice contribute to tissue remodeling that is characteristic of the progression to asthma. The specific activity of MO was examined in vitro utilizing an air-liquid interface culture involving the human U937 monocyte cell line, confirming the CCL production trend observed in the mice while further implicating matrix metalloproteinase-7 (MMP7) and MMP9 as further drivers of ECM destruction. Additionally, the enduring AM and neutrophilia presence in the 3-week old mice could further contribute to continued CCL production and general ECM dysregulation due to the nature of these cells. This data suggests that the immune profile of the juvenile lung during acute RSV infection facilitates unproductive ECM remodeling that could facilitate the conditions which support the development of asthma.

   



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  IL-27p28 production by DCs predicts adjuvant-elicited CD8+ T cell responses
  Presenter: Augustus Kilgore
  All Authors:Augustus Kilgore, Seth Welsh, Benjamin J. Kedl, Nathan D. Pennock, Christopher A. Hunter, Ross M. Kedl
  University of Colorado Denver Anschutz Medical Campus, School of Medicine, Department of Immunology and Microbiology
   
 

Generating effective T cell responses to subunit immunization is one of the major unsolved challenges of vaccine development. Attempts to address this challenge have succeeded in creating robust inflammatory environments but have failed to produce the level of T cell response needed for protective or therapeutic immunity. We previously reported that T cell responses induced by subunit vaccination were dependent on the cytokine IL-27. Using a novel IL-27p28-GFP reporter mouse, we show here that the degree to which an adjuvant induces IL-27p28 production from dendritic cells and monocytes directly predicts the subsequent magnitude of the T cell response. These data are the first to identify a concrete innate correlate of vaccine-elicited cellular immunity. Of note, our reporter indicates that IL-27 production by DCs in response to adjuvant administration is limited to 6-18 hours post vaccination, a time frame that is highly amenable to evaluation by multiphoton microscopy. We have thus utilized this technique to evaluate by live imaging the influence of IL-27 on T cell localization, migration and interaction with DCs during the earliest periods of the subunit vaccine-induced T cell response. Our data continue to support the assertion that divergent biological mechanisms guide subunit vaccine-elicited immunity versus infection-generated immunity.

   



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  Genetic and pharmaceutical targeting of OCA-B/Pou2af1 blocks T1D in mouse models
  Presenter: Heejoo Kim
  All Authors:Heejoo Kim, Arvind Shakya, Jelena Perovanovic, Andrea Ibarra, Brian Evavold, Danny H.-C. Chou, Xiao He, Peter E Jensen and Dean R Tantin
  University of Utah
   
 

Treatments for autoimmunity aim to inhibit autoreactivity while preserving normal immune function. The transcriptional coregulator OCA-B (gene symbol Pou2af1) is induced in stimulated naïve CD4+ T cells, where docks with transcription factor Oct1 to regulate genes such as Il2 and Ifng. Oct1 and OCA-B only regulate their targets in cases of persistent antigen exposure, a common feature of autoimmunity. We hypothesized that T cell- specific OCA-B deletion would protect mice from autoimmune diabetes, and that pharmacologic OCA-B inhibition would similarly protect diabetes-prone mice. We generated an Ocab(Pou2af1) conditional allele onto a NOD/ShiLtJ strain background. OCA-B loss in T cells had no effect on the naïve T cell receptor repertoire or gene expression in the pancreatic lymph nodes, but protected NOD mice from both mice spontaneous and induced T1D. In scRNA-seq data with pancreatic leukocytes, dominant CD8 TCR clones known to participate in disease failed to emerge in the islets of OCA-B deficient mice. Protection was also associated with reduced islet neutrophil and macrophage infiltration and reduced neutrophil activation. The protective effect of OCA-B loss was diminished, or even eliminated, using monoclonal TCR models with high affinity to artificial or neoantigens. Finally, we developed rationally-designed membrane-penetrating peptide inhibitors of the interaction between OCA-B and its downstream target, Jmjd1a. Pharmacologic OCA-B inhibition normalized glucose levels, and reduced T cell infiltration and proinflammatory cytokine expression, in newly-diabetic NOD mice. Together, the results indicate that OCA-B is a potent autoimmune regulator and a promising target for pharmacologic inhibition.

   



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  Role of a single self-peptide in the prevention of organ-specific autoimmunity
  Presenter: David E.J. Klawon
  All Authors:David E.J. Klawon, Dana C. Gilmore*, John D. Leonard, Jaime L. Chao, Ryan K. Duncombe, Erin J. Adams, Peter A. Savage
  Department of Pathology, University of Chicago; Department of Biochemistry and Molecular Biology, University of Chicago
   
 

The display of agonist peptide ligands in the thymus, driven in part by Aire, can promote both the clonal deletion of antigen-specific T cells and the differentiation of such cells into the Treg cell lineage. Through the identification and study of T cells reactive to two natural Aire-dependent self-peptide ligands derived from the prostatic protein Tcaf3, and using gene-targeted mice lacking one of these epitopes, we define the role of cognate antigen expression in directing these alternate cell fates and preventing prostatic infiltration by antigen-specific T cells. We show that expression of a single peptide ligand in the thymus is required to direct antigen-specific monoclonal cells into the Treg lineage, and has a negligible role in driving concurrent clonal deletion. Moreover, T cell transfer experiments reveal that skewing of this single specificity to the Treg compartment is critical for the prevention of prostate-specific T cell infiltration. Thus, the protection of a peripheral organ from autoimmune attack can be dependent on T cell selection against a single self-peptide ligand.

   



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  Naïve CD8 T cell IFN[g] responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5
  Presenter: Angel K. Kongsomboonvech
  All Authors:Angel K. Kongsomboonvech, Felipe Rodriguez, Anh L. Diep, Brandon M. Justice, Brayan E. Castallanos, Ana Camejo, Debanjan Mukhopadhyay, Gregory A. Taylor, Masahiro Yamamoto, Jeroen P.J. Saeij, Michael L. Reese, Kirk D.C. Jensen
  University of California, Merced
   
 

Host resistance to Toxoplasma gondii infections rely on CD8 T cell IFNγ responses. Since manipulation of CD8 T cell IFNγ responses may influence T. gondii’s ability to achieve chronic infection, we investigated host and parasite’s requirements for eliciting this response. To this end, naïve CD8 T cell IFNγ responses to the endogenous T. gondii vacuolar resident antigen, TGD057, were analyzed. TGD057 antigen-specific CD8 T cells (T57), isolated from naïve transnuclear mice, responded to T. gondii-infected bone marrow-derived macrophages in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 antigen were independent of the parasite’s protein export machinery MYR1 and ASP5. Instead, host immunity pathways downstream of regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D T. gondii strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected cells. T57 response differences between clade A and B, C, D, F T. gondii strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to T. gondii. Instead macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. In contrast, other members of the conventional inflammasome cascade are only partly required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11 and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, and NLR-dependent but inflammasome-independent pathways determine full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

   



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  Defining the role of Plasmodium-specific memory B cells
  Presenter: Akshay Krishnamurty
  All Authors:Akshay Krishnamurty, Marion Pepper
  University of Washington
   
 

Malaria, caused by Plasmodium parasites, is an infection that kills almost 1 million people per year. The parasite life cycle consists of an early, asymptomatic hepatocyte-residing stage and a later, symptomatic erythrocyte-residing stage. A lack of understanding of the immune mechanisms associated with protection has hindered the development of an effective vaccine. B cells are known to be critical mediators of immunity against the blood stage of Plasmodium infection but little is known about the development or function of protective malaria-specific memory B cells. We have generated a novel B cell tetramer reagent to identify B cells specific for the C-terminal region of Merozoite Surface Protein 1 (MSP1), a parasite surface protein expressed in the blood-stage of infection. Using this tetramer and a magnetic bead-based enrichment method, we can track and analyze MSP1-specific B cells during blood stage infection in a well-characterized rodent model of malaria, Plasmodium chabaudi chabaudi. This approach allows us to directly assess how Plasmodium-specific memory B cells develop during blood stage infection and subsequently respond during a secondary infection. MSP1-specific B cells expand within days of infection and as the parasite is cleared and germinal centers form, we observe the formation of a MSP1-specific memory B cell population. As the GC reaction eventually terminates, remarkably persisting for at least 165 days post infection, a stable population of MSP1-specific memory B cells continues to persist at high levels up to 265 days post infection, made up of IgG+ MBCs and more recently described IgM+ MBCs. We are currently assessing the specific functions of these memory populations in an effort to understand how memory B cells contribute to protection during a secondary Plasmodium infection. These studies represent the first glimpse of the endogenous memory B cell response to Plasmodium with the hopes of defining what constitutes optimally protective anti-malarial B cell immunity.

   



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  Germinal-center derived, somatically hypermutated Plasmodium-specific IgM+ memory B cells are optimally responsive to a secondary blood stage malaria infection
  Presenter: Akshay Krishnamurty
  All Authors:Akshay Krishnamurty, Chris Thouvenel, Gladys Keitany, Brian Hondowicz, Peter D. Crompton, David J. Rawlings, and Marion Pepper
  University of Washington
   
 

Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells (LLPCs) and rapidly reactive memory B cells (MBCs). Classically defined MBCs express class-switched, somatically hypermutated BCRs after undergoing a germinal center (GC) reaction to produce high affinity antibodies within days of infection, making these cells the gold standard target of most vaccine platforms. More recently, this homogenous view of MBCs has been challenged and it is now recognized that diverse MBC subsets exist in both mice and humans. We hypothesized that the development of a system to visualize endogenous populations of distinct MBC subsets that form in response to infection could be used to elucidate the identity and function of various MBC subsets. In this study, we chose to focus on the development of the humoral immune response to Plasmodium, the causative agent of malaria, in which B cells play a critical role in immune protection. To clarify roles for various MBC subsets during malaria infection, novel B cells tetramers were generated containing the Plasmodium-specific protein Merozoite Surface Protein 1 (MSP1) to provide the first direct ex vivo visualization of antigen-specific MBC development and function during blood stage malaria infection in a murine model of malaria, Plasmodium chabaudi. Using these novel reagents we identified distinct long-lived MSP1-specific MBC subsets exist including classically defined, somatically hypermutated, isotype-switched IgG+ MBCs that express CD73 and CD80 and a GC-independent IgMloIgD+ (IgD+) unswitched population that more closely resemble naïve B cells. Interestingly, a third IgM+IgDlo (IgM+) unswitched MBC population also persisted, expressed both CD73 and CD80 and somatically hypermutated BCRs, and was also derived in a GC-dependent manner, more closely resembling IgG+ MBCs. Within three days of a secondary malaria infection, in a T cell dependent manner, MSP1-specific IgM+ MBCs outcompeted clonally related IgG+ MBCs to rapidly form antibody-secreting plasmablasts resulting in an increase in serum IgM antibody levels prior to any IgG+ antibodies. These studies therefore demonstrate that a previously unrecognized population of malaria-specific, somatically hypermutated IgM+, germinal center-derived MBCs can outcompete switched MBC subsets in response to a secondary malaria infection and should an important population targeted by anti-malarial vaccine strategies.

   



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  Metabolic Adaptation of Tissue-resident Macrophages in Cancer
  Presenter: Alexandra Kuhlmann
  All Authors:Alexandra Kuhlmann, Camila Robles-Otieza, Deb Ayeni, Ziyan Xu, Curtis Perry, Katerina Politi, and Susan Kaech
  Yale University Department of Immunobiology
   
 

Tissue resident immune cells play a critical role in maintaining homeostasis and orchestrating host defense. Resident macrophages represent cell populations that are uniquely adapted to their specific niche, integrating multiple complex environmental cues to generate a maximally beneficial response for their client epithelia. We propose a model in which the deregulated proliferation of malignant epithelia co-opts resident macrophage homeostatic networks to favor tumorigenesis. Using a genetically inducible mouse model of lung adenocarcinoma we observed a striking expansion in the resident cells of the alveolus, the alveolar macrophage(AM). By engaging AM lipid metabolism—a metabolic program known to skew macrophages towards an M2 like state—AM homeostatic clearance of the lipid-rich alveolar surfactant aids in the maintenance of airway tolerance, critically important in the lungs where there is a high cost to inflammation. As tumors progressed, AMs up-regulated surface expression of markers associated with alternatively activated macrophages, decreased their production of inflammatory cytokines, while increasing their lipid uptake and storage. AMs had functionally altered metabolic states with tumor-associated AMs having significantly increased rates of basal respiration as well as mitochondrial uncoupling. Induction of the transcription factor PPARg is necessary for AM maturation, integrating AM surfactant clearance and consequently lipid catabolism with their tolerant immune state. Pharmacological inhibition of PPARg delayed tumor progression, reduced AM recruitment, while restoring their inflammatory cytokine production. Our data suggests that rewiring of AM metabolism, specifically via antagonism of PPARg has the potential to be a novel therapeutic target in the treatment of lung cancer.

   



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  Factors that influence autoreactive thymocyte fate
  Presenter: Nadia Kurd
  All Authors:Nadia Kurd, Ashley Hoover, Jenny Yoon, Ellen Robey
  University of California Berkeley
   
 

The processes of negative selection and agonist selection both make important contributions to establishing central tolerance. However, the factors that determine whether an autoreactive thymocyte will undergo negative selection or agonist selection following antigen encounter remain ambiguous. Here, we make use of a thymic slice system in which class I-restricted thymocytes undergo both negative selection and agonist selection to investigate these factors. We demonstrate that interactions with phagocytic antigen presenting cells (APCs) promote negative selection, and highlight a previously unappreciated role for phagocytes as “executioners” of autoreactive thymocytes. In contrast, the cytokine IL-15, which is produced by thymic epithelial cells, protects against death and promotes agonist selection. Taken together, our data suggest that the ability of a thymic APC to phagocytose or deliver cytokines could be a key factor in determining the fate of an autoreactive thymocyte.

   



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  Enhanced Th2 cell differentiation and function without NADPH oxidase 2 (Nox2)
  Presenter: Bo-In Kwon
  All Authors:Bo-In Kwon, Seung-Hyo Lee
  KAIST
   
 

Nox2/gp91phox, one of phagocyte NADPH oxidase subunits, is mainly expressed in phagocytes. They transport electrons across the plasma membranes and kill engulfed pathogens by producing ROS. Many evidences have shown that Nox2 deficiency causes susceptibility to infections and immunosuppression. Recent studies have identified functional phagocyte-type Nox2 is also expressed in helper T cells through T cell receptor (TCR) stimulation. Thereafter, many studies attempted to define the role of Nox2 on Th1 and Th17 cell differentiation, but the exact function of Nox2 in Th2 differentiation remains unclear. To elucidate this, we adopted experimental allergic asthma that Th2 cells have been considered as a pivotal mediator for pathogenesis of asthma. Here, we showed that Nox2 deficient mice exhibited enhanced allergic asthma phenotypes including increased airway hyper responsiveness, airway eosinophilia, glycoprotein and IL-4 secretion in bronchoalveolar lavage through augmented Th2 differentiation, regardless of challenge routes and allergen types. Surprisingly, we identified that in early phase of TCR stimulation, mitochondrial ROS, which is a TCR signaling enhancer, was upregulated in Nox2 deficiency, in contrast with decreased cytosolic ROS production. Thus, increased mitochondrial ROS production could be an underlined mechanism of enhanced Th2 cell differentiation and function. These results are consistent with previous studies that mitochondrial ROS in CD4 T cell is critical for not only TCR mediated signaling but also IL-2 and IL-4 production. In addition, AKT (T308), which plays a critical role in mediating T cell differentiation, proliferation and survival, was highly phosphorylated with anti-CD3 and CD28 antibody stimulation. Based on our results, we conclude that Nox2 could be a molecular target for controlling Th2 cell differentiation and allergic asthma.

   



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  TLR adaptors MyD88 and TRIF are critical for extrafollicular B cell responses to influenza
  Presenter: Jonathan Lam
  All Authors:Jonathan Lam, Nicole Baumgarth
  University of California, Davis
   
 

Antibodies produced during primary influenza infection can be protective and form rapidly. This occurs independently of germinal centers (GCs) in extrafollicular B cell (EF) responses, where antibody-secreting cells (ASCs) blast and produce antibodies in the medulla of the mediastinal lymph node. While the EF response to influenza is known to be mostly T-dependent, it is unknown why B cells are shunted away from a GC fate and what factors lead to generation of EF ASCs. We show that absence of both Toll-like receptor (TLR) adaptors MyD88 and TRIF (double knockout, DKO) cause severe, B cell-intrinsic reductions in EF responses to influenza. Observing a reduction of the B cell differentiation factor IRF4 in DKO plasmablasts in vivo, we used in vitro culture systems to assess BCR signaling and T cell help by stimulating B cells with anti-IgM(Fab)2 and CD40L and/or BAFF, respectively. DKO B cells showed reduced survival in culture, which was rescued by providing either CD40L or BAFF. Strikingly, DKO B cells barely proliferated in response to any dose of anti-IgM, with or without CD40L/BAFF. High-dose BCR stimulation further reduced the viability of DKO B cells, which correlated with reduced IRF4 induction and altered NFkB signaling. Specifically, we show by image flow cytometry reduced nuclear localization of NFkB c-Rel, a positive regulator of IRF4, following BCR stimulation of DKO compared to wild type B cells. We propose that EF responses during influenza infection require not only antigen and T cell help, but also MyD88 and/or TRIF signaling to drive activation of c-Rel. This enhances antigen-specific B cell survival, proliferation, and the strong upregulation of IRF4 required for plasmablast differentiation.

   



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  Mitochondrial Pyruvate Import Promotes Long- Term Survival of Antibody-Secreting Plasma Cells
  Presenter: Wing Y Lam
  All Authors:Wing Y Lam, Amy M. Becker, Krista M. Kennerly, Rachel Wong, Jonathan D. Curtis, Elizabeth M. Payne, Kyle S. McCommis, Johannes Fahrmann, Hannah A. Pizzato, Ryan M. Nunley, Jieun Lee, Michael J. Wolfgang, Gary J. Patti, Brian N. Finck, Erika L. Pearce, and Deepta Bhattacharya
  Washington University in St.Louis School of Medicine
   
 

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and loss of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to provide enduring antibody-mediated immunity.

   



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  Distinct functions of hematopoietic and nonhematopoietic PD-L1 on the antitumor CD8+ T cell response
  Presenter: Ryan Lane
  All Authors:Ryan Lane, Amanda W. Lund
  Oregon Health & Science University
   
 

PD-L1 has been an effective target for immunotherapy to treat metastatic melanoma. While it is assumed that tumor and myeloid cell-specific PD-L1 induce CD8 T cell exhaustion in the tumor, other cells in the tumor microenvironment also express PD-L1 and their role in T cell exhaustion has not been ruled out. We have identified novel PD-L1 expression on nonhematopoietic components of the tumor microenvironment, including lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblasts, indicating a potential role in local CD8 T cell suppression. Importantly, in chronic LCMV infection nonhematopoietic PD-L1 controls local T cell function to prevent immunopathology and hematopoietic PD-L1 suppresses T cell activation. In this work we test the hypothesis that, similar to LCMV infection, nonhematopoietic PD-L1 suppresses local T cell function in the tumor microenvironment and hematopoietic PD-L1 limits CD8 T cell activation. Here we use B16F10 murine melanoma to investigate the role of PD-L1 on the antitumor immune response by implantation into PD-L1 KO mice. In tumor-bearing animals, endogenous CD8 T cells had increased numbers and markers of activation (CD44, core-2 O-linked glycosylation, and PD-1) in tumor draining lymph nodes (LN) and spleens of PD-L1 KO compared to WT mice. Additionally, tumor infiltrating CD8 T cells demonstrate increased IFNg production upon 3 hr ex vivo stimulation with PMA/ionomycin in PD-L1 KO mice compared to WT controls. To determine the contribution of hematopoietic and nonhematopoietic PD-L1 to either the reduced activation in secondary lymphoid organs or rather the impaired cytotoxic function in the tumor microenvironment we generated PD-L1 KO bone marrow chimeras. Mice lacking hematopoietic PD-L1 (PD-L1 KO-->WT) have increased CD8 T cell counts in tumor draining LNs and increased markers of activation, compared to WT-->WT controls. Importantly, mice lacking either hematopoietic (PD-L1 KO-->WT) or nonhematopoietic PD-L1 (WT-->PD-L1 KO) had increased numbers of CD8 tumor infiltrates as well as increased activation markers compared to WT-->WT controls. This work indicates that hematopoietic cell PD-L1 may limit antitumor T cell expansion in lymph nodes as well as local CD8 T cell function, and that tissue resident nonhematopoietic cells may suppress local antitumor effector T cell function.

   



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  Developing CD1d Multimers to Study Lipid-Reactive Natural Killer T Cells
  Presenter: Irene Lau
  All Authors:Irene Lau, Taylor Sicard, Irene Lin, Tao Chan, James Lazarovits, Warren Chan, Jean-Philippe Julien, Thierry Mallevaey
  University of Toronto
   
 

Natural Killer T (NKT) cells are unconventional T cells that respond to lipid antigens presented by the major histocompatibility complex (MHC) Class Ib molecule, CD1d. Two types of NKT cells exist in mammals: type I or invariant (i) and type II or diverse (d) NKT cells. Both iNKT and dNKT cells recognize a variety of mammalian and microbe-derived lipid antigens, but there is no antigen cross-reactivity between the two cell types. iNKT cells respond to lipids such as a-galactosylceramide (aGC), whereas dNKT cells recognize sulfatide. Although iNKT cells have been described to play potent immunomodulatory functions in various diseases, such as infection, autoimmunity and cancer, the biology of dNKT cells remains elusive, due to the lack of potent reagents to track them. Lipid-loaded CD1d tetramers have been successful in studies characterizing iNKT cells, but not dNKT cells, due to the low affinity T cell receptor-CD1d interaction. Thus, increasing the avidity of lipid-loaded CD1d multimers may overcome this obstacle. We have developed two types of novel multimers, taking advantage of 1) self-assembling CD1d fusion proteins, and 2) CD1d-coated gold nanoparticles (AuNPs). We show that such aGC-loaded fluorescently-labelled CD1d multimers specifically bind primary iNKT cells and iNKT cell lines. We are now comparing the staining efficiency of these CD1d multimers with conventional CD1d tetramers, and will evaluate their ability to activate iNKT cells in vitro and in vivo. In parallel, we will evaluate the ability of sulfatide-loaded CD1d multimers to stain established dNKT cell lines, and attempt to identify dNKT cells in vivo. In summary, these novel reagents may prove to be an efficient alternative to CD1d tetramers, and may ultimately allow for further elucidating the biology and functional roles of dNKT cells, and other elusive T cell populations.

   



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  DNA tumor virus oncogenes antagonize the interferon stimulatory DNA pathway
  Presenter: Laura Lau
  All Authors:Laura Lau, Dan Stetson
  University of Washington
   
 

Innate immune sensors of foreign nucleic acids are essential for immunity to virus infection. Detection of intracellular DNA activates the interferon-stimulatory DNA (ISD) pathway, which signals through the adapter protein STING (TMEM173) to initiate an antiviral response to DNA viruses and retroviruses. These viruses have likely developed means to prevent activation of the ISD pathway, but such virus-encoded antagonists remain largely unknown. We identify the viral oncogenes of the DNA tumor viruses, including E7 from human papillomavirus (HPV) and E1A from adenovirus, as potent and specific inhibitors of the ISD pathway. We show that the LXCXE motif of these oncoproteins, which is essential for blockade of the retinoblastoma tumor suppressor, is also important for ISD pathway antagonism. We find that E1A and E7 bind to STING, and that silencing of these oncogenes in human tumor cells restores ISD pathway signaling. Our findings reveal a host-virus conflict that may have shaped the evolution of viral oncogenes, with implications for the origins of the DNA viruses that cause cancer in humans.

   



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  Diet-induced obesity impairs induction of Experimental Autoimmune Encephalomyelitis and is restored by PD-1 blockade
  Presenter: Catherine Le
  All Authors:Catherine Le, Lam Khuat, Athena Soulika, and William J. Murphy
  University of California, Davis
   
 

There is an increasing prevalence of obesity in the U.S. reaching pandemic proportions. Obesity has marked effects on immune function. Increased adiposity and accumulation of visceral fat results in continuous, low-level systemic inflammation that induces or exacerbates several chronic conditions. Obesity is associated with increased susceptibility to infection and decreased adaptive immune response to vaccination. We were interested in examining the effects of obesity on adaptive autoimmune immune responses using a mouse model of diet-induced obesity. The pathogenesis of experimental autoimmune encephalomyelitis (EAE), a model for autoimmune demyelinating disease of the central nervous system (CNS) such as multiple sclerosis, is mediated by autoreactive CD4+ T cells, particularly Th1 and Th17 subsets. We immunized 8-month-old C57Bl/6 male mice who were fed an open-source purified diet consisting of either 60% fat or 10% fat as a control diet starting at 8 weeks of age at which time there was notable differences in accumulated body fat. The groups were then immunized with 300 μg of rodent MOG35-55 peptide in complete Freund’s adjuvant (CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis, which has been demonstrated to induce EAE symptoms and pathology in susceptible strains. Lean mice had significantly greater body weight loss compared to diet-induced obese mice on day 12 (22% vs 10% body weight loss) and day 13 (28% vs 11% body weight loss) post-induction. Lean mice also exhibited earlier onset of severe clinical symptoms of hind limb paralysis by day 10 post-induction. Clinical scores were correlated with greater number of MOG-specific tetramer-positive CD4 T cells and greater number of IFN-gamma and IL-17a-expressing CD4 T cells in the CNS of lean mice. Diet-induced obese mice eventually developed clinical symptoms comparable to lean mice with a 4-day delay in the induction of severe clinical symptoms. Decreased functional cytokine expression called into question the exhaustive nature of T cells in obese mice through the up-regulation of the PD-1/PD-L1 signaling, a pathway that has been implicated as a negative regulator of EAE. We induced lean and obese mice with EAE and treated with anti-PD-1 blockade at 500 μg on Day 0 and 250 μg on Day 2, 4, 6, and 8. The delay in induction was partially rescued by PD-1 blockade treatment, resulting in earlier and faster induction of EAE in obese mice in both clinical score and number of pathologic CD4 T cells in the CNS. The kinetics of induction did not differ with anti-PD-1 blockade in lean mice. Our findings may indicate an impairment of the adaptive arm of the immune system through the PD-1/PD-L1/PD-L2 pathway in obesity, indicating an augmentation of the checkpoint pathway in settings of systemic inflammation and autoimmunity.

   



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  Microanatomical organization of CD4+ T cell responses to vaccines
  Presenter: Joseph Leal
  All Authors:Joseph Leal , Brandy Olin, Karan Kohli, Michael Gerner
  Department of Immunology, University of Washington
   
 

The cellular immune response to foreign antigens is orchestrated through interactions between innate antigen presenting cells, dendritic cells (DCs), and T lymphocytes. Activated T cells are polarized into distinct helper cell subsets, promoting generation of highly tailored protection to distinct pathogens. Further, multiple subsets of DCs exist with non-overlapping functions with respect to T cell activation and effector cell differentiation. Utilizing a novel quantitative imaging approach, our laboratory has shown that these DC subsets occupy different spatial locations within secondary lymphoid tissues during the steady state. Here we investigated whether the spatial localization of DC subsets influences T cell priming using mouse models of immunization. As anticipated, distinct vaccine adjuvants elicited divergent programs of T cell effector differentiation. Intriguingly, we found that different adjuvants also induced divergent spatial patterning of DCs and CD4+ T cells in draining lymph nodes, and that such positioning correlated with effector T cell polarization. Ongoing and future studies will examine the hypothesis that distinct adjuvants induce formation of discrete tissue microenvironments that are functionally specialized in generating specific programs of T cell immunity.

   



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  Intranodal dendritic cell relocalization during inflammation impacts T cell immunity
  Presenter: Joseph Leal
  All Authors:Joseph Leal, Brandy Olin, Michael Gerner
  University of Washington, Department of Immunology
   
 

Conventional dendritic cells (cDCs) are the critical antigen-presenting cells that initiate adaptive immunity in response to external inflammatory stimuli. This process of cDC initiated immunity occurs in lymph nodes (LNs): highly organized immune organs that funnel antigen and immune cells together to drive immunity. We have previously shown that LN-resident cDC2s, a DC subset critical for generating CD4 T cell responses, reside in regions highly proximal to the lymphatic sinuses, enabling superior acquisition of draining soluble and particulate antigens. However, given that this peripheral positioning is distal from the deep LN paracortex and the T cell zone, we hypothesized that for optimal generation of adaptive immunity, LN-resident cDC2s must migrate intranodally from peripheral sites into the deep T zone in order to engage in cognate DC-T cell interactions during priming. Here, we show that s.c immunization with TLR-agonists, as well as infection with West Nile Virus, induces rapid relocalization of cDC2s into the deep T zone of the LN. This cDC2 relocalization requires Type 1 interferon in some, but not all, adjuvant immunizations, and is predominantly driven by increased CCR7 expression on cDCs after maturation. When lacking CCR7, LN-resident cDC2s failed to reposition to the T cell zone across multiple adjuvant comparisons, and this in turn led to a marked reduction in the activation and proliferation of CD4+ T cells. A decrease in the differentiation of Tfh cells was also observed, suggesting a possible impact on humoral immunity. Collectively, this work identifies a novel CCR7-mediated spatial reorganization of LN-resident cDC2s during inflammation, independent of peripheral migratory DCs, and its role in the generation of adaptive immunity.

   



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  Innate immune signaling and intranodal relocalization of dendritic cells during inflammation promotes T cell immunity
  Presenter: Joseph Leal
  All Authors:Joseph Leal, Jessica Huang, Miranda Lyons-Cohen, Michael Gale Jr., Michael Y Gerner
  University of Washington
   
 

Microanatomical organization of innate immune cells within lymph nodes (LNs) is critical for the generation of adaptive responses. In particular, steady-state LN-resident dendritic cells (Res cDCs) are strategically localized to intercept lymph-draining antigens. How this organization changes in response to inflammation and whether that impacts the generation of immune responses in LNs is unknown. Here, we report that after TLR agonist immunization or West Nile virus infection, but not during Type-II inflammation, antigen-presenting Res cDCs underwent intranodal relocalization from the LN periphery into the deep T cell zone (TZ). This process occurred rapidly and was associated with a concomitant increase in expression of the chemotactic receptor CCR7. In the absence of CCR7, Res cDCs could not relocalize to the TZ and instead preferentially occupied the B cell follicles and the medullary sinuses. Failure to relocalize to the TZ during vaccination led to profound defects in the priming and clonal expansion of antigen-specific CD4+ and CD8+ T cells, suggesting a critical role for inflammation-induced Res cDC relocalization in draining LNs in eliciting adaptive immune responses. Res cDC relocalization and upregulation of CCR7 was accompanied by enhanced expression of costimulatory molecules and MHC-II, as well as increased cellularity, all in a cDC subset and TLR agonist-specific manner. Investigation of the involved molecular pathways revealed partial dependencies on both direct TLR signaling via MyD88 as well as indirect cytokine sensing in a cDC subset dependent manner, indicating complex interplay of both direct and indirect signaling during inflammation. Together, these findings reveal that Type-I inflammation elicits a profound spatial, phenotypic, and numerical remodeling of the Res cDC compartment to promote generation of adaptive responses to vaccines and infections.

   



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  Roles of HDAC4 in T-Cell Development & Effector Function
  Presenter: Intelly Lee
  All Authors:Intelly Lee, Herbert Kasler, Hyungwook Lim, Eric Verdin
  University of California - San Francisco
   
 

The Class IIA HDACs are a highly conserved family of transcriptional regulators in metazoans that regulate signal-dependent developmental programs in many organ systems and tissue types, but their functional roles in T-cells remain poorly understood. HDAC4 is one of two highly expressed Class IIA family members found in developing thymocytes with continued expression in mature peripheral cells, and we sought to elucidate specific functions for HDAC4 in T-cells through a reverse genetic approach. In contrast to the profound dysregulation of positive and negative selection that results from loss of HDAC7, the other highly expressed Class IIA HDAC in T-cells, T-cell specific deletion of HDAC4 had limited effect on the thymic compartment in knockout animals. In mixed bone marrow chimeras, though, we uncovered a reduction of thymocyte numbers (~5 fold) starting at the DP stage with limited further effect at later stages. We find that at rest (before positive selection), a significant fraction of HDAC4 is found in the nuclear compartment of DP thymocytes, but after strong stimulation in vitro becomes strongly nuclear excluded in a PKD-dependent fashion. Microarray analysis of DP thymocytes reveal that loss of HDAC4 leads to only minor changes in transcriptome profiles (~70 genes more than 1.5x down- or upregulated), with many gene targets significantly overlapping with HDAC7 and highly enriched in Type I Interferon signaling. Although HDAC4 remains primarily nuclear-excluded in mature peripheral T-cells in a variety of conditions (resting, activated, anergic), using an adoptive transfer OVA-driven model of skin-specific autoimmune disease, we find that HDAC4KO T-cells, despite appropriately recognizing antigen and dividing in draining lymph nodes, display impaired trafficking back to the periphery and mount a diminished Th1 response, leading to lessened disease severity. Together, our results suggest that although HDAC4 biochemically and genetically serves overlapping roles with the more abundant HDAC7 in thymocyte development, it is not entirely redundant and may play a specific role in controlling effector T-cell function in the periphery.

   



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  Origin and Potentiation of Tissue-Resident Type 2 Innate Lymphoid Cells
  Presenter: Jinwoo Lee
  All Authors:Jinwoo Lee, Christoph Schneider, Jesse C. Nussbaum, Hong-Erh Liang, Roberto Ricardo-Gonzalez, Steven Van Dyken, Lucas K. Smith, Saul A. Villeda, Richard M. Locksley
  University of California, San Francisco
   
 

Tissue-resident type 2 innate lymphoid cells (ILC2s) are key factors in initiating and sustaining type 2 immune responses, which characterize conditions such as allergy, asthma, and parasitic infections. However, the ontogeny of ILC2s has not been well-characterized. Our results from parabiosis experiments suggest a long tissue lifespan and limited circulation of ILC2s. In addition, using a lineage-tracing approach, we find that de novo adult-derived ILC2s contribute minimally to the ILC2 pool in tissues at steady state, consistent with the results from parabiosis. Lineage-tracing also allowed us to identify three waves of ILC2s populating peripheral tissues throughout the lifespan of the mouse. Prenatal ILC2s can be detected in embryonic tissues and give rise to a significant fraction of adult ILC2s. However, the most substantial fraction of adult ILC2s are derived from ILC2s that are generated shortly after birth, a period during which ILC2s are rapidly proliferating and becoming activated. Interestingly, in long-term lineage-tracing experiments in which fate-mapped ILC2s were followed out for over 1 year, we observe tissue-dependent heterogeneity with regard to the proportions of adult-derived versus early-life-derived ILC2s, suggesting that the tissue environment determines the rate of replacement of established ILC2s. In conclusion, we find that resident ILC2 populations are established in various tissues during a perinatal window while undergoing vigorous activation and proliferation, and we find that ILC2 heterogeneity among tissues may partially be due to differences in origin.

   



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  Identification of Mechanisms of Immune Regulation and Exhaustion in Patients with Advanced Non-Small Cell Lung Cancer
  Presenter: Diego Lema
  All Authors:Diego Lema, Jankowska-Gan E, Burlingham W, Leal T
  University of Wisconsin-Madison, Dept of Surgery, Transplant Division
   
 

Background: The success of PD-1/PD-L1 inhibition is clearly dependent on optimal patient selection. Developing validated biomarkers that identify patients with an increased probability of response to these antibodies remains a challenge. DTH responses are a standard method to evaluate cellular immune responses in animal and human immunotherapy trials. The trans-vivo delayed-type hypersensitivity (tvDTH) methodology, using the SCID mouse footpad swelling response to human PBMC (peripheral blood mononuclear cells) + antigen injection, and using antibodies to block the regulatory pathways, was developed in our laboratory and has been used to characterize regulation systems in tolerant transplant recipients and more recently in prostate cancer. Methods: In this pilot project we aim to investigate the mechanisms of regulatory immune responses in patients with advanced NSCLC receiving anti-cancer therapies using the tvDTH, and to correlate tvDTH results with responses to checkpoint inhibitor blockade therapy. So far we have enrolled 4 NSCLC patients. PBMCs are obtained by blood draw and lung cancer antigen (LCA) is prepared from malignant pleural effusion cellular pellet or fresh frozen tumor. 1 of 4 patients’ sample was negative for malignancy Results: Our preliminary data suggests that all NSCLC patients with pleural effusions positive for malignancy had a PD-1-dependent exhausted immune response towards their LCA. Remarkably, 1 of these patients not only showed PD-1-dependent exhaustion, but also PD-1-dependent bystander suppression, whereby a positive control immune response was reduced by co-injection of LCA, suggesting the existence of immunosuppressive mechanisms. Unlike our previous results with prostate cancer, no patient showed CTLA-4-dependent exhaustion or suppression. Importantly, an antigen preparation from a NSCLC that showed no malignant cells by cytology did not induce a swelling response. Conclusions: We plan to expand our study patient population and follow up our patients in order to correlate tvDTH results with clinical outcomes.

   



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  Regulation of tissue-resident macrophage development by IL-7R signaling
  Presenter: Gabriel Leung
  All Authors:Gabriel Leung, Taylor McCann, Camilla Forsberg, Anna Beaudin
  University of California, Merced; University of California, Santa Cruz
   
 

In addition to their duties in immune surveillance, specific tissue-resident macrophages play critical roles in normal tissue homeostasis and disease. Many populations of tissue-resident macrophages derive from fetal progenitors and self-maintain across the lifespan through in situ proliferation, independent of bone marrow hematopoiesis. However, the developmental mechanisms that specify the unique function of fetal-derived tissue-resident macrophages is poorly understood. Here we have identified a novel cytokine regulating tissue-resident macrophage development using an IL7Ra-cre lineage tracing model. Adult tissue resident macrophages in the brain (microglia), skin (Langerhans cells), liver (Kupffer cells), and lung (alveolar macrophages) were extensively labeled by IL7ra-cre, in the absence of IL7ra message or protein expression. To gain insight into developmental expression of IL7ra, we profiled IL7Ra surface expression, mRNA expression, and IL7ra-cre-driven labeling during fetal myeloid development. During fetal liver-stage hematopoiesis, macrophages derived from the yolk sac are thought to already be present in the tissues but may be replaced or replenished by incoming fetal liver-derived monocytes. We observed rapid upregulation of IL7ra surface expression during a limited window of tissue macrophage establishment, and dynamic regulation of IL7ra message and protein levels in monocyte precursors, suggesting that IL7ra regulated the transition from fetal liver monocytes into tissue macrophages. Blockade of the IL7R using an antagonistic monoclonal antibody significantly decreased cellularity of lung and skin tissue-resident macrophages at birth, with a concomitant increase in the number of fetal liver monocytes. These data suggest that during late gestation, IL7Ra is upregulated as fetal monocytes exit the fetal liver and differentiate into macrophages, and also provides evidence that IL-7R signaling directly regulates fetal tissue-resident macrophage development. Ongoing work addresses the function of IL-7 signaling in myeloid development, in vivo and ex vivo, as well as the distinct function of IL-7R-marked macrophages in adult immunity.

   



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  IL7R regulates fetal tissue resident macrophage development by facilitating cell survival
  Presenter: Gabriel Leung
  All Authors:Gabriel Leung, Clint Valencia, Anna E. Beaudin
  University of California Merced
   
 

Tissue-resident macrophages (TRMs) play critical roles in tissue homeostasis and disease. Many populations of TRMs derive from fetal progenitors and independently self-maintain across the lifespan through in situ proliferation. Previously, we have identified the interleukin-7 receptor (IL7R) as a novel regulator of TRM development. We have shown that antibody blockade of the IL7R during gestation impaired liver, lung, and epidermal TRM cellularity at birth. Here we show that in vivo fetal rIL-7 stimulation of the IL7R increased neonatal liver and lung TRM. In order to determine how IL7R signaling regulates fetal macrophage development, apoptosis was measured after late gestation IL7R blockade. Apoptosis in the macrophages and monocytes of the liver and lung was dramatically increased after IL7R blockade, suggesting that increases in neonatal cellularity after rIL-7 treatment may be due to increased survival signaling in these tissues. Our previous analysis also revealed dynamic regulation of IL7R mRNA surface protein expression as fetal monocytes differentiate into CD64+ macrophages during fetal development. We performed intracellular staining for IL7Ra protein to determine that monocytes contain intracellular IL7R protein that is not expressed on the surface until differentiation, suggesting that regulation of IL7R expression occurs at the level of surface expression. These data reveal dynamic regulation of IL7Ra expression in TRMs and TRM precursors during late gestation and provide evidence that IL-7/IL7R signaling regulates fetal TRM development by facilitating cell survival. Ongoing work addresses downstream signaling and other developmental processes regulated by IL7R signaling during fetal TRM development.

   



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  Regulatory T cells adopt a tissue-repair phenotype in response to cell-intrinsic TLR7 signaling
  Presenter: Nicholas Lind
  All Authors:Nicholas Lind, Shaina Carroll, Gregory Barton
  UC Berkeley
   
 

Foxp3+ regulatory T cells (Tregs) in non-lymphoid tissues are capable of promoting repair following injury or infection. This “tissue repair” phenotype in Tregs is thought to be driven by signaling through the IL-18 and/or IL-33 receptors. However, the stimuli that cause Tregs to upregulate these receptors, as well as drive their accumulation in damaged tissue, is not well understood. Here, we identify Toll-like receptor 7 (TLR7) as a key mediator that primes Tregs to participate in tissue repair. Using TLR reporter mice, we have determined that TLR7 is the only TLR expressed by Tregs. Indeed, all Tregs express TLR7 upon exit from the thymus, and that expression is actively modulated in the periphery. Expression of TLR7 on Tregs in the lung is particularly high when compared to other non-lymphoid tissues. Treg-intrinsic TLR7 signaling has no impact on suppressive capacity, but instead results in proliferation, enhanced IL-18 receptor expression, and elevated levels of amphiregulin production. We hypothesize that in vivo, Tregs use TLR7 as a sensor of endogenous RNA nucleotides that are released following tissue damage, and subsequently adjust their function to facilitate repair. To test this theory directly, we have generated mice with a Treg-specific deletion of TLR7. These mice have reduced numbers of lung-resident Tregs at steady-state, and ongoing experiments are testing whether they show impaired recovery following lung damage. Collectively, our data demonstrates that TLR7 signaling can directly modulate Treg function, revealing a new pathway capable of shaping Treg responses to tissue damage.

   



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  Characterization of the implication of the Idd2 locus in type 1 diabetes development
  Presenter: Felix Lombard-Vadnais
  All Authors:Felix Lombard-Vadnais, Roxanne Collin, Genevieve Chabot-Roy, Sylvie Lesage
  McGill University
   
 

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of the beta cells of the pancreas. Genetic susceptibility to T1D is conferred by complex traits. More than 20 genetic loci, termed Idd for “insulin dependent diabetes”, are linked to T1D in the NOD mice, a model which spontaneously develops T1D. The second genetic locus, namely Idd2, was identified in the 1980s, but its contribution to T1D susceptibility was never validated. We generated a NOD.B10-Chr9 congenic mouse, where the Chr9 locus encompasses part of the Idd2 locus, enabling us to assess the specific contribution of Idd2 in T1D development. Using this congenic strain, we validate that Idd2 confers resistance to insulitis and, consequently, to T1D in the NOD mouse. By generating bone marrow chimeras, we find that the resistance is conferred in a bone marrow-intrinsic manner. Moreover, we demonstrate that Idd2 contributes to the formation of germinal centers as well as the expression of the major histocompatibility complex (MHC) on B cells. Together, these data not only validate Idd2 as a genetic locus contributing to T1D susceptibility, it strongly supports recent evidence of the contribution of the humoral response in T1D susceptibility.

   



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  A critical period of innate-immune development: Fetal origins of allergic asthma susceptibility
  Presenter: Diego A. López
  All Authors:Diego A. López, Anna E. Beaudin
  University of California, Merced
   
 

Our lab has previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that only exists during fetal development and specifically gives rise to innate-like lymphocytes that persist across the lifespan. The identification of a developmentally-limited cell of origin for innate-like immune cells that persist across the lifespan defines a “critical window” for immune development, in which the phenotype of the developing immune system can be shaped via extrinsic inputs. To test how developmental perturbation during this critical window drives immune dysfunction, we examined underlying changes to innate lymphoid cells (ILCs) in the lung and susceptibility to airway dysfunction following maternal immune stimulation. ILCs are a recently identified family of fetal-derived innate-like lymphocytes that mimic the adaptive T-helper arm of our immune system. Many ILCs are tissue-resident and play essential roles in tissue development, homeostasis and protection during infection through cytokine secretion in response to tissue damage and immune activation. In the lung, type-2 innate lymphoid cells (ILC2s) are recognized as potent producers of IL5 and IL13, cytokines important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, maternal immune stimulation via a single low-dose injection of poly (i:c) at mid-gestation robustly increased proliferative capacity and cellularity of lung ILC2s in offspring at postnatal day (P)9 and P14, respectively, concomitant with drHSC and common-helper innate lymphoid progenitor (ChILP) cell expansion during fetal development. Additionally, lung ILC2s exhibited heightened IL5 and IL13 production upon in-vitro stimulation with PMA and ionomycin in poly (i:c) perturbed offspring. Ongoing experiments will examine how heightened ILC2 functionality, as a result of developmental perturbation, underlie persistent changes at the level of the ILC-progenitor, and how this may alter susceptibility to allergic asthma in response to secondary house-dust mite immune stimulation. Together, our data suggest immune perturbation of the transient progenitors during fetal development may impact the ChILP immune trajectory, altering the establishment and function of neonatal lung ILC2s, and ultimately contributing to allergic asthma susceptibility into adulthood.

   



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  Regulation of TLR Signaling by Integrin [a]v[b]3 in Plasmacytoid Dendritic Cells
  Presenter: Alina Lorant
  All Authors:Alina Lorant, Adam Lacy-Hulbert
  University of Washington
   
 

Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies against nucleic acids and associated nuclear proteins. The nucleic acid-sensing Toll-like receptors (TLR7/9) have been heavily implicated in the development of SLE in humans and in lupus-like autoimmunity in mouse models, suggesting that SLE is triggered by innate immune responses to self-derived nucleic acids. In addition, SLE is strongly associated with high levels of type I interferon (IFN), largely produced by plasmacytoid DCs (pDCs) after TLR7/9 stimulation, contributing to autoimmunity. Understanding how pDC production of IFN is regulated, and how this breaks down during autoimmunity and other inflammatory disorders, is an important unanswered question. Although TLRs function as critical sensors of pathogens through recognition of microbial, fungal and viral ligands, TLR signaling also relies on a wide range of co-receptors and accessory proteins, which present ligands for TLR binding, amplify signaling, and promote internalization of ligands to endosomal compartments where they can engage endosomal TLRs. As an example, engagement of pDC Fc receptors by DNA-containing immune complexes promotes endosomal maturation, allowing activation of IRF7 and production of IFN downstream of TLR9. I hypothesize that co-receptors for self-derived TLR ligands may likewise regulate TLR signaling in pDCs, promoting ‘tolerogenic’ responses, and that loss of these regulatory signals will predispose to autoimmunity. This hypothesis is supported by two major findings from many laboratories, including my mentor Dr. Lacy-Hulbert. First, engagement of apoptotic cells, a likely source of autoantigens in SLE, inhibits pro-inflammatory cytokine production and promotes development of a regulatory phenotype in macrophages or DCs, characterized by production of IL-10 and TGF-β. Second, recognition and phagocytosis of dying cells is impaired in SLE, and disruption of this process in mice leads to SLE-like autoimmune disease. However, the exact mechanisms underlying this process, and whether they operate in pDCs, remain unclear. Recently, my mentor’s laboratory has identified a novel role for αvβ3 integrin in regulating TLR signaling in B cells, through a mechanism involving endosomal maturation and non-canonical autophagy. αvβ3 and the related integrin αvβ5 serve as internalization receptors for apoptotic cells, and we therefore speculate that this represents a mechanism to regulate TLR signaling in response to self-antigens derived from dying cells. Supporting this, deletion of αv or β3 integrins in B cells results in increased production of autoantibodies. In this project, I will investigate how αv integrins function to regulate TLR signaling and IFN-I in pDCs first in vitro, and then in the context of autoimmunity. So far I have seen altered cytokine responses to TLR9 stimulation in αv-KO cells compared to wild-type. Decreased interferon responses in αv-KO mice suggest the integrin could be limiting pDCs response to when self-derived stimuli are present.

   



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  The Role of CX3CR1+ T cells in Helminth Infections
  Presenter: Denis Loredan
  All Authors:Denis Loredan , Natasha M. Girgis, P'ng Loke
  New York University
   
 

CD4+ T cell help is required for immunological resistance and tolerance to parasitic helminth infections. However, the exact subsets of T cells involved and their differing roles in the immune response remain to be elucidated. The chemokine receptor CX3CR1 is expressed on monocytes, natural killer cells, and some memory and effector CD4+/CD8+ T cells respectively, and binds to a single known ligand, CX3CL1. The interaction of this receptor with its ligand has been shown to exacerbate Th2-mediated pathology in mouse modes of asthma and atopic dermatitis, although no role has been described yet for these cells in Th2-mediated immune responses to helminth infections. Using a CX3CR1-EYFP reporter mouse we have identified a subset of CD4+ T cells that are CX3CR1+ during chronic infection with Schistosoma mansoni and acute infection with Nippostrongylus brasilinesis. We have characterized these cells as activated CD44+ CD25+ ST2+ T cells present at the main sites of tissue damage, but not the secondary lymphoid organs. Our lab is making use of a CX3CR1-CreERT-IRES-YFP Rosa26-tdTomato fate-mapping mouse model to track the kinetics of this particular subset over time and determine their potential for memory cell formation. This approach has enabled us to observe a population of T cells labeled with the tdTomato fluorophore yet not actively expressing CX3CR1 in the spleens of mice infected with the rodent hookworm N. brasilinesis, indicating potential for recirculation. It remains to be determined if CX3CR1+ CD4+ T cells are a general component of type-2 immunity. As well, understanding the adaptive immune response to helminth infection can inform not just our understanding of pathology, but also attempts to develop an effective vaccine.

   



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  Examining the Role of Lymph Node Eosinophils during the Generation of Type 2 Immune Responses
  Presenter: Miranda Lyons-Cohen
  All Authors:Miranda Lyons-Cohen, Michael Gerner
  University of Washington
   
 

Eosinophils are short-lived innate granulocytes associated with type 2 inflammation, capable of promoting protective immunity against helminth infection, while also exacerbating allergy and asthma. Eosinophils develop in the bone marrow and circulate throughout the blood, maintaining a steady state population in peripheral organs, such as the thymus, ovary, and uterus, and intestinal tissues. During type 2 inflammation, eosinophils are recruited into peripheral tissues, such as the lung, where they can mediate their effector functions. However, the presence and function of eosinophils in draining lymph nodes (LNs) during the early generation of type 2 responses has not been thoroughly investigated. Here, we have uncovered a population of SiglecF+ST2+Sirpα+GR1+ eosinophils that were present in LNs at both steady state and after immunization with a type 2 adjuvant, papain. LN eosinophils were readily identifiable by confocal microscopy and localized primarily in the outer T cell zone, T-B border, and medullary regions. Using an IL-4 mRNA reporter mouse strain, we found that LN eosinophils made copious IL-4 message and made up the majority of total IL-4 competent cells in the steady state and after papain administration. Preliminary studies using an IL-4 protein reporter strain, however, revealed that LN eosinophils did not produce detectable IL-4 protein on days 2 or 5 after papain immunization. Collectively, these studies uncover a previously unrecognized population of eosinophils in LNs that are poised for IL-4 production, suggesting a potentially important role for this immune subset in the generation or maintenance of type 2 immune responses. Determining the function, role, life cycle, and trafficking of these cells to and within the LN will be critical for understanding the mechanisms governing the generation of type 2 responses and protective immunity against parasitic helminths or during allergy and asthma.

   



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  Microanatomical Organization of Type 2 Immune Responses in the Lymph node
  Presenter: Miranda R. Lyons-Cohen
  All Authors:Miranda R. Lyons-Cohen, Michael Y. Gerner
  University of Washington, Department of Immunology
   
 

Type 2 helper T (Th2) cells orchestrate a diverse range of type 2 immune responses and are critical for protection against parasitic helminths, as well as being involved in inappropriate inflammation during allergy and asthma. Substantial efforts have gone into identifying the inflammatory stimuli and in vivo mechanisms leading to the generation of Th2 cells in lymph nodes (LNs) during priming. However, these studies have largely ignored the importance of cellular localization and microenvironments in these highly organized lymphoid tissues. Here, we used highly-multiplexed quantitative imaging to study the spatial organization of early Th2 cell differentiation in skin draining LNs after Papain immunization or infection with Nippostrongylus brasiliensis. Using an IL-4 mRNA reporter (4get) OVA-specific (OT-II) adoptive transfer model, we find that Papain immunization led to extensive clustering of early Th2 cells in discrete zones located at the border of the T cell zone and B cell follicles. Similar clustering was observed for polyclonal early-Th2 cells during Nippostrongylus brasiliensis infection. Such extensive cellular aggregation was distinct from that observed during formation of Th1 responses, which instead were associated with formation of smaller T cell aggregates distributed equally throughout the T cell zone. In addition, we find that formation of Th2 zones is dependent on the site of skin immunization. Administration of Papain in the footpads elicited significantly reduced T cell clustering and Th2 differentiation in draining LNs as compared to immunization of other cutaneous sites. Collectively, these studies identify existence of dedicated microenvironments in draining LNs that support Th2 differentiation, as well as reveal that formation of these microenvironments is context-dependent, with the site of skin immunization being a critical factor in the generation of Type-2 immunity.

   



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  Cross-dressed dendritic cells prime anti-tumor CD8+ T cell responses
  Presenter: Brendan MacNabb
  All Authors:Brendan MacNabb, Douglas E Kline, Xiufen Chen, Sravya Tumuluru, Justin Kline
  University of Chicago
   
 

CD103+ dendritic cells (DCs) orchestrate anti-tumor CD8+ T cell responses by presenting tumor antigens on MHCI molecules and providing costimulatory signals in the tumor-draining lymph node (tdLN). Tumor antigen presentation is widely assumed to require classical antigen cross-presentation. However, recent studies have demonstrated that DCs can also acquire and present intact exogenous peptide:MHC (p:MHC) molecules through a phenomenon known as cross-dressing. To determine the contribution of cross-dressing and classical cross-presentation to spontaneous anti-tumor CD8+ T cell priming in vivo, the murine MHCI molecule, H2-Kb, was deleted in syngeneic B16 melanoma and C1498 leukemia cell lines. Engraftment of B16 and C1498 tumors expressing Kb-restricted model antigens in wildtype, Batf3-/-, Tap1-/-, and KbDb-/- mice has revealed three key observations: 1) Kb expression by cancer cells is required for optimal CD8+ T cell priming against Kb restricted model antigens, to the extent that there was no discernable CD8+ T cell response against Kb-deficient C1498 tumors; 2) abundant tumor-derived MHCI molecules are observed on the surface of tumor-resident DCs and macrophages; and 3) presentation of tumor-derived p:MHCI molecules by migratory DCs in the tdLN is sufficient for ex vivo CD8+ T cell priming in the absence of classical cross-presentation. Mechanisms by which DC cross-dressing occurs are being explored both in vivo and through in vitro studies of MHCI-sufficient or deficient tumor cells and bone marrow-derived DCs. In contrast with current dogma, our results identify cross-dressing as a critical mechanism of tumor antigen presentation, and a potential determinant of anti-tumor CD8+ T cell responses in mice and man.

   



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  Human DNA-PK activates a STING-independent DNA sensing pathway
  Presenter: Joanna Maltbaek
  All Authors:Joanna Maltbaek, Katie Burleigh, Stephanie Cambier, Daniel B. Stetson
  University of Washington
   
 

Detection of foreign nucleic acids is critical for antiviral defense. The cGAS-STING pathway has, until now, been considered the sole driver of the antiviral response to intracellular DNA. Activation of cGAS by the sugar-phosphate backbone of DNA triggers STING signaling that rapidly induces a potent type I interferon (IFN) response. This pathway is broadly required across cell types and species, and its relevance for viral infection, autoimmunity, and cancer has been thoroughly established. We have discovered that there is a second, STING-independent DNA sensing pathway (SIDSP) in human cells, but not in mouse cells. This response is delayed by several hours compared to cGAS-STING. RNA-Seq analysis revealed that the SIDSP was virtually indistinguishable from the cGAS-STING pathway in terms of robustness and breadth of the antiviral response. In order to identify components of the SIDSP, we took a candidate approach using both CRISPR and highly specific kinase inhibitors. We identified DNA-PK as the sensor of the SIDSP, along with its DNA-binding partner proteins, Ku70/Ku80. Moreover, we have identified HSPA8 as a novel and specific target of the DNA-PK-SIDSP. Future experiments will expand upon the mechanism by which DNA-PK activates a STING-independent type I IFN response and address the biological relevance of the SIDSP in the context of infection and autoimmunity.

   



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  Regulation of the cGAS-STING pathway through export and ENPP1-mediated degradation of cGAMP
  Presenter: Joanna Maltbaek
  All Authors:Joanna Maltbaek, Daniel B. Stetson
  University of Washington Department of Immunology
   
 

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. The intracellular DNA sensor cGAS binds to double-stranded DNA and synthesizes the endogenous second messenger cyclic GMP-AMP (cGAMP), which activates STING and initiates type I interferon production. While many negative regulators of this pathway have been described, the mechanisms of cGAMP regulation and degradation are poorly understood. The only identified hydrolase of cGAMP is the extracellular enzyme ENPP1, but whether cGAMP is exported to the extracellular space to enable its degradation is unclear. Congruent with this, the biological relevance of ENPP1 as a potential negative regulator of type I interferon production remains completely unexplored. Our preliminary data demonstrate that live cells potently export cGAMP in a STING-independent manner following cGAS activation. We further demonstrate that cGAMP export enables its degradation by extracellular ENPP1. Finally, we show that following acute in vivo cGAS activation, mice treated with an ENPP1 inhibitor have increased expression of interferon-stimulated genes in peripheral blood. Our ongoing studies are aimed at identifying the mechanism of cGAMP export and exploring whether ENPP1 inhibition potentiates antiviral and/or antitumor immunity. These findings will have clinical relevance for harnessing agonism of the cGAS-STING pathway for improved vaccine adjuvant design and tumor immunotherapy.

   



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  Kindlin-3 is required for nonclassical monocyte patrolling and enables protection of lung tissue from metastatic seeding of B16 melanoma.
  Presenter: Paola Marcovecchio
  All Authors:Paola Marcovecchio, Tacke R, Hanna R, Wu R, Yoakum D, McArdle S, Liikanen I, Goldrath AW, Ley K, Hedrick CC
  La Jolla Institute, UCSD, MedImmune, Fate Therapeutics
   
 

Nonclassical monocytes are effective in preventing metastatic tumor cells from seeding in lung tissue. Nonclassical monocytes patrol the vasculature to aid in maintaining vascular homeostasis and to respond to inflammatory signals. However, whether patrolling is required for nonclassical monocytes to prevent tumor metastasis is unclear. Using an inducible cre-lox mouse model and intravital microscopy, we show that Kindlin-3, an intracellular adaptor protein capable of activating integrins to their high-affinity conformation, is essential for driving the patrolling activity of nonclassical monocytes. Without Kindlin-3, nonclassical monocytes cannot patrol along the vascular endothelium, and are only able to roll and occasionally arrest. We hypothesized that nonclassical monocytes require patrolling to prevent metastases to the lung. When B16 melanoma tumor cells were injected intravenously into wild-type mice or mice with Kindlin-3 deficient monocytes, there was a significant 4-fold increase in B16 tumor metastases in the lungs of Kindlin-3 deficient mice compared to wild-type mice. Kindlin-3-/- nonclassical monocytes showed defective migration to the lung after intravenous tumor cell injection. Further investigation revealed that Kindlin-3-/- nonclassical monocytes failed to take up tumor material compared to wild-type monocytes. We found that this was due to lack of integrin activation in the absence of Kindlin-3. We conclude that the adaptor protein Kindlin-3 is essential for driving the nonclassical monocyte patrolling response to tumor metastasis in the lung.

   



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  Functions of the STING pathway in Nematostella vectensis
  Presenter: Shally Margolis
  All Authors:Shally Margolis, Stephen C. Wilson, Russell E. Vance
  UC Berkeley, HHMI
   
 

Nucleic acid sensors are critical players in innate immunity, as they allow cells to detect and respond to infection. In vertebrates, cytosolic DNA from pathogens or tissue damage is sensed by cGAS, leading to cGAMP production and STING activation. This leads to the production of type I interferons, the major cytokines that control antiviral responses. Interestingly, however, core components of the STING pathway predate the emergence of type I interferons in the vertebrate lineage. Our lab has previously demonstrated that the genome of the model cnidarian Nematostella vectensis, with which we shared our most recent common ancestor over 600 million years ago, contains functional components of the STING pathway. In this study, we aim to understand the function of this pathway in Nematostella. We have found that Nematostella express STING throughout life, and treatment of animals with cGAMP leads to the induction of putative immune genes. Some of these genes encode proteins with antibacterial activity, and their induction is dependent on the transcription factor NF- κB. In addition, some encode proteins with antiviral activity, and a subset of these seem to depend on STAT signaling. This work indicates that the ancestral functions of this pathway included both bacterial and viral control, even in the absence of interferons.

   



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  A novel model of ADAR1-driven autoinflammation
  Presenter: Megan Maurano
  All Authors:Megan Maurano, Damion Winship, Daniel Stetson
  University of Washington
   
 

The RIG-I like receptors (RLRs) detect viral RNA and initiate the interferon response that is essential for antiviral defense. However, these pathways can be activated by our own RNA, resulting in autoimmunity. Adenosine Deaminase Acting on Double-stranded RNA (ADAR1) “edits” double-stranded RNAs (dsRNA) by converting adenosines to inosines. Editing of endogenous RNA is required to prevent the activation of the RLR MDA5, aberrant interferon (IFN) production, and death. Loss of function mutations in ADAR cause aberrant recognition of self-RNA and result in the autoinflammatory disease, Aicardi-Goutieres Syndrome (AGS). 61% of patients with ADAR1-driven AGS have one P193A allele and a frameshift or deaminase domain mutation on their second allele. The prevalence of P193A far exceeds the that of any other ADAR allele in either patients or the healthy population, though no patients homozygous for P193A have been identified. P193A (P195A in mice) is in the z-alpha domain of ADAR1, whose role in ADAR1 function is unclear. To determine how P193A impairs ADAR1 function and contributes to disease, we developed a mouse model of P193A by inserting this mutation into the murine Adar locus. This new Adar P195A mouse model more faithfully recapitulates the genotype of AGS patients than current ADAR1 loss of function models, all three of which eliminate ADAR1 editing activity and require total loss of MDA5 signaling for the mice to be viable. We have found that homozygous Adar P195A/P195A mice are born at the normal Mendelian ratios. However, mice with one P195A allele combined with a null allele of either the interferon-inducible isoform of ADAR1 (p150 P195A/-) or both isoforms of ADAR1 (Adar P195A/-) have a dramatic survival defect with 100% mortality by day 120 and day 40, respectively. Prior to death, these mice are runted, with lower weights and delayed development relative to littermates of other genotypes. The survival defect and runting are MDA5 dependent: p150 P195A/- and ADAR P195A/- are partially rescued on an MDA5+/- background while mortality and runting are entirely prevented on an MDA5-/- background. Surprisingly, these mice do not have a robust IFN or ISG signature, as seen in current models, even after abrupt tamoxifen-induced deletion of the remaining Adar allele in Adar fl/P195A mice, implying that MDA5 functions other than cytokine induction are critical to the deleterious effects of ADAR1 loss of function in vivo. Future studies will focus on how the P195A mutation affects ADAR1 localization and editing of endogenous and viral RNAs.

   



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  Uncovering the innate immune signalling pathways regulated by ADAR1 editing
  Presenter: Megan Maurano
  All Authors:Megan Maurano, Daniel B. Stetson
  University of Washington
   
 

The RIG-I like receptors (RLRs) are essential to initiate the antiviral Type I Interferon response upon detection of viral RNA. However, these pathways can also be activated by endogenous RNA, resulting in autoimmunity from chronic interferon in the absence of infection. Adenosine Deaminase Acting on Double-stranded RNA (ADAR1) “edits” double-stranded RNAs (dsRNA) by converting adenosines to inosines, disrupting the double stranded structure and preventing aberrant MDA5 activation, interferon (IFN) production, and death. Loss of function mutations in ADAR cause aberrant recognition of self-RNA and result in the autoinflammatory disease, Aicardi-Goutieres Syndrome (AGS). 61% of patients with ADAR1-driven AGS have a P193A allele combined with a frameshift or deaminase domain mutation on the other allele. P193A (P195A in mice) is in the z-alpha domain of ADAR1, whose role in ADAR1 function is unclear. Interestingly, the prevalence of P193A far exceeds that of any other ADAR allele. To determine how P193A impairs ADAR1 function and contributes to disease, we developed a mouse model of P193A, inserting this mutation into the murine Adar locus. This new P195A mouse model more faithfully recapitulates the genotype of AGS patients than current ADAR1 loss of function models. Though homozygous AdarP195A/P195A mice are born at the normal Mendelian ratios, and survive as well as WT littermates, mice with one P195A allele combined with a null allele of either the interferon-inducible isoform of ADAR1 (AdarP195A/p150- or both isoforms of ADAR1 (AdarP195A/Adar-) have a dramatic survival defect with 100% mortality by day 120 and day 40, respectively. Prior to death AdarP195A/p150- mice are runted and have severe liver and kidney defects. AdarP195A/p150- mice are entirely rescued on an Ifih1-/- background, demonstrating that P195A specifically impacts regulation of MDA5, leaving other functions of ADAR1 intact. The survival defect and runting are entirely MDA5 dependent: AdarP195A/p150- and AdarP195A/Adar- mice are partially rescued on an Ifih1+/- background while mortality and runting are entirely prevented on an Ifih1-/- background. These mice do not have a robust IFN or ISG signature, as seen in current models, even after abrupt tamoxifen-induced deletion of the remaining Adar allele in Adarfl/P195A mice. The more subtle phenotype of these mice – full knock outs of Adar and p150 are embryonic lethal - better resembles AGS, and offers opportunities to understand what pathways downstream and in parallel to MDA5 contribute to disease. Future studies will focus on how the P195A mutation affects ADAR1 localization and editing of endogenous and viral RNAs.

   



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  Cyclooxygenase-2 dependent prostaglandin E2 signaling through the EP3 receptor is required for cell-mediated immunity to Listeria monocytogenes
  Presenter: Courtney E McDougal
  All Authors:Courtney E McDougal, Erin M Theisen, John-Demian Sauer
  University of Wisconsin-Madison
   
 

Listeria monocytogenes is an intracellular bacterium currently being developed as a cancer immunotherapeutic platform due to its ability to elicit robust CD8+ T-cell responses. Though the role of cytokines in Listeria-stimulated immunity has been extensively studied, the influence of lipid modulators of inflammation, known as eicosanoids, is less clear. Understanding how eicosanoids impact immunity is critical as current L. monocytogenes based immunotherapy trials utilize modulators of eicosanoid signaling, common nonsteroidal anti-inflammatory drugs (NSAIDs), as analgesics to alleviate patient discomfort following administration of L. monocytogenes. We hypothesized that eicosanoids, particularly those produced downstream of cyclooxygenases, influence the response to L. monocytogenes. Use of the non-selective cyclooxygenase inhibitor indomethacin demonstrated a clear role for cyclooxygenases in L. monocytogenes-stimulated immunity as antigen specific T-cells and protective immunity were decreased following treatment with indomethacin. Using Cox-1-/- mice we found that cyclooxygenase-1 activity was detrimental to immunity, whereas the use of a Cox-2 selective inhibitor demonstrated that cyclooxygenase-2 activity was required for immunity. Analysis of eicosanoid levels in the spleen revealed an acute and short-lived upregulation of the eicosanoid prostaglandin E2 (PGE2) twelve hours post immunization. PGE2 induction is critical for immunity as mice deficient in microsomal PGE2 synthase-1 showed impaired cell-mediated immune responses. Furthermore, administration of PGE2 restored immunity during Cox-2 inhibition, demonstrating that it is both necessary and sufficient. Finally, preliminary data suggests that PGE2 signaling through the EP3 receptor is critical for mediating immunity, as use of an EP3 antagonist diminished cell-mediated immune responses to L. monocytogenes. Understanding which cells produce PGE2 following L. monocytogenes infection will be essential to designing optimized platforms for Listeria-based immunotherapy. In addition, determining signaling pathways induced by PGE2 during Listeria-stimulated immunity will be key to developing a fully optimized therapeutic platform.

   



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  microRNA-132 regulates hematopoietic stem cell function and survival through FOXO3
  Presenter: Arnav Mehta
  All Authors:Arnav Mehta, David Baltimore
  California Institute of Technology
   
 

A complex network of interactions tightly regulates hematopoiesis to ensure balanced and appropriate output of blood cells, both under normal and stressful conditions. This network includes a novel class of RNA molecules, called microRNAs, that do not code for proteins, but instead negatively regulate the expression of genes. These molecules serve as “fine-tuners” of gene expression, and when dysregulated, can drastically alter the balance of hematopoiesis, potentially leading to cancer. We have identified microRNA-132, an LPS-inducible microRNA in macrophages, to be enriched in certain hematopoietic compartments. Importantly, ectopic expression of microRNA-132 leads to proliferation of hematopoietic stem cells (HSCs) and early progenitors, with eventual exhaustion and extra medullary hematopoiesis. Deletion of microRNA-132 leads to increased survival of HSCs, especially in conditions of continuous inflammatory stress. We have determined that this phenotype is primarily mediated by the microRNA-132 target FOXO3, a key transcription factor in regulating HSC proliferation and protective autophagy of aged HSCs. We have thus identified microRNA-132 as an important regulator of HSC self-renewal and function, with therapeutic implications in hematopoietic aging and cancer.

   



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  Presenter: mhcmgythu mhcmgythu
  All Authors:mhcmgythu mhcmgythu, mhcmgythu
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  Diet-induced weight loss restores T cell number and normalizes lipid droplets in the intestinal epithelium of obese mice
  Presenter: Nadia Miranda
  All Authors:Nadia Miranda, Nadia Miranda, Natalie Limon, Christa Park, Kitty Cheung, Dr. Julie Jameson
  CSU San Marcos
   
 

Diet-induced weight loss restores T cell number and normalizes lipid droplets in the intestinal epithelium of obese mice Obesity and type 2 diabetes are an increasing epidemic in the United States. Studies show that obesity causes complications such as barrier dysfunction and chronic non-healing wounds. Our laboratory has demonstrated that obesity and type 2 diabetes negatively affects the function and number of T cells in the epithelial layer of the small intestine. However, little is known about whether these T cells can be restored in number and if there is a change in lipid droplets in the intestine after a diet-induced weight loss. In this study, mice were separated into three cohorts. The control lean cohort consumed a 10% kcal Normal Chow Diet (NCD) for 14 weeks. The obese cohort consumed a 60% kcal high fat diet (HFD) for 14 weeks and the diet-induced weight loss cohort consumed a 60% kcal HFD for 7 weeks followed by a 10% kcal diet for the remaining 7 weeks. The small intestine was examined by freezing the tissue, cutting 5um sections with a cryostat machine, staining the tissue sections with antibodies specific for T cells as well as a Bodipy stain for lipid droplets, and examining the sections under an immunofluorescent microscope. Images were examined and quantified using Photoshop and ImageJ to calculate the number of T cells per villi in the small intestine and lipid droplet droplets per .7um diameter of an epithelial cell. Here we demonstrate that diet-induced weight loss restored T cell numbers and lipid droplets in the small intestine were at the same levels as a NCD cohort. This data is published and funding was provided by CSUPERB Presidents' Commission Scholars Program.

   



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  ICOS as a unique source of PI3K signaling in regulatory T cells
  Presenter: Kristen Mittelsteadt
  All Authors:Kristen Mittelsteadt, Daniel J. Campbell
  University of Washington, Benaroya Research Institute
   
 

Foxp3+ regulatory T cells (Treg) are critical for maintaining immune tolerance and preventing inflammatory disease at different tissue sites. Recent work suggests that signaling through the co-stimulatory receptor ICOS is required to control Treg cell abundance and function in vivo in peripheral tissues and sites of inflammation. ICOS signaling primarily results in strong PI3K activation. Though Treg are thought to employ mechanisms to suppress PI3K signaling downstream of the TCR and cytokine receptors compared to conventional T cells (Tconv), mice expressing catalytically inactive PI3K exhibit severe defects in Treg maintenance and suppressive function. Taken together, we hypothesize that ICOS is an important source of PI3K signaling in Treg. Indeed, we find that while Tconv are capable of activating PI3K in response to multiple inputs, stimulation of sorted Treg through ICOS results in potent phosphorylation of AKT downstream of PI3K without any synergistic effect of additional stimuli. Mice lacking ICOS (KO) or carrying a knock-in mutation that specifically abolishes ICOS-dependent PI3K signaling (YF) have reduced Treg frequencies in lymphoid and non-lymphoid tissues. YF and KO Treg have impaired regulation of downstream PI3K targets, including decreased phosphorylation of AKT and S6 and altered expression of the lymphoid homing molecule CD62L. In mixed bone marrow chimeras, YF and KO Treg preferentially repopulate lymphoid organs and exhibit a competitive disadvantage in reconstituting non-lymphoid tissues compared to WT Treg. Furthermore, YF and KO mice are unable to recover from both active and passive transfer experimental autoimmune encephalomyelitis, correlating with lower frequencies of activated Treg at effector sites. Taken together, we suggest an important role for ICOS-mediated PI3K activation in the maintenance and function of Tregs.

   



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  ICOS-dependent PI3K signaling in regulatory T cell development and function
  Presenter: Kristen Mittelsteadt
  All Authors:Kristen Mittelsteadt, Daniel J. Campbell
  University of Washington, Benaroya Research Institute
   
 

Foxp3+ regulatory T cells (Treg) are critical for maintaining immune tolerance and preventing inflammatory disease at different tissue sites. The co-stimulatory receptor ICOS is an important regulator of T cell function. Recent work suggests that ICOS signaling is required to control Treg cell abundance and function in vivo in peripheral tissues and sites of inflammation. Although the mechanisms by which ICOS supports peripheral Tregs is unknown, they may involve its ability to potently activate PI3K signaling. In order to study the role of ICOS-dependent PI3K activation in Treg homeostasis and function, we obtained mice that carry a knock-in mutation in the Icos gene that alters the cytoplasmic tail motif of the ICOS protein and specifically abolishes ICOS-dependent PI3K signaling (YF mice). Indeed, Treg frequencies and proliferation are reduced in these mice in lymphoid organs. YF Tregs have impaired regulation of downstream PI3K targets, including decreased phosphorylation of AKT and S6, and altered expression of the lymphoid homing molecule CD62L. In mixed bone marrow chimeras, YF Tregs exhibit a competitive disadvantage in populating non-lymphoid tissue. Furthermore, YF mice are unable to recover from both active and passive transfer experimental autoimmune encephalomyelitis, correlating with lower frequencies of activated Tregs at effector sites. Taken together, we suggest an important role for ICOS-mediated PI3K activation in the maintenance and function of Tregs.

   



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  ICOS-dependent PI3K signaling in regulatory T cell development and function
  Presenter: Kristen Mittelsteadt
  All Authors:Kristen Mittelsteadt, Jenna Sullivan, Michael Stolley, Daniel J Campbell
  University of Washington, Benaroya Research Institute
   
 

Foxp3+ regulatory T cells (Treg) are critical for maintaining immune tolerance and preventing inflammatory disease. Given their potent immunosuppressive capabilities, manipulation of Treg function and/or abundance is a promising therapeutic strategy to either augment or inhibit immune responses in the context of disease. A detailed understanding of the key factors that control Treg development, homeostasis, and function is essential for the successful application of Treg-based therapies. Recent work suggests that inducible T cell costimulator (ICOS) signaling is required for maintenance of effector Treg (eTreg) cells, which migrate to peripheral sites of inflammation. ICOS ligation is most notably a potent activator of phosphatidylinositol 3-kinase (PI3K), however the key signaling pathways downstream of ICOS that support eTreg have not been investigated. In order to study the role of ICOS-dependent PI3K signaling, we obtained mice that carry a knock-in mutation in the Icos gene that alters the cytoplasmic tail motif of the ICOS protein, thereby specifically abolishing ICOS-mediated PI3K activation (IcosY181F mice). The frequency and number of Treg is reduced in these mice, as is the proliferation of ICOShi Treg in the spleen. Early thymic frequencies are altered in IcosY181F thymii, suggesting a developmental bottleneck at the early stages of T cell development in the absence of ICOS-dependent PI3K activation. Furthermore, IcosY181F hematopoietic cells exhibit a selective disadvantage in repopulating peripheral T cell compartments as well as early bulk thymocyte populations compared to WT cells in mixed bone marrow chimeras. To assess function of IcosY181F Treg, we subjected mice to a model of experimental autoimmune encephalomyelitis. IcosY181F mice develop more severe disease earlier than WT controls and have lower eTreg frequencies in the brain and spinal cord. Taken together, preliminary data from our lab suggests an important role for ICOS-mediated PI3K activation in the development, maintenance, and function of Treg.

   



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  HnRNPA2B1 Role as an Immune Gene Regulator
  Presenter: Mays Mohammed Salih
  All Authors:Mays Mohammed Salih, Susan Carpenter
  University of California/ Santa Cruz
   
 

The innate immune system is the first line of defense against pathogens; it functions through various pattern recognition receptors (PRRs) that recognize microbial products or danger signals leading to the activation of signaling pathways which initiate transcription of inflammatory genes. Activation of the innate immune response is essential to resolve infections, however, its dysregulation can result in pathological inflammation, contributing to an array of diseases, such as atherosclerosis, autoimmunity and cancer. Our understanding of the mechanisms and genes that regulate this response is not yet complete. We have recently found hnRNPA2/B1, a ubiquitous RNA processing protein, to be a negative regulator of interferon stimulated genes (ISGs) where knocking it out in macrophages resulted in an altered immune response state. We hypothesize that hnRNPA2B1 is critical for regulation of immune genes in vivo and modulates their expression in response to an infection or a stimulus. To test whether hnRNPA2B1’s recently discovered role as an immune gene regulator is conserved in humans, I will knock it down in macrophages differentiated from human monocyte cell line (THP) using siRNA and subject these cells to an immune challenge. Additionally, to further characterize its role in vivo specifically in macrophages – integral early responders to inflammatory challenges- we recently generated a mouse where hnRNPA2B1 is exclusively knocked out in myeloid cells using a cre-lox system. Absence of hnRNPA2B1 in macrophages from mouse and human origins could result in an altered transcriptome and cytokine profile and lead to an altered ability to fight off microbes. To investigate the mechanism by which hnRNPA2B1 exerts its control on immune genes I will identify binding partners using Co-IP experiments on cells from human and mouse origins. This study will advance our understanding of how the inflammatory response is coordinated and help us better understand inflammatory and infectious diseases. Furthermore, this study will advance the field by shedding light on the functions of a poorly studied RNA processing protein that’s implicated in an array of biological processes and enhance our understanding of hnRNPs in general.

   



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  Investigating the effect of bendamustine on the STAT3-Flt3 signaling pathway in DCs
  Presenter: Megan Molina
  All Authors:Megan Molina, Jessica Stokes, Emely Hoffman, Richard J. Simpson, Emmnauel Katsanis
  University of Arizona
   
 

Our laboratory has previously demonstrated that bendamustine conditioning is a safer alternative to cyclophosphamide in an MHC-mismatched murine bone marrow transplantation (BMT) model of graft-versus-host disease (GvHD). Bendamustine is a hybrid molecule, containing alkylating groups and a purine analog, conferring unknown anti-metabolite functions. It is known to bind directly to the SH2 domain of STAT3 and inhibit its canonical signaling. STAT3 is indispensible for Flt3 Ligand (Flt3L)-dependent dendritic cell (DC) differentiation. Flt3L-derived DCs have been shown to have unique cytokine production and trafficking ability, which may have significant implications for GvHD pathogenesis. Here we sought to investigate the effect of bendamustine on host DC differentiation in vivo and in vitro. We hypothesize that by inhibiting STAT3, bendamustine triggers a positive feedback loop that promotes Flt3 signaling in host DCs. To test this hypothesis in vivo, BALB/c mice were given equivalent doses of bendamustine (40 mg/kg BEN i.v.) or cyclophosphamide (200 mg/kg CY i.p.) on day -2 before BMT. On day 0, when the conditioned recipient would normally be transplanted, pan-DCs from the recipient spleen were isolated via magnetic selection and then characterized by flow cytometry. For in vitro experiments, bone marrow was collected from BALB/c mice and cultured in the presence of Flt3L to generate bone marrow-derived DCs (BMDCs). BMDCs were cultured in the presence of increasing concentrations of bendamustine for 6 days, and LPS was added to activate DCs for the final 18 hours. Additional BMDC experiments were performed in the presence of increasing concentrations of STAT3 inhibitor (JSI-124) and Flt3 inhibitor (A220). On day 6 of culture, BMDCs were harvested for characterization. Flow cytometry was performed to measure CCR7 and Flt3 receptor (CD135) expression on the various subsets of DCs both in vivo and in vitro. Bendamustine treatment resulted in a significant increase in cDCs and significant decrease in pDCs in vivo as compared to cyclophosphamide. Bendamustine treated DCs also showed a pattern of increased CCR7 and Flt3 receptor expression, which was significant in some DC subsets. These findings advocate a potential role of Flt3L-dependent DCs in the mechanism by which BEN alters GvHD pathogenesis to limit morbidity and mortality. Thus, BEN may have advantages as an alternative agent to CY for pre-transplant conditioning in allogeneic BMT.

   



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  CD8[a]+ Type 1 Conventional Dendritic Cells And Their Immediate Precursor Exhibit Phenotypic Differences And Induce Distinct Allogeneic CD8+ T-Cell Responses
  Presenter: Megan Stanley Molina
  All Authors:Megan Stanley Molina, Jessica Stokes, Emely Hoffman, Richard J. Simpson, Emmanuel Katsanis
  University of Arizona
   
 

The type 1 conventional murine dendritic cell (DC) subset, CD8α+ cDC1, has been identified as a strong inducer of anti-cancer responses through cross-presentation of tumor antigens to CD8+ T-cells. CD8α+ cDC1s have also been identified as suppressors of graft-versus-host disease (GvHD) in the context of allogeneic bone marrow transplantation (BMT). The immediate precursor to this DC subset, pre-cDC1, has been identified as CD24highCD8α-. The few studies characterizing this precursor population have found that pre-cDC1s more potently induce memory CD8+ T-cells in response to viral infection compared to CD8α+ cDC1, indicating fundamental functional differences. However, this precursor population has never been studied in the context of alloreactivity as compared to CD8α+ cDC1s. We therefore used flow cytometry to evaluate the expression of activation markers and inhibitory signaling molecules on pre-cDC1 as compared to CD8α+ cDC1 in naïve mice and in mice treated with sub-lethal total body irradiation (TBI). We further compared the ability of pre-cDC1 and CD8α+ cDC1 to stimulate allogeneic, naive total T-cells and allogeneic, naïve CD8+ T-cells. We report that pre-cDC1s exhibit significant differences in their expression of the activation and inhibitory molecules PD-L1, CD70, ICOSL, and PIR-B compared to CD8α+ cDC1s. Further, we report that stimulation of allogeneic T-cells with pre-cDC1 compared to CD8α+ cDC1s results in significantly more total T-cell proliferation measured by tritiated thymidine uptake. When we further analyzed the ability of pre-cDC1s to stimulate allogeneic CD8+ T-cells compared to CD8α+ cDC1s, we find that stimulation with pre-cDC1s result in significantly higher expression of CD44, a marker for effector memory T-cells, and significantly higher expression of CD122 (IL-2RB). Additionally, stimulation with pre-cDC1s results in a trend of greater expression of TIM-3, a marker of T-cell exhaustion. These results indicate significant phenotypic and functional differences between these two subsets of murine DCs, though further studies are required to determine the role pre-cDC1s may play in the context of GvHD and anti-cancer responses.

   



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  A septin requirement differentiates autonomous and contact-facilitated T cell proliferation
  Presenter: Adriana Mujal
  All Authors:Adriana Mujal, Julia Gilden, Audrey Gerard, Makoto Kinoshita, Matthew Krummel
  UCSF
   
 

An efficacious immune response requires both rapid antigen-specific expansion of activated T cells, as well as homeostatic turnover and maintenance of naïve and memory T cells. Although these proliferative processes are driven by distinct or synergistic cues of T cell receptor (TCR) signaling or cytokines, it has been thought that equivalent cellular machinery is used to undergo cell division. In particular, the septin cytoskeleton, which consists of a family of GTP-binding proteins, is highly conserved in its role in cytokinesis. One striking exception to the requirement of septins for mammalian cytokinesis, however, has been T cells. Yet, in investigating T cells that lack the septin cytoskeleton, we found that successful cell division has discrete septin –dependent and –independent pathways. Septin-deficient CD8+ T cells undergo robust proliferation when activated by antigen-presenting cells (APCs), but exhibit cytokinetic failure following cytokine-driven division. Surprisingly, APCs facilitate septin-independent cell division through cell-cell contacts and phosphoinositide 3-kinase (PI3K) signaling provided by co-stimulatory and integrin molecules. We could differentiate cytokine- versus antigen-driven proliferation in vivo and thus uncover the potential to selectively target detrimental bystander or homeostatic cytokine-driven proliferation without impacting conventional antigen-specific T cell expansion.

   



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  T cell signaling and Treg dysfunction define disease kinetics in IL-2Ra-KO autoimmune mice
  Presenter: Genevieve Mullins
  All Authors:Genevieve Mullins, Kristen M. Valentine, Mufadhal Al-Kuhlani, Dan Davini, Kirk D.C. Jensen, Katrina K. Hoyer
  UC Merced
   
 

IL-2Rα is required to generate the high affinity receptor for IL-2, a cytokine important in immune proliferation, activation, and regulation. Mice deficient in IL-2Rα (IL-2Rα-KO) develop systemic autoimmune disease and die from severe anemia between 18-80 days of age. These mice develop kinetically distinct autoimmune progression, with approximately a quarter dying by 21 days of age and half dying after 30 days. This research aims to define immune parameters and signaling that distinguish cohorts of IL-2Rα-KO mice that develop early- versus late-stage autoimmune disease. To investigate these differences, we evaluated complete blood counts (CBC), antibody binding of RBCs, T cell numbers and activation, hematopoietic progenitor changes, and signaling kinetics, during autoimmune hemolytic anemia (AIHA) and bone marrow failure. Using a simple CBC we were able to predict disease kinetics to explore mechanisms underlying early and late disease. We identified several alterations, that combined, contribute to disease kinetics. Early onset disease correlates with anti-RBC antibodies, lower hematocrit, reduced IL-7 signaling, and increased CD8 T cell expansion. CD8 regulatory T cells (Tregs) lost IL-10 expression and had enhanced apoptosis in early disease. Further, CD8 Tregs maintained a higher suppressive capacity and presence in delayed disease. IL-2Rα-KO disease pathology rates are driven by T cell signaling that skew effector T cell activation and expansion, and Treg dysfunction. Altered cytokine and TCR signaling, from IL-2Rß upregulation and increased IL-2 production, along with decreased suppression from CD8 Tregs, may in turn promote increased CD8 T cell expansion, resulting in the elevated CD8 T cells and effector/memory fate choices that skew disease kinetics.

   



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  Signaling responsiveness driving autoimmune disease pathology and kinetics
  Presenter: Genevieve Mullins
  All Authors:Genevieve Mullins, Kristen M. Valentine, Mufadhal Al-Kuhlani, Dan Davini, and Katrina K. Hoyer
  Univeristy of California, Merced
   
 

IL-2Rα is the high affinity receptor for IL-2, a cytokine important in immune proliferation, activation, and regulation. Mice deficient in IL-2Rα (IL-2Rα-KO) develop systemic autoimmune disease and die from severe anemia between 18-80 days of age. These mice develop kinetically differing autoimmune disease, with approximately a quarter dying by 21 days and half dying after 30 days. This research aims to evaluate the kinetic and phenotypic differences between early- and late-stage autoimmune disease in IL-2Rα-KO mice. To investigate the kinetic differences and the extent of peripheral autoimmune hemolytic anemia and bone marrow failure, we evaluated complete blood counts, antibody binding of RBCs, T cell numbers and activation, and hematopoietic stem cell (HSC) lineage changes. Our data indicate that IL-2Rα-KO mice develop both disease phenotypes largely regardless of disease kinetics, although autoimmune hemolytic anemia is more severe in mice predicted to die early. Upon further exploration, we identified differences in CD4 and CD8 T cell capacity to produce and respond to the cytokines IL-2, IL-7, and IFNγ. We observed differences in expression of these cytokine receptors and in phosphorylation of downstream signaling pathways. We are currently exploring the role of these signaling differences in the disease kinetics and pathologies.

   



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  Regulation of succinate-driven type 2 immune responses in the small intestine
  Presenter: Marija Nadjsombati
  All Authors:Marija Nadjsombati, John McGinty, Natalie Niepoth, Andrés Bendesky, Jakob von Moltke
  University of Washington
   
 

In the small intestine, type 2 immune responses can be initiated through a tuft-ILC2 circuit in which tuft cell-derived IL-25 activates group 2 innate lymphoid cells (ILC2s) in the lamina propria. Recently, our group and others reported that tuft cells in the small intestine detect Tritrichomonas protists via their secreted metabolite succinate. Succinate binding to its receptor, SUCNR1, on tuft cells initiates a signaling cascade sufficient to induce a robust type 2 immune response, particularly in the distal small intestine (i.e. ileum). However, while studying succinate sensing by tuft cells, we observed that activation of the tuft-ILC2 circuit, as measured by tuft cell hyperplasia, varies among different strains of mice. Balb/c mice do not develop tuft cell hyperplasia when given succinate yet are capable of responding to succinate if first ‘primed’ with recombinant IL-25. This suggests all components of the succinate-sensing tuft-ILC2 circuit are functional, yet differentially regulated between B6 and Balb/c mice. We demonstrate that this differential regulation is genetically encoded. Balb/c x B6 F1 progeny all develop tuft cell hyperplasia following succinate treatment and Balb/c x B6 F2 progeny have varied responses with approximately a 3 to 1 ratio of responder to non-responder mice. Furthermore, quantitative trait loci (QTL) mapping of succinate response phenotypes of Balb/c x B6 F2 mice identified a single robust QTL associated with variations in succinate responsiveness. This QTL does not contain any genes currently implicated in the tuft-ILC2 circuit. Together, these results indicate that a single recessive locus in Balb/c mice regulates succinate-mediated activation of the tuft-ILC2 circuit. We hypothesize that there is a novel regulator of the succinate-tuft-ILC2 circuit and future work will aim to define the mechanism by which this regulation occurs.

   



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  IL-4 and IL-21 reciprocally regulate a unique T- BET driven phenotype in B cells
  Presenter: Martin Naradikian
  All Authors:Martin Naradikian, Susanne Linderman, Lucas Woods, Daniel Beiting, Rosanne Spolski, E. John Wherry, Christopher Hunter, Scott Hensley, Warren J. Leonard, and Michael P. Cancro
  University of Pennsylvania
   
 

In B cells, T-BET and CD11c expression are linked with IgG2c isotype switching, propensity for humoral autoimmunity, and the formation of age-associated B cells. Prior studies implicate IFN-γ as a mediator of these effector fates. Here we show that T-BET and CD11c are induced by IL-21 in the context of TLR7 or TLR9 signals, and can be blocked by concomitant IL-4 stimulation. This differs from IFN-γ driven T-BET expression because IFN-γ neither induces CD11c nor is blocked by IL-4. Moreover, IFN-γ and IL-21 establish similar but distinct transcriptional profiles in the resulting T-BET+ B cells. Consistent with our in vitro observations, IL-21 overexpression in vivo is sufficient to promote disproportionate accumulation of T-BET+CD11c+ B cells. To further dissect the interplay of IL-21, IL-4, and IFN-γ in controlling the T-BET+CD11c+ B cell fate, we used two infection models. Mice infected with either influenza or H. Polygyrus accumulated T-BET+CD11c+ B cells preferentially into the memory B (BMEM) cell pools in the absence of IL-4 and IFN-γ. Further, the predominant antibody isotype generated against H. polygyrus changes from IgG1 to IgG2c in IL-4 deficient mice and is independent of IFN-γ. Thus, while the magnitude of the germinal center B, plasma cell, BMEM, and T follicular helper cell responses remain largely comparable across genotypes, the quality of the immune response changes based on the relative prevalence of these three cytokines.

   



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  Regulation of Myeloid Immune Cell Activation and the Type I IFN Response by Interleukin Enhancer Binding Factor 3 (ILF3)
  Presenter: Rudy Nazitto
  All Authors:Rudy Nazitto, Jarrod S Johnson, Lynn M Amon, Alan Aderem
  University of Washington/Center for Infectious Disease Research
   
 

Type I interferon (IFN) acts as a potent antiviral weapon. Almost all cells can produce type I IFN, but innate immune cells, such as myeloid dendritic cells (DCs), play key roles in IFN signaling as they are positioned to “sense” pathogens and program adaptive immune responses. It is critical that we understand IFN regulation, since dysregulation occurs in disorders such as autoimmune disease, cancer, and chronic infections. In particular, HIV-1 is known to evade early detection in myeloid DCs, but as the disease progresses, IFN responses become exacerbated and correlate with the degree of pathogenesis. Using systems biology approaches, we have uncovered that Interleukin Enhancer Binding Factor 3 (ILF3) acts as a negative regulator of HIV-driven innate immune responses. ILF3 is known to have a role in regulating stress responses and host mRNA in ribonucleoprotein (RNP) complexes, and our work suggests an additional role in regulating cytoplasmic DNA sensing. Here we find that shRNA knockdown of ILF3 in MDDCs led to increased basal activation and significantly potentiated maturation (CD86, HLA-DR, CD40) and IFN production during HIV-1 infection. Inhibition of HIV-1 reverse transcription completely abrogated maturation and IFN induction, consistent with the role for cytoplasmic DNA as a trigger for innate sensing. Surprisingly, when HIV integration was blocked, cells deficient in ILF3 still exhibited potentiated IFN responses and increased maturation, suggesting ILF3 may contribute to regulation of cytoplasmic cDNA sensing through the established DNA sensor for HIV-1, cGAS. Transcriptomic analysis of ILF3-deficient MDDCs paired with motif scanning of differentially regulated transcripts has given us insight into how ILF3 might regulate signaling thresholds in innate immunity. This work uniquely links host RNA-binding complexes to modulation of innate sensing, antiviral responses, and myeloid maturation.

   



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  Helios predisposes human fetal CD4+ naïve T cells towards regulatory T cell differentiation
  Presenter: Melissa Ng
  All Authors:Melissa Ng, Melissa S. F. Ng, Theodore L. Roth, Ventura F. Mendoza, Alexander Marson, Trevor D. Burt
  University of California, San Francisco
   
 

Activation of naïve CD4+ T cells by T cell receptor (TCR) stimulation and cytokine cues lead to differentiation into effector T cell populations with distinct pro-inflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells uniquely differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signalling. This facility for Treg differentiation is crucial for generating tolerance in utero; however, the mechanisms underlying this fetal cell-intrinsic predisposition towards the Treg cell fate are largely unknown. Here, we reveal FOXP3-independent transcriptional and epigenetic programs shared between fetal naive T cells and committed adult Treg cells that are inactive in adult naive T cells. We show that a subset of adult Treg-specific super-enhancers is active within fetal naive T cells, including two active super-enhancers at Helios, a signature thymic Treg gene. Helios is only expressed in fetal naive T cells, but not in adult naïve T cells, and only fetal-derived induced Treg (iTreg) cells continue to express Helios. Fetal, but not adult iTreg cells, have suppressed IL-2 production, which is regulated by Helios in committed Treg cells. CRISPR-Cas9 ablation of Helios in fetal naive T cells subsequently resulted in increased IL-2 production by fetal iTreg cells. Crucially, the loss of Helios expression in fetal naïve T cells impaired their subsequent differentiation into Treg cells upon TCR stimulation, indicating Helios as a critical contributor to cell-intrinsic predisposition of fetal naive T cells for Treg cell differentiation. The Treg-biased transcriptional and epigenetic programs within fetal naive T cells identified here could thus be utilized to engineer enhanced iTreg populations from adult naïve T cells for adoptive cellular therapies.

   



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  Characterization of T Follicular Helper Cells and T Follicular Regulatory Cells in HIV-Infected and Non-Infected Patients
  Presenter: Minh Quan Nguyen
  All Authors:Minh Quan Nguyen, Minh Quan T. Nguyen, Rachel S. Resop, Brent Gordon, Marta Epeldegui, Otoniel Martinez-Maza, Christel H. Uittenbogaart
  UCLA
   
 

Background: The humoral response is important in fighting bacteria and other intracellular pathogens by the production of specific antibodies by B cells. In the germinal center, T follicular helper (gcTFH) cells provide important help to B cell antibody production. gcTFH have been shown to contribute to HIV persistence and act as an HIV reservoir. Yet, the recently discovered germinal center T follicular regulatory (gcTFR) cells, which inhibit the function of T follicular helper cells, express very similar surface markers as gcTFH. FOXP3 is the only marker that distinguishes gcTFR from gcTFH. Thus, it is unknown whether the increase in gcTFH which has been observed in HIV infection is also due an increase in gcTFR and whether gcTFR, like gcTFH can also contribute to HIV persistence. Methods: Using multicolor flow cytometry, peripheral blood mononuclear cells (PBMC) were stained for intracellular TFR markers: FOXP3 and BCL6, in combination with other cell surface markers including CD4, CD3, CD8, CD25, TIGIT, PD1 and ICOS as well as the HIV co-receptor, CCR5. We used available cryopreserved PBMC from HIV-infected and non-infected individuals in the UCLA Multicenter AIDS Cohort Study (MACS), as opposed to the scarcer lymph nodes. Results: In our study, we have characterized and identified the presence of CD3+CXCR5+CD8-BCL6+ peripheral blood T follicular helper (pTFH) and CD3+CXCR5+CD8-FOXP3+ peripheral blood T regulatory cells (pTFR) in PBMC. Our results show that unlike gcTFR, pTFR do not express B cell lymphoma 6 (BCL6), a TFH master regulator. In HIV-infected individuals, the percentage of pTFH is higher. However, the percentage of pTFR is similar in HIV-infected and non-infected individuals. Our results show that there is a statistically significant difference between the levels of ICOS expressed on pTFR from HIV-infected than non-infected individuals. ICOS is expressed on activated T cells and its constitutive expression was shown on regulatory T cells. We noticed constitutive expression of ICOS on pTFR from non-infected individuals and that HIV infection increased its expression on pTFR. The expression of Programmed Cell Death Protein 1 (PD1) is increased on both pTFH and pTFR in HIV-infected individuals. pTFR expressed higher levels of TIGIT than pTFH, but there was no difference between the expression of TIGIT on cells from non-infected and HIV-infected individuals. Both pTFH and pTFR express from 2% to 42% of CCR5, indicating their susceptibility to CCR5-tropic HIV and a potential to contribute to HIV reservoir. Conclusion: In summary, our data show that pTFH and pTFR can be detected in peripheral blood of HIV-infected and non-infected individuals and potentially serve as a representative measure of gcTFH and gcTFR inside the lymph nodes. Although the percentage of pTFR is not elevated in HIV-infected individuals, we do not yet know whether their function is affected or whether they harbor HIV and thereby contribute to HIV persistence. Experiments are planned to address these questions. Further understanding of cells contributing to HIV persistence will be beneficial to the discovery of a functional cure for HIV infection.

   



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  The Contribution of T Follicular Regulatory Cells in HIV Persistence
  Presenter: Minh Quan Nguyen
  All Authors:Minh Quan Nguyen, Rachel S. Resop, Brent Gordon, Marta Epeldegui, Otoniel Martinez-Maza, Christel H. Uittenbogaart
  UCLA
   
 

The HIV reservoir presents a major challenge in finding a functional cure for HIV. In lymphoid tissue, T follicular helper cells in the germinal center (GC) have been previously found to be contributors to the viral reservoir. Recently, a subset of regulatory T cells was identified in the GC of the mouse lymphoid tissues and was named T follicular regulatory cells (TFR). TFR express a similar set of markers to that of T follicular helper cells such as CD4, CXCR5, and BCL6. In addition, they express T regulatory cells markers: FoxP3 and CD25. Our data shows that cells with a TFR phenotype (CD3+, FoxP3+, CXCR5+, BCL6+) are present in the human tonsils and can also be detected in peripheral blood. This leads to our hypothesis in which TFR may play a role in HIV pathogenesis and in the maintenance of the HIV reservoir, as T follicular helper cells are known to form part of the HIV reservoir. It remains unknown whether only T follicular helper cells in the lymph nodes harbor HIV in HIV-infected individuals, or whether TFR present in lymph nodes and peripheral blood also contribute to HIV persistence. Using multicolor flow cytometry, peripheral blood mononuclear cells (PBMC) were stained for intracellular TFR markers: FoxP3 and BCL6, in combination with other cell surface markers including CD4, CD3, CD8, CD25, TIGIT, PD1 and ICOS as well as the HIV co-receptors, CCR5 and CXCR4. We examined the expression profile of peripheral blood T follicular helper cells and TFR in fresh and frozen PBMC from healthy individuals and in frozen PBMC from HIV-infected individuals, obtained from the Multicenter AIDS Cohort Study (MACS). We found that a subset of CD3+CD4+CXCR5+ T cells expresses CD25 and FoxP3 in fresh PBMC. These CD4+CXCR5+CD25+FoxP3+ T cells are identified as TFR, which are distinct from the CXCR5+FoxP3- T follicular helper cells. Unlike the expression profile in lymphoid tissue, BCL6 expression could not be detected in peripheral blood TFR. Thus, peripheral blood TFR may differ from lymphoid tissue TFR in that they do not express BCL6. TFR also expressed CD62L, a lymphoid tissue homing receptor, suggesting that peripheral blood TFR may be the pre-cursors of lymphoid tissue TFR. CD62L expression, however, was found to be unstable upon freezing, so CD62L would not be useful for immunotyping of frozen PBMC, which is currently underway. Lastly, peripheral blood CD4+CXCR5+FoxP3+CD25+ TFR expressed the HIV co-receptors, CCR5 and CXCR4. This indicates that TFR can potentially be infected by CCR5-tropic and CXCR4-tropic HIV and that TFR have the potential to serve as a viral reservoir. Overall, these initial findings haven given insights into the immunophenotype of TFR and demonstrate that these cells have the potential to contribute to HIV persistence. In the future, we will be exploring the expression profile of TFR in HIV-infected individuals. Peripheral TFR are a unique T cell subset that expresses Foxp3 and CD25, in addition to T follicular helper cell markers, and can potentially contribute to HIV persistence.

   



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  Cell-type specific control of immune responses mediated by inflamamsomes in vivo
  Presenter: Randilea Nichols
  All Authors:Randilea Nichols, Jakob von Moltke, and Russell E. Vance
  University of California, Berkeley
   
 

Upon pathogen recognition, certain cytosolic nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) form multi-protein complexes called inflammasomes, which activate CASPASE-1. CASP1 initiates inflammation by processing IL-1beta and -18 into their active forms, and by inducing a rapid, lytic cell death called pyroptosis. The NLRC4 inflammasome responds to the cytosolic presence of specific bacterial proteins, such as flagellin, via NAIPs. A fundamental question is whether and how activation of innate immune responses triggers pathogen-specific adaptive immune responses. We seek to test the hypothesis that cell-type-specific activation of the inflammasome is sufficient to induce inflammation and adaptive immune responses in vivo. To test our hypothesis, we are using a novel ‘knock-in’ mouse strain, which can selectively activate the NLRC4 inflammasome. This mouse contains a gene encoding chicken ovalbumin (OVA) fused to the C-terminal 166 amino acids of Legionella pneumophila flagellin (Fla). The OVA-Fla fusion is inserted into the constitutively expressed Rosa26 locus, downstream of a loxP-flanked transcriptional STOP cassette, which prevents OVA-Fla expression unless Cre recombinase excises the STOP cassette. OVA-Fla:LysM-Cre mice, which express the OVA-Fla fusion protein in neutrophils, monocytes, and macrophages, display a visible inflammatory phenotype at 4-5 weeks of age. OVA-Fla:LysM-Cre mice suffer from anemia, increased serum cytokines/chemokines, and cellular changes in the lymph nodes and spleen. Histology shows bone erosion, neutrophil infiltration, and swelling of the tibiotarsal joint, consistent with an arthritis-like disease. Our preliminary data indicate extensive NLRC4-dependent inflammatory disease in mice expressing OVA-Fla in LysM-positive cells.

   



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  Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family
  Presenter: Kouta Niizuma
  All Authors:Kouta Niizuma, Satoko Tahara-Hanaoka, Emiko Noguchi, Akira Shibuya
  University of Tsukuba, Japan
   
 

Recruitment of circulating monocytes and neutrophils to infection sites is essential for host defense against infections. Here, we identified a previously unannotated gene that encodes an immunoglobulin-like receptor, designated CD300H, which is located in the CD300 gene cluster. CD300H has a short cytoplasmic tail and associates with the signaling adaptor proteins, DAP12 and DAP10. CD300H is expressed on CD16+ monocytes and myeloid dendritic cells. Ligation of CD300H on CD16+ monocytes and myeloid dendritic cells with anti-CD300H monoclonal antibody induced the production of neutrophil chemoattractants. Interestingly, CD300H expression varied among healthy subjects, who could be classified into two groups according to “positive” and “negative” expression. Genomic sequence analysis revealed a single-nucleotide substitution (rs905709 (G/A)) at a splice donor site on intron 1 on either one or both alleles. The International HapMap Project database has demonstrated that homozygosity for the A allele of single nucleotide polymorphism (SNP) rs905709 (“negative” expression) is highly frequent in Han Chinese in Beijing, Japanese in Tokyo, and Europeans (A/A genotype frequencies 0.349, 0.167, and 0.138, respectively) but extremely rare in Sub-Saharan African populations. Together, these results suggest that CD300H may play an important role in innate immunity, at least in populations that carry the G/G or G/A genotype of CD300H.

   



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  ICOS signaling suppresses an excessive T cell response in the CNS during chronic T. gondii infection
  Presenter: Carleigh O'Brien
  All Authors:Carleigh O'Brien, Tajie Harris
  University of Virginia
   
 

CNS infection with the parasite Toxoplasma gondii results in ongoing T cell recruitment and chronic inflammation in the brain, which is required for control of the parasite. It has also been shown that immunosuppressive cytokine production during this chronic inflammation is important in preventing severe immunopathology associated with this chronic effector response, however, very little is known about the specific mechanisms promoting regulation of long-term inflammatory responses in the CNS. ICOS (inducible T cell costimulator) is expressed on activated T cells and has been shown to both promote inflammatory effector T cell responses in acute infection models, as well contribute to immunoregulation through induction of IL-10. The role of ICOS signaling during the maintenance of long-term immune responses to chronic inflammation still remains poorly understood. We find ICOS expressing T cells in the brain during chronic T. gondii infection, and blocking ICOS signaling at this stage of infection unexpectedly leads to increased effector T cell responses and increased immunopathology in the CNS. This effect appears to be limited to the inflamed CNS, where the increased effector T cell response seen with ICOS-ligand blockade is associated with higher CD25 and pSTAT5 levels, as well as increased proliferation and Bcl-2 expression. Together, these data suggest that ICOS signaling in the context of chronic inflammation negatively regulates IL-2 signaling in effector T cells. Surprisingly, no changes in IL-10 production are seen with ICOS-ligand blockade, suggesting that ICOS signaling promotes regulation of effector T cell responses independent of the effects of IL-10. Indeed, blockade of IL-10R during chronic T. gondii infection does not increase levels of CD25 or Bcl-2 in effector T cells. Overall, the excessive T cell response seen in the absence of ICOS signaling suggests an important role for ICOS-ICOSL costimulation in maintaining local immune suppression in the CNS during chronic infection.

   



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  Pak2: An essential regulator for regulatory T cell development and function
  Presenter: Kyle O'Hagan
  All Authors:Kyle O'Hagan, Hyewon Phee
  Northwestern University
   
 

Although significant effort has been devoted to understanding the thymic development of Foxp3+ regulatory T cells (Tregs), the precise signaling pathways that govern their lineage commitment remain enigmatic. Our work has shown a novel role for the actin cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), in Treg development, homeostasis and function. We have reported that Pak2 was necessary for generating the high-affinity TCR- and IL-2–mediated signals that are required by developing Tregs for their lineage commitment. Furthermore, deletion of Pak2 in Tregs specifically resulted in a loss of the characteristic Treg phenotype and the onset of a lethal multi-organ autoimmune phenotype, suggestive of a loss in Treg function. Our findings are the first to link Pak2 as an essential regulator of Treg biology.

   



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  Rig-I-Like Receptors (RLRs) regulate humoral immunity to West Nile Virus (WNV) infection
  Presenter: Marvin Oketch
  All Authors:Marvin Oketch, Dominik Schenten
  Department of Immunobiology, College of Medicine, The University of Arizona
   
 

The activation of pattern recognition receptors (PRRs) is a major regulatory checkpoint for the generation of adaptive immunity. Rig-I-like Receptors (RLRs) comprise a PRR family that includes the RNA helicases RIG-I and MDA-5, which recognize microbial RNA in the cytosol. RLR activation induces an anti-viral state in infected cells and leads to the release of proinflammatory cytokines and interferons. RLRs are therefore important mediators of innate immunity to many viral infections. However, the role of RLRs in the regulation of adaptive immunity is still poorly understood. Infection of mice deficient of MAVS, the essential signaling adaptor for RLRs, with West Nile Virus (WNV) results in a defective adaptive immune response. While this finding suggests a role for RLRs in the regulation of adaptive immunity to WNV, it is difficult to interpret due a high WNV viremia in the absence of a MAVS-dependent innate immune response. In order to overcome this caveat, we have infected MAVS-deficient mice with a mutant form of WNV that is unable to assemble newly infectious virions and is therefore limited to a single round of infection. Here we show that MAVS-deficient mice display increased numbers of antigen-specific CD4 T cells as well as an enlarged germinal center (GC) B cell compartment. Importantly, these mice fail to produce an effective neutralizing antibody response to WNV despite normal levels of WNV-specific antibodies. Together, these findings suggest that RLR-dependent signals regulate humoral immunity to WNV.

   



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  Innate immune recognition of HIV RNA in latent cells
  Presenter: Rebecca Olson
  All Authors:Rebecca Olson, Sunil Thomas, Michael Gale Jr.
  University of Washington
   
 

Over 37 million people are currently infected with HIV worldwide. There is no way to completely clear virus from an HIV-infected individual, in large part due to a population of latently infected cells that silently harbor viral DNA while retaining the ability to produce infectious virions upon cell activation. Designing a therapy to eliminate the HIV latent reservoir while sparing uninfected cells remains a major challenge. This task might be accomplished by targeting intracellular innate immune pathways, several of which are known to recognize HIV nucleic acids and induce type 1 interferon in acutely infected cells. However, the establishment of latent infection is associated with innate immune silence and it is not known if HIV products can activate these pathways in latent cells. We are addressing this critical gap in knowledge using the J-Lat CD4+ T cell line model of HIV latent infection. We have found that latent cells have intact type 1 interferon responses that can be triggered by unrelated viruses but not by reactivation of HIV infection alone. We have also identified a unique immune signature of TNFα production and CD59 expression in latent cells transcribing HIV RNA, which is absent in activated cells lacking HIV. This response has not previously been described in the context of HIV latent infection and suggests host recognition of HIV RNA through an NFκB-dependent pathway. TNFα production in latent cells was diminished with the onset of HIV translation, supporting a role for HIV proteins in subverting this response. We are further exploring what innate immune pathways are triggered by HIV RNA upon reactivation of latent infection. Our goal is to define the HIV products that stimulate innate immune signaling and to reveal how HIV proteins regulate these pathways. These studies will provide insight on the host response to HIV infection and will inform new therapies that harness intracellular innate immunity to eradicate the latent reservoir.

   



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  Differentiation and maturation of bone marrow-derived dendritic cells to Coccidioides
  Presenter: Kelly Otsuka
  All Authors:Kelly Otsuka, Anh Diep, Katrina Hoyer
  UC Merced
   
 

Coccidioidomycosis is a respiratory fungal infection caused by inhalation of Coccidioides immitis or Coccidioides posadasii. Individuals can be asymptomatic and can naturally resolve infection; conversely, disseminated coccidioidomycosis can progress into severe pulmonary disease. Individuals that resolve infection mount a Th1 and Th17 response and develop cell-mediated immunity. As a bridge between the innate and adaptive immune response, dendritic cells can activate and modulate lymphocytes towards protective Th1 and Th17 responses. However, little is known about the contribution of dendritic cells on immune memory in response to Coccidioides. Our lab utilizes in vitro culture to elucidate the differentiation and maturation of bone marrow-derived dendritic cells to Coccidioides and how these interactions influence adaptive immune cell recruitment and function in nonimmune individuals.

   



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  A novel factor regulating cross-presentation in CD8+ dendritic cells
  Presenter: Pengju Ou
  All Authors:Pengju Ou, Xiaoli Liu, Lifen Weng, Ling Wang, Chaofei Su, Hailiang Wu, Xiaoling Huang, Hai Ni, Cliff Y Yang
  Zhongshan School of Medicine, Sun Yat-Sen University
   
 

Cross-presentation is an important antigen presentation pathway to prime CD8+ T cells to eliminate pathogens and tumors. Although there is enormous interest in the molecular mechanism of cross-presentation, it is unclear how the cross-presented antigen is trafficked through endosomal compartments. According to our preliminary data, PPT1 (Palmitoyl-protein thioesterase 1) is specifically expressed in CD8 + dendritic cells at a high level, and knockout mice have a greatly enhanced ability to cross-prime CD8+ T cells. Thus, we hypothesize it may play an important role in the cross-presentation ability of CD8+ dendritic cells. Using transgenic mouse models, we have examined the function of this protein in cross-presentation ability of CD8+ dendritic cells under steady state and inflammatory conditions, evaluated the importance of this protein in immune response with infection and analyzed the molecular mechanism of how it controls cross-presentation in CD8+ dendritic cells. This project will not only further our understanding of the molecular mechanism of cross-presentation, it will also facilitate the design of a novel immunotherapy against infectious diseases and cancer by modulating its protein activity.

   



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  The prostaglandin D2 receptor CRTH2 promotes IL-33-induced ILC2 accumulation in the lung
  Presenter: Oyesola Oyebola
  All Authors:Oyesola Oyebola, Carolina Duque, Linda C. Huang, Elisabeth M. Larson, Simon P. Fr?h, Lauren M. Webb, Seth A. Peng, and Elia D. Tait Wojno
  Baker Institute for Animal Health and Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine; Department of Immunology, University of Washington
   
 

ILC2s are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine interleukin-33 (IL-33), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D2 (PGD2). The IL-33-ST2 and the PGD2-CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. Here, we show that ILC2 accumulation and associated type 2 inflammation in the murine lung in response to systemic IL-33 treatment were partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. While donor wild type ILC2s preferentially accumulated in the lungs compared to CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD2-CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation.

   



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  Vitamins in Host Defense Against an Enteric Pathogen
  Presenter: Alexandria Palaferri Schieber
  All Authors:Alexandria Palaferri Schieber, Janelle Ayres
  The Salk Institute for Biological Sciences; University of California, San Diego
   
 

Upon infection, the host mounts multifactorial responses to defend against disease. Host defense can be categorized as resistance, disease tolerance, or anti-virulence defenses. While resistance mechanisms encoded by the immune system kill the pathogen, disease tolerance and anti-virulence defenses promote host health while having a neutral to positive effect on pathogen fitness and are executed by the cooperative defense system. Anti- virulence mechanisms are physiological responses of the host that prevent or neutralize pathogenic signals, whether from the pathogen or from the host response to the pathogen. Disease tolerance involves mechanisms that induce health by decreasing tissue susceptibility to these pathogenic signals. One known regulator of immunity is the calcitriol receptor, commonly known as the Vitamin D receptor (VDR). Utilizing multiple murine models to investigate the role of VDR during infection with an attaching and effacing (A/E) pathogen, our data implicate the VDR in disease tolerance or anti-virulence defenses in response to infection. Intact Calcitriol receptor promotes host health during infection but does not alter pathogen burden. We are currently investigating how this pathway mediates cooperative defenses against infection focusing on VDRs role in glucose metabolism and organ homeostasis.

   



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  Dissecting the role of the CD4 transmembrane domain in T cell activation
  Presenter: Heather Parrish
  All Authors:Heather Parrish, Madeline Keenen, Neha Deshpande, Michael Kuhns
  University of Arizona
   
 

CD4 is essential for CD4+ T cell fate decisions and effector functions. It contributes to the transduction of information from a peptide antigen embedded within Major Histocompatibility Complex (MHC) molecules on the surface of antigen presenting cells (APCs) to the T cell’s intracellular signaling machinery via the T cell receptor (TCR). The TCR is a disulfide-bonded heterodimer that lacks intrinsic signaling activity. Instead, it is associated with the CD3δΕ, CD3γΕ, and CD3ζζ signaling modules that contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains. These ITAMs serve as substrates for the Src kinase Lck, which is constitutively associated with the CD4 co-receptor. The crystal structure of CD4 and experiments using Förster’s resonance energy transfer (FRET) suggest that CD4 homodimerizes on T cell membranes. Furthermore, functional studies in which CD4 was mutated at residues of the membrane proximal D4 domain (K318E and Q344E) thought to mediate CD4 dimerization significantly reduce T cell activation. Interestingly, CD4 contains a series of GXXXG motifs in its transmembrane domain that are known to form a transmembrane dimerization interface in other proteins. We have found that mutating glycine residues in these motifs in the CD4 transmembrane domain results in impaired TCR-CD3 signaling. Surprisingly, using two distinct proximity assays we have seen no evidence for CD4 dimerization when TCR is not engaged with pMHC. This suggests that the CD4 transmembrane domain is mediating dimerization only upon TCR-pMHC engagement or that CD4 interacts heterotypically with a different molecule that is important for its function in T cell activation. Because CD4 plays an important role in recruiting Lck to the TCR-CD3 complex, a thorough understanding of how CD4 functions is critical to understanding how different antigenic signals result in T cell fate decisions.

   



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  Functional evidence for TCR-intrinsic specificity for MHCII
  Presenter: Heather Parrish
  All Authors:Heather Parrish, Neha Deshpande, Jelena Vasic, Michael Kuhns
  University of Arizona
   
 

T cells must discriminate between a diverse array of antigenic peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells via clonotypic T cell receptors (TCR) to initiate signaling events that direct T cell fate decisions. The TCR is composed of subunits that are encoded by rearranged gene segments that then must be screened in the thymus for their ability to bind pMHC. During thymocyte development, the affinity of interactions between TCR and pMHCI or pMHCII determines whether a thymocyte will be positively selected to become a mature CD8+ or CD4+ T cell, respectively. Once selected, TCRs on mature peripheral T cells must be able to recognize self-pMHC molecules to receive maintenance signals, as well as bind cognate pMHC molecules presenting antigenic peptide to initiate T cell activation. At present, it remains unclear whether TCRs possess a sub-threshold intrinsic ability to scan the contents of MHC molecules to facilitate the surveillance and discrimination of self and foreign peptides during positive selection and T cell activation, or whether TCR-pMHC interactions are more analogous to antibody-antigen recognition. Additionally, it has recently been suggested that the frequency of CD4 association with the Src kinase Lck determines whether TCR-pMHC interactions are of sufficient duration to result in functional outcomes such as thymocyte selection. We hypothesized that genetically increasing the frequency of CD4 association with Lck would allow for detection of TCR-pMHCII interactions that are normally too weak to measure. Here, we used such a tuned system to assess the ability of ten distinct TCRs to recognize and functionally respond to MHCII. We found that three MHCII-restricted TCRs responded to selecting and non-selecting MHCII in an allele-independent and peptide sequence-independent manner that was dependent on both TCR-MHC and CD4-MHC interactions. Additionally, two MHCI-restricted TCRs responded to MHCII in an allele and peptide sequence-independent, but TCR-MHC and CD4-MHC dependent manner. Finally, five in vitro generated TCRs of unknown restriction and specificity displayed allele-independent and peptide sequence-independent reactivity to MHCII. Altogether, our data provide functional evidence for intrinsic TCR specificity for MHCII that is MHC allele-independent and peptide-sequence-independent – we interpret this as evidence for TCR scanning of MHC.

   



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  Differential ASC Requirements Reveal a Key Role for Neutrophils to the Acute IL-1[b] Response to Pseudomonas aeruginosa.
  Presenter: Yash R. Patankar
  All Authors:Yash R. Patankar, Rodwell Mabaera, Brent Berwin
  Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon NH 03756
   
 

Infection of macrophages and dendritic cells with Pseudomonas aeruginosa infection leads to the release of the potent pro-inflammatory cytokine IL-1β through the activation of the NLRC4 inflammasome complex. Previous genetic studies have shown that the molecular components of this complex, including NLRC4, ASC and Caspase-1, are required for the IL-1β response by macrophages and dendritic cells to P. aeruginosa infection. Surprisingly, ASC-/- mice were not deficient in mounting an IL-1β response in vivo to an acute P. aeruginosa infection. Upon further investigation of this discrepancy of the ASC requirement for IL-1β release in vitro but not in vivo, we identified that neutrophils are the predominant cell type that is initially recruited upon acute peritoneal or pulmonary P. aeruginosa infection and, concomitantly, these cells upregulate pro-IL-1β expression and release the mature IL-1β cytokine. Importantly, depletion of neutrophils in vivo leads to significantly lower IL-1β release in ASC-/- mice as compared to WT mice. These results were validated with purified murine and human neutrophils, which secreted IL-1β in response to P. aeruginosa. In corroboration of the in vivo phenotype, the murine neutrophil IL-1β response was predominantly ASC-independent. Interestingly, there appear to be multiple levels of regulation of the NLRC4 inflammasome complex in neutrophils, since Pseudomonas and Salmonella typhimurium, both of which are bacterial pathogens that trigger the NLRC4 inflammasome, have differential requirements of ASC, NLRC4 and Caspase-1 for IL-1β release. Our findings identify murine and human neutrophils as a significant and important cellular source of in vivo IL-1β production in response to P. aeruginosa, and these findings reconcile the observed discrepancy between previous in vitro experiments with macrophages and dendritic cells and the observed in vivo phenotype.

   



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  CD169+ marginal zone macrophages orchestrate innate immune responses to bacterial infection
  Presenter: Oriana Perez
  All Authors:Oriana Perez, Zhijuan Qiu, Pablo Romagnoli, Alexandre P. Bénéchet, Leigh Maher, Kamal M. Khanna
  University of Connecticut Health Center
   
 

Spleen is an important site for generating protective immune responses against pathogens. Immune cells in the spleen undergo rapid reorganization to initiate and maintain local inflammatory responses against pathogens. How the spatial dynamics of cellular communication are regulated in the spleen after infection remains unclear. Respectively, CD169+ macrophages are a subpopulation of tissue resident macrophages that are positioned in the marginal zone to rapidly encounter invading pathogens. However, their role during infections is not well understood. Here we show that splenic CD169+ macrophages serve as a primary cellular host to blood-borne bacteria Listeria monocytogens (Lm) and are also essential for the clearance of other bacterial and viral pathogens. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrated a second phase of innate protection by mediating the transport of Lm to the splenic T cell zones and driving the subsequent reorganization of neutrophils, monocytes and NK cells into hierarchical clusters. Specifically our study revealed that bacterial transport to the T cell zones was mediated by trans-infection of closely interacting CD8α+ DCs with Lm-infected CD169+ macrophages. Although, CD8α+ DCs are required for a productive Lm infection in the spleen, to our surprise mice that lacked both CD8α+ DCs and CD169+ macrophages were unable to control Lm infection. These results demonstrated that CD169+ macrophages regulated bacterial access to the pathogen favorable CD8α+ DC niche, ultimately providing a rationale for reassessing the current paradigm regarding the temporal role of CD8α+ DCs during Lm infection. Furthermore, these results provide valuable insight into how lymphoid organ structure and function are related at a fundamental level and how the anatomical positioning of a pathogen is critical for generating protective innate immune responses in vivo.

   



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  The role of ADAR1 in nucleic acid sensing
  Presenter: Kathleen Pestal
  All Authors:Kathleen Pestal, Dan Stetson
  University of Washington
   
 

Adenosine deaminases that act on dsRNAs (ADARs) deaminate adenosines in pre-mRNA, noncoding RNAs and viral RNAs to create inosines. Inosine can alter codons and splice forms, resulting in multiple protein isoforms from the same gene. Additionally, inosine binds to uracil with less affinity as compared to adenosine, leading to instability in the structure of the target RNA. Recently, mutations in ADAR1 have been shown to cause Aicardi-Goutières Syndrome (AGS), a genetically determined inflammatory disorder of the brain and skin. Previously identified AGS mutations occur in several enzymes that act on a common pathway of retroelement metabolism, where loss of their individual functions results in an overstimulation of innate DNA sensing. However, the mechanism by which the loss of ADAR1 results in an upregulation of Type I interferon signaling is currently unknown. Mice that are deficient in ADAR1 do not survive past embryonic day 12 due, in part, to a failure of the hematopoietic system. Interestingly, when we crossed Adar-/- mice to MAVS-deficient mice, we rescued the interferon (IFN) signature in embryos. Furthermore, we have now generated the first live Adar-/- mice, as the IFN signature is only partially responsible for embryonic lethality. The rescue of the IFN signature and the live births of Adar/Mavs DKO mice enabled the first analysis of the role of ADAR1 in development independent from the pathological IFN response. Together, our preliminary findings reveal a novel disease mechanism that may contribute to AGS caused by ADAR mutations: dysregulation of the MAVS-dependent antiviral response. Moreover, we show that ADAR1 is required for the normal development of kidneys, gastrointestinal tract, and B cells, revealing a function in development that is distinguishable from ADAR1 regulation of the MAVS pathway. Based on these data, we hypothesize that ADAR1 has two independent functions: regulation of the MAVS-dependent antiviral response, and control of organ development. We further hypothesize that AGS is caused by defects in the first function of ADAR1, not the second.

   



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  The Effect of the VHL/HIF Pathway on CTL Memory Differentiation and Function
  Presenter: Anthony Phan
  All Authors:Anthony Phan, Andrew L. Doedens, Ananda W. Goldrath
  University of California San Diego
   
 

During response to infection, T cells become activated in lymphoid tissues and traffic to a myriad of host tissues in order to perform effector functions. Upon entering peripheral tissues, environmental signals, such as cytokines and nutrient availability, likely instruct responding T cells thereby tailoring effector responses. CD8+ cytotoxic T lymphocytes (CTL) are essential for the clearance of intracellular pathogens, such as viruses, and likely utilize environmental signals to modulate their cytolytic function. We have found that the von-Hippel-Lindau/Hypoxia Inducible Factor (VHL/HIF) pathway, the central transcriptional response to hypoxia, is a critical regulator of CTL differentiation and function. Strikingly, in response to acute infection, conditional deletion of VHL in CD8+ T cells results in CTL that expand poorly and fail to upregulate KLRG1, a marker of shorter-lived CTL, in comparison to wildtype CTL. Despite poor expansion and inability to form this shorter-lived CTL population, VHL-deficient CTL appear to form populations of long-lived memory cells that respond to secondary challenge despite alterations in expression of many transcription factors involved in effector and memory differentiation and perturbation of CD8+ T cell metabolism. These effects are likely due to alterations in expression of HIF targets as well as indirect effects due to HIF-dependent metabolic changes. We find that constitutive HIF activity due to VHL deletion in CD8+ T cells has profound effects on differentiation, maintenance, and function of long-lived memory CD8+ T cells following acute infection impacting protective long-term immunity.

   



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  Constitutive Glycolytic Metabolism Promotes CD8+ T cell Effector Memory
  Presenter: Anthony Phan
  All Authors:Anthony Phan, Andrew Doedens, Kitty Cheung, and Ananda Goldrath
  University of California San Diego
   
 

Memory CD8+ T cells can provide enduring protection against intracellular pathogens and tumors. As such, eliciting memory T cells is a primary objective of vaccination strategies. Extensive metabolic changes accompany T cell activation: amongst these are an increase in glycolytic energy production and biosynthesis. Recent studies suggest that reliance on fatty acid oxidation (FAO) and oxidative phosphorylation as well as generation of increased spare respiratory capacity (SRC) following clearance of pathogen is essential for the formation of memory CD8+ T cells. Strikingly, we find that constitutive glycolytic metabolism and active suppression of oxidative phosphorylation due to HIF transcriptional activity, enhances generation of long-lived memory CD8+ T cells. In spite of HIF-driven glycolysis, CD8+ effector cells generate sufficient levels of ATP as memory precursor cells emerge, upregulate IL-7 receptor and key transcription factors associated with the generation of long-lived memory cells, and show a heightened response to secondary challenge. Importantly, increased glycolysis favors formation of effector memory CD8+ T cells, which can provide rapid protection from reinfection. These data clarify the role of cellular metabolism in the differentiation of CD8+ T cell memory, showing that constitutive reliance on glycolytic metabolism does not necessarily hinder formation of memory CD8+ T cells. This suggests that memory subset heterogeneity may be driven by both transcriptional programs and alterations in cellular metabolism.

   



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  Dynamic, whole-lymph node imaging and single cell tracking using sample-adaptive two photon microscopy
  Presenter: Henry Pinkard
  All Authors:Henry Pinkard, Adriana Mujal, Hratch Baghdassarian, Daniel Friedman, Ivana Malenica, Adam Fries, Daniel Fletcher, Laura Waller, Matthew Krummel
  UCSF, UC Berkeley
   
 

Dynamic imaging of lymph nodes using two-photon microscopy has been a major driver of our understanding of the spatiotemporal sequence of cell-cell interactions that drive immune responses. However, such experiments present two major technical challenges, which has limited their applicability to small subvolumes of lymph nodes with several orders of magnitude higher than physiological clonal cell frequencies: 1) The challenge of imaging large volumes of intact, living lymph nodes 2) The challenge of identifying individual cells within such a volume and tracking their movements. In this work, we demonstrate the solutions to each of these problems. Taking inspiration from the technique of standard candles in astronomy, we demonstrate that fluorescent lymphocytes can be used as markers that enable a machine learning algorithm to automatically control excitation laser power in response to the local morphology of the tissue. This enables two-photon imaging to be scaled to the whole organ level. Next, we show an algorithmic pipeline that uses 3D segmentation algorithms to identify potential cells, and interactive machine learning to efficiently label informative subsets of these potential cells. This allows populations of labelled cells to be identified based on their morphology and fluorescent spectra with minimal manual effort. Together these technologies enable a major advance in our ability to visualize immune dynamics over large areas of space in physiologically realistic conditions.

   



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  Mitochondrial Pyruvate Import is Necessary for the Development of GMP-derived Cells
  Presenter: Hannah A. Pizzato
  All Authors:Hannah A. Pizzato, Wing Y. Lam, Brian N. Finck, and Deepta Bhattacharya
  Washington University in Saint Louis School of Medicine
   
 

As hematopoietic stem cells differentiate, they gradually lose the potential to generate specific blood cell types until commitment to a single lineage is achieved. As hematopoietic stem cells are exceedingly rare, their differentiation occurs concomitantly with a progressive expansion of downstream progenitors to ensure the sufficient production of mature cells. How this expansion occurs remains unknown but may involve metabolic switches. Here we demonstrate that mitochondrial pyruvate deficiency via the deletion of an essential subunit of the mitochondrial pyruvate carrier (Mpc2) leads to a significant reduction in neutrophils and monocytes, yet spares naïve B and T cells. This defect begins at the transition between the common myeloid progenitor (CMP) and the granulocyte-monocyte progenitor (GMP). In wild type mice, glucose uptake increases during the CMP to GMP transition, suggesting the existence of an essential metabolic switch dependent on glycolysis and mitochondrial respiration. In contrast, glucose uptake remains relatively low in mature neutrophils. Mpc2-deficient neutrophils can be rescued by retroviral transduction of the long-chain fatty acid transporter Slc27a1, which can provide alternate respiratory substrates and/or compensate for defective fatty acid synthesis. Ongoing work is designed to identify extrinsic cues that enact these metabolic switches. Taken together, these data demonstrate a requirement for mitochondrial pyruvate for the expansion of GMPs and downstream myeloid cells.

   



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  Determining the Mechanism of TNF-Mediated Defense Against Legionella pneumophila Infection
  Presenter: Tzvi Pollock
  All Authors:Tzvi Pollock, Sunny Shin
  University of Pennsylvania
   
 

Intracellular bacterial pathogens are responsible for significant disease burden every year. Successful control of these organisms by the host depends on the inflammatory cytokine Tumor Necrosis Factor (TNF). While TNF is known to protect against many intracellular bacterial infections, and the molecular mechanisms of TNF signaling are understood in sterile contexts, the precise modes through which TNF can defend the host against bacterial infection remain unclear. The following research aims to elucidate the mechanism of TNF in the context of intracellular infection. In this project, the gram-negative bacterium Legionella pneumophila, the causative agent of Legionnaire’s Disease, acts as a model intracellular pathogen. Infection of bone marrow derived macrophages and live mice are both used to explore the effect of TNF signaling on control of infection. Thus far we have shown that TNF is required for restriction of bacterial replication both in vitro and in vivo. In addition, the cysteine protease caspase 8 appears to be necessary for control of Legionella infection in vivo, however its pro-apoptotic auto-processing does not. Preliminary data suggest that caspase 8 still mediates a form of TNF-dependent cell death in response to infection. Thus, a function of caspase 8 other than classical apoptosis may therefore be the mechanism through which TNF restricts bacterial replication.

   



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  Bacteria in human intestine promote mucosal immune development in utero
  Presenter: Elze Rackaityte
  All Authors:Elze Rackaityte, Rackaityte E, Halkias J, Fukui EM, Mendoza VF, Crawford ED, Fujimura KE, Burt TD, Lynch SV*
  UCSF
   
 

Mucosal immunity influences host-microbial interactions and is evident in the human fetal intestine by 11-14 weeks of gestation; the developing intestine is populated by lymphoid aggregates, memory T cells and dendritic cells capable of responding to microbial stimuli. Recent evidence for bacterial presence in utero comes from DNA-based, culture-independent studies of the placenta and neonatal meconium, the latter comprised of amniotic fluid ingested during gestation. However, whether intestinal encounters with viable microbes occur in utero and shape immune maturation has not been investigated. Here, using culture-independent methods, we identified subsets of fetal meconium relatively enriched for Lactobacillus or Micrococcus that related to divergent epithelial cell layer transcriptomes and proportions of lamina propria innate-like PLZF+ CD161+ CD4+ T cells in paired intestinal samples. Mimicking conditions in the fetal intestine permitted isolation of viable Lactobacillus and Micrococcus strains from fetal meconium and, in contrast with phylogenetically related reference strains, fetal isolates utilized placental hormones for growth, remained viable within macrophages, and exhibited species-specific capacity to promote immune tolerance. Fetal Lactobacillus isolates reduced activation of antigen presenting cells and promoted IL17F production in T cell co-cultures; the Micrococcus isolate reduced TNFα production in antigen presenting cells and inhibited IFNγ production by memory PLZF+ T cells. Whole genome sequencing of fetal isolates identified them as strains of Micrococcus luteus or Lactobacillus jensenii and phylogenetically unique genes for intracellular survival, immune modulation, and steroid uptake and metabolism were identified. These data suggest that pre-natal immunity is influenced by bacterial exposure in the fetal intestine, identifying a previously unknown component of human mucosal immune development.

   



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  NLRC4 inflammasome activation in neutrophils is sufficient to cause systemic inflammatory disease
  Presenter: Nichols Randilea
  All Authors:Nichols Randilea, Jakob von Moltke, Russell E. Vance
  University of California, Berkeley
   
 

Inflammasomes are cytosolic multi-protein complexes that initiate inflammation in response to infection or other stimuli. Inflammasomes are broadly expressed in hematopoietic and non-hematopoietic cells but tools to dissect cell type-specific functions of inflammasomes in vivo have been lacking. Here we describe a novel mouse model in which the endogenous NLRC4 inflammasome is selectively activated in specific cell types using Cre drivers. NLRC4 activation in Lysozyme2+ cells (monocytes, macrophages, neutrophils) in vivo causes a severe systemic inflammatory disease, reminiscent of human patients that carry mutant auto-active NLRC4 alleles. Interestingly, specific NLRC4 activation in Mrp8+ neutrophils (but not Cd11c+ cells) was also sufficient to cause severe inflammatory disease. Disease required the inflammasome adaptor ASC and could be ameliorated by therapeutic injections of anti-IL1 receptor antibody. Our results identify a functional role for NLRC4 inflammasome activation in neutrophils in mediating auto-inflammatory disease in vivo.

   



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  Adipose tissue physiology in host-parasite interactions
  Presenter: Samuel Redford
  All Authors:Samuel Redford, Samuel Redford, Janelle Ayres
  Salk, UCSD
   
 

Traditionally, white adipose tissue was believed to function solely as an energy storage tissue, however, evidence from recent studies have shown WAT is a critical regulator of immune responses. This is best appreciated in the context of metabolic diseases where the morbidities such as hyperinsulinemia, hyperglycemia, and chronic low-grade inflammation, can be associated with improper immune activation within the WAT. A role for adipose tissue during infections is less well understood. Multiple pathogens have been found to localize in adipose tissue during chronic infection including parasites, bacteria, and viruses. We are interested in why pathogens localize to the adipose tissue for a chronic infection. To address our goal, we are utilizing the eukaryotic, single cellular parasite, Trypanosoma brucei. T. brucei is the causative agent of African sleeping sickness in humans and trypanosomiasis in animals. The parasite has previously been thought to mainly reside in the blood before entering the central nervous system before causing death. However, it has recently been found to localize to adipose tissue and has more parasites in the adipose tissue than in any other tissue, including blood, once the disease enters the chronic phase. Here I describe our initial studies characterizing the host metabolic responses to T. brucei over the course of the infection.

   



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  Transcriptional Regulation of Durable Antibody Mediated Immunity
  Presenter: Tyler Ripperger
  All Authors:Tyler Ripperger, Yinan Wang, Arijita Jash, Lucas D'Souza, Deepta Bhattacharya
  University of Arizona
   
 

Durable antibody-mediated immunity is maintained by memory B cells and long-lived plasma cells. The length of protection varies greatly depending on the specific vaccine or infection. Defining the transcriptional profiles required for sustained antibody-mediated immunity may help explain these differences and provide guidance on how to improve vaccine responses. Previously, our lab demonstrated two members from the BTB/POZ family of transcription factors exert opposite effects on the duration of antibody responses and plasma cell longevity. ZBTB20 is required for long-term primary antibody responses and plasma cell longevity, while the related ZBTB32 suppresses the lifespan of plasma cells in recall responses. We hypothesize ZBTB20 and ZBTB32 regulate the magnitude of antibody responses and plasma cell lifespan through temporal cell type specific mechanisms. We will test our hypothesis by: 1) Establishing the B cell stages in which ZBTB20 is required; 2) Determining direct targets and genes regulated by ZBTB20 and ZBTB32. These experiments will define essential genetic programs that promote and antagonize plasma cell lifespan.

   



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  Inflammation drives alternative first exon usage to regulate immune genes including Aim2
  Presenter: Elektra Robinson
  All Authors:Elektra Robinson, Pratibha Jagannatha, Sergio Covarrubias, Matthew Cattle, Rojin Safavi, Ran Song, Kasthuribai Viswanathan, Barbara Shapleigh, Robin Abu-Shumays, Miten Jain, Suzanne Cloonan, Edward Wakeland, Mark Akeson, Angela N. Brooks and Susan Carpenter
  University of California, Santa Cruz
   
 

Macrophages are critical effector cells of the innate immune system essential for controlling infection and maintaining tissue homeostasis. At the cellular level, pathogen-response involves recognition by classes of receptors expressed on the cell surface and intracellularly, once activated they initiate complex signaling cascades that result in the induction of an inflammatory program. Perturbations to these signaling pathways can have devastating consequences, leading to autoimmune diseases and cancer. The contribution of alternative splicing to the regulation of innate immune responses remains poorly studied. Through the analysis of differential splicing using both short read and long-read RNA sequencing of human and mouse macrophages, we have identified that alternative first exon (AFE) changes are a prominent widespread event during inflammatory activation. Of these AFE events, we have identified 50 unannotated transcriptional start sites (TSS) in mouse bone marrow-derived macrophages (BMDMs) using Oxford Nanopore technology, one of those being Aim2. The protein Aim2 (Absent in Melanoma) is the cytosolic receptor for dsDNA within the cytosol. Once Aim2 is activated by dsDNA it results in the formation of the inflammasome complex leading to the production of the pro-inflammatory cytokines IL1b and IL18. From a combination of high throughput approaches including ChIP-seq, ATAC-seq, and RNA-seq we characterized the unannotated promoter and TSS of Aim2 as inflammatory specific. Next, we examined the 5’UTR of the unannotated Aim2 isoform and identified an iron response element (IRE). Our functional data of polysome profiling, GFP reporter system and western blot analysis indicate that this unannotated inflammatory activated isoform is translated at a lower level compared to the canonical isoform. This result is also reversible through the manipulation of iron or the removal of the IRE. This could be a mechanism required in order to limit activation of the Aim2 pathway so that following inflammation Aim2 protein activation is tightly controlled and rapidly switched off. This novel isoform of Aim2 is conserved between human and mouse. We are now determining the exact mechanisms of action and regulation of this isoform and its importance in autoimmune diseases such as Systemic Lupus Erythematosus.

   



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  Characterization of an Inflammatory Specific Isoform of Aim2 that is Regulated by Iron
  Presenter: Elektra Robinson
  All Authors:Elektra Robinson, Sergio Covarrubias, Barbara Shapleigh, Rojin Safavi, Ran Song, Edward K Wakeland, Mark Akeson, Susan Carpenter
  UC Santa Cruz
   
 

Macrophages are critical effector cells of the innate immune system essential for controlling infection and maintaining tissue homeostasis. At the cellular level, pathogen-response involves recognition by classes of receptors expressed on the cell surface, and intracellularly, that once activate initiate complex signaling cascades resulting in induction of an inflammatory program. Perturbations to these signaling pathways can have devastating consequences, leading to autoimmune diseases and cancer. An inducible gene expression program is a critical component of the host inflammatory response. The protein Aim2 (Absent in Melanoma) is the cytosolic receptor for dsDNA within the cytosol. Once Aim2 is activated by DNA it results in the formation of the inflammasome complex leading to the production of the proinflammatory cytokines Il1b and IL18. From a combination of high throughput approaches including ChIP-seq, ATAC-seq and RNA-seq we identified an active promoter and transcript 1kb upstream of the annotated promoter and transcription start site (TSS) for Aim2. Interestingly this region is more active following inflammatory activation compared with the canonical TSS. Initially we investigated this region as a possible long noncoding RNA but following direct RNA-sequencing of macrophages before and after inflammatory activation using Oxford Nanopore we found that this region is in fact an alternative 1st exon for Aim2. Long read sequencing provided us with the essential isoform information from this locus, something that was missed with our previous approaches. Our functional data G. indicates that this inflammatory activated isoform is translated at a lower level compared to the canonical mRNA isoform. Translation is controlled through an iron response element (IRE) that is present within the alternative 1st exon. Using reporter assays we confirm that indeed the IRE is the critical element controlling the translational efficiency as removal of this element or treating the cells with iron reverses this effect. This novel mRNA isoform of Aim2 is conserved between human and mice. Future work will aim to uncover the importance of this alternative isoform switch in the context of human diseass such as Systemic Lupus Erythematosus.

   



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  Investigating the Regulatory Role of LncRNA-Aim2 in Mouse Macrophages
  Presenter: Elektra Robinson
  All Authors:Elektra Robinson, Sergio Covarrubias, Vanille J. Greiner, Michael T. McManus, Susan Carpenter
  University of California, Santa Cruz
   
 

Advancements in next generation sequencing has provided us with an unprecedented view of the human genome. One of the most fascinating findings is that less than 3% of the genome codes for protein coding exons, yet more than 85% of the genome is transcribed. We are now faced with the challenge of understanding what these RNA genes do and whether they play key roles in biological processes. The largest group of RNA produced from the genome is Long noncoding RNA (lncRNA). LncRNAs are described as transcripts greater than 200 nucleotides in length that do not code for protein. To date there is experimental data available on approximately 1% of known lncRNAs. Our lab is investigating how lncRNAs act as key regulators during inflammation and host defense against infection. Here we describe a new inflammatory inducible lncRNA we have identified upstream of the protein coding gene absent in melanoma 2 (Aim2) on chromosome 1. Aim2 is a critical component of the innate immune response to DNA. It is highly activated by the type 1 Interferon (IFN) pathway. Aim2 forms an inflammasome complex in response to cytosolic DNA stimulation and results in the activation and processing of the critical proinflammatory proteins IL1ß and IL18. We identified lncRNA-Aim2 through RNA-sequencing of murine macrophages. We see that it is co-expressed with Aim2 following inflammatory stimulation. We have successfully used CRISPRi to knockdown expression of lncRNA-Aim2 in macrophages. Our preliminary data shows that lncRNA-Aim2 does not function in cis to regulate Aim2. We are now carrying out RNA-seq to determine if lncRNA-Am2 functions to regulate other immune genes.

   



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  CXCL12-expressing reticular cells are a novel stromal cell type in the germinal center dark zone
  Presenter: Lauren B. Rodda
  All Authors:Lauren B. Rodda, Oliver Bannard and Jason G. Cyster
  Department of Microbiology and Immunology, University of California, San Francisco
   
 

Germinal centers (GCs) are tightly organized into a light zone (LZ) and a dark zone (DZ). GC B cells respond to CXCL12 to access the DZ niche and undergo efficient mutation and selection, but the source of this critical chemokine is unknown. Using CXCL12-gfp reporter mice, we identified a new subset of stromal cells we termed CXCL12-expressing reticular cells (CRCs) in the GC DZ and T zone proximal region of the primary follicle. These cells form tight, irregular nets in spleen and peripheral lymph node GCs in response to LCMV infection, flu infection and SRBC immunization. CRCs are also present in chronic GCs in mesenteric lymph nodes and Peyer’s patches. Distinct from follicular dendritic cells (FDCs) and fibroblastic reticular cells (FRCs), CRCs are low or negative for CD35, CD16/32, Mfge8, FDCM2, ER-TR7 and Type IV collagen. Unlike FDCs, CRCs are not dependent on LTa1ß2 and TNF signaling for short-term maintenance. CXCR4 signaling was required for CRC distribution through the DZ, likely reflecting a dependence on GC B cell proximity for their structural maintenance. Our findings identify CRCs as a major stromal cell type in the GC DZ and suggest they support critical activities of GC B cells in the DZ niche.

   



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  Differential regulation of lncRNAs during effector and memory CTL development
  Presenter: Walter Rogal
  All Authors:Walter Rogal, Nicholas Bild, Gustavo Martinez, Matthew Pipkin
  The Scripps Research Institute
   
 

The mammalian genome encodes thousands of long non-coding RNAs (lncRNAs). However, the functions in cellular differentiation of most lncRNAs is unclear, and their role(s) in the development of effector and memory cytotoxic T lymphocytes (CTL) from naïve CD8 T cells is essentially unexplored. Using several RNA-seq approaches to analyze effector and memory CTL development in a cell culture system that is modulated by IL-2 receptor stimulation in vitro, and in bona fide memory precursor effector cells (KLRG1- CD127+) and short-lived effector cells (KLRG1+ CD127-) responding to lymphocytic choriomeningitis virus (LCMV) infection in vivo, we have identified thousands of differentially expressed lncRNAs that might contribute to CTL differentiation. A substantial fraction of differentially expressed lncRNAs from ex vivo effector and memory CTL were regulated by the strength of IL-2 receptor stimulation in vitro, and many were controlled transcriptionally by these signals. Our data is consistent with the possibility that lncRNAs promote CTL differentiation. We are currently applying retroviral-mediated transgene expression and RNAi approaches to decipher the function(s) of differentially expressed lncRNAs during CD8 T cell differentiation.

   



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  Dietary iron-induced lipolysis reduces colitis severity during Citrobacter rodentium infection in C57BL/6 mice
  Presenter: Alicia Romero
  All Authors:Alicia Romero, Janelle Ayres
  UCSD/Salk Institute
   
 

Iron is an essential nutrient for both host and pathogen. During infection, hosts employ an ensemble of resistance defenses that ultimately limit a pathogen’s access to iron— a process referred to as nutritional immunity. Likewise, pathogens have evolved a plethora of counter strategies to overcome nutritional immunity and acquire iron within their hosts. Altogether, the principle of nutritional immunity holds that during infection, a surplus of iron will promote pathogen proliferation and decrease host health and survival. And while nutritional immunity thoroughly describes the consequence of excess iron during infection, it does not address how excess iron influences host physiology, and how these changes may independently affect infection dynamics. Our lab has recently described one such model where C3H/hEJ mice fed an iron rich diet become transiently insulin resistant and exhibit increased glucose availability in the gut. This iron-dependent physiological response leads to attenuation of Citrobacter rodentium—thus, demonstrating that nutritional supplementation can give rise to physiological responses that dramatically affect infection dynamics. However, this model does not extend to other strains of mice, highlighting that genetic background profoundly influences physiological responses to nutrient supplementation. Here, we describe an infection model where the genetically distinct murine strain, C57BL/6, challenged with C. rodentium exhibit increased resistance defenses when fed an iron rich diet. Interestingly, increased resistance does not confer an increase in host health—owing to the independent effects of excess iron on host physiology. This study examines the effects of dietary iron on C57BL/6 physiology, and how these physiological shifts enhance host resistance defenses during C. rodentium infection.

   



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  Muscle wasting is a maladaptive host metabolic response to infection
  Presenter: Alicia Romero
  All Authors:Alicia Romero, Alicia Romero, Janelle Ayres
  University of California San Diego
   
 

During infection, hosts endure significant metabolic changes that can dramatically influence host defense responses and pathogen behavior. Muscle wasting is one of the most highly conserved metabolic changes observed across a wide range of infections and across the animal kingdom. The function of muscle wasting during infection, if any, is not understood. It has been proposed that muscle wasting is a host metabolic response that modulates energy allocation during infection. However, current experimental and clinical evidence show that muscle wasting decreases host survival and impairs recovery from severe infections. Using a transgenic murine model lacking a critical mediator of muscle atrophy (FoxO1) in skeletal muscle, we will determine why and how muscle wasting leads to a more severe outcome of infection in the context of Salmonella Typhimurium infection. Mice deficient for muscle Foxo1 had increased survival when challenged with a lethal dose of S. Typhimurium. Consistent with our previous report, we found that the protection from muscle wasting was not accompanied by changes in pathogen burden. This suggests that muscle wasting increases disease severity by promoting pathogen virulence or impairing a disease tolerance mechanism rather than host resistance.

   



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  NAIP-NLRC4 deficient mice are a new model of Shigella Pathogenesis
  Presenter: JL* Roncaioli
  All Authors:JL* Roncaioli, Mitchell PS*, Rauch I, Chavez RA, Lee AY, Bergen I, Vance RE
  UC Berkeley
   
 

Shigella flexneri is a human-specific bacterial pathogen that invades and replicates within the colonic and rectal epithelium, causing severe and often bloody diarrhea which claims >200,000 lives each year. A challenge in studying and combating Shigella-induced disease is the lack of genetically tractable in vivo models that accurately reflect human infection. It is not clear why mice are resistant to Shigellosis, but we hypothesized that protection might be mediated by the NAIP-NLRC4 inflammasome, a cytosolic innate immune sensor that recognizes components of Shigella’s type three secretion system (T3SS). We have found that mice pre-treated with oral streptomycin and deficient for the NAIP-NLRC4 inflammasome are acutely susceptible to oral Shigella challenge and display hallmarks of human Shigellosis. Among these, we observe weight loss, malaise, diarrhea, and blood and neutrophils in the stool. Upon histological examination, infected NAIP-NLRC4 knockout mice exhibit significant edema, epithelial damage, hyperplasia, and inflammatory infiltrate in both the cecum and the colon relative to infected wild-type mice. Inflammation depends on both T3SS-dependent epithelial cell invasion and IcsA-dependent cell-to-cell spread in the absence of NAIP-NLRC4. Furthermore, mice that express NAIP-NLRC4 only in the intestinal epithelium phenocopy wild-type mice, suggesting that activation of the inflammasome specifically in the epithelium is sufficient to protect against epithelial cell invasion, spread, and resulting disease. Our findings provide the first oral infection mouse model of Shigella. Our work highlights the importance of epithelial cell inflammasomes in defense against enteric pathogens and provides insight into the species-specific differences that control resistance to Shigella infection.

   



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  Age Associated B Cells Represent a Tissue Resident Memory B Cell Subset
  Presenter: Rebecca Rosenthal
  All Authors:Rebecca Rosenthal, Arpita Myles (Co-First Author), Elinor Willis, Scott Hensley, Mike Cancro
  University of Pennsylvania
   
 

Age Associated B Cells (ABCs) are a newly described memory B cell subset characterized by TBET expression. The ABC subset enlarges with age and is associated with both viral immunity and humoral autoimmunity. Our lab recently showed that a triad of signaling events – TLR engagement, TFH cytokine milieu, and BCR signaling – act in concert to govern ABC development. Furthermore, we have shown that Tbet+ B cells use a diverse array of VH and VL genes, are somatically mutated, and when induced by viral infection, persist indefinitely. Based on these and other findings, our current working model is that Tbet+ B cells are a memory B cell subset that arises during germinal center reactions driven by nucleic acid containing antigens. Surprisingly, following PR8 influenza infection ABCs are largely restricted to the spleen and are entirely absent from the draining lymph nodes. This intriguing observation raised the question of whether ABCs are a splenic tissue resident B cell subset or if they migrate under basal conditions. To address this question, our lab undertook a parabiosis based approach to investigate ABC residency. We parabiosed B6 mice 30+ days post PR8 infection with naïve B6.SJL congenic partners. We observed that while PR8 specific TBET- B cells achieve significant mixing in parabiotic pairs, TBET+ PR8 specific B cells remain in the infected partner and are not seen in the naïve conjoined mouse, showing that the TBET+ ABC population is tissue resident in the spleen.

   



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  Infection with respiratory syncytial virus drives an enhanced allergen-specific Th2 response following subsequent house dust mite exposure
  Presenter: Mikel Ruterbusch
  All Authors:Mikel Ruterbusch, Marion Pepper
  University of Washington
   
 

Allergic asthma is a global health burden, affecting over 300 million individuals worldwide. Even with treatment, these individuals are at risk of hospitalization and death from acute asthma-related complications. Improving our understanding of how allergic asthma develops and is maintained is essential to further treat and prevent this disease with novel therapeutic and environmental interventions. Among several other risk factors, children are more likely to develop atopic asthma throughout the remainder of childhood if they contract a severe respiratory syncytial virus (RSV) infection during infancy. However, it is still unclear how severe RSV infections are linked to the development and exacerbation of allergic asthma long after the virus has cleared. We have identified that in contrast to influenza A virus, intranasal infection with RSV drives the production of viral-specific CD4+ T cells in the lung that produce the type 2 cytokines IL-13 and IL-5 at eight days post-infection. Furthermore, we have found that compared to mice treated with house dust mite (HDM) alone, mice infected with RSV three weeks prior to allergic sensitization with HDM possess more Th2-differentiated CD4+ T cells specific for the HDM-derived protein Der p 1 in the lung early after allergic challenge. Our findings suggest that infection with RSV can induce viral-specific CD4+ Th2 cells and leads to increased numbers of Der p 1-specific CD4+ Th2 cells in the lung upon subsequent allergic sensitization and challenge with HDM. We are currently investigating the mechanisms underlying these observations to improve our understanding of how RSV infection impacts the development of asthma which may inform new therapeutic strategies to treat or prevent allergic asthmatic disease.

   



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  Uncovering the phenotype of pathogen-reactive innate-like B cells
  Presenter: Hannah Savage
  All Authors:Hannah Savage, Nicole Baumgarth
  UC Davis
   
 

B-1 cells, an innate-like subset of B lymphocytes, generate protective IgM antibodies to many viral and bacterial pathogens. The respective contributions by the two known subsets of B-1 cells, CD5+ (B-1a) and CD5neg (B-1b), to IgM production are unclear. We showed previously that B-1a, but not B-1b cells, migrate to the respiratory tract draining mediastinal lymph node (MedLN) in response to influenza infection, resulting in increased B-1 derived IgM-production at that site. In contrast, following infection with Streptococcus pneumonia, Borrelia hermsii and Salmonella spp, others reported increased secretion of IgM by B-1b cells. Here we aimed to resolve the reasons underlying the apparent discrepancies in responsiveness of B-1a and B-1b cells to the various pathogens. Given the observed restrictions on B-1a migration to the MedLN after influenza infection, we first determined the phenotype of the IgM-secreting B-1 cells in the MedLN. Surprisingly, the results showed that CD5 neg B-1 cells were the main source of B-1-derived IgM on days 3, 5, and 7 after influenza infection, suggesting that CD5 expression of B-1a cells is lost after differentiation to an IgM-secreting cell. In support, in vitro studies demonstrated that three-day cultures of highly FACS-purified B-1a (CD5+) cells stimulated with LPS contained mainly CD5neg cells. This was not due to a preferential outgrowth of contaminating B-1b cells, as B-1a and B-1b cells showed similar rates of cell death, spiking of cultures with B-1b cells did not significantly affect numbers of CD5neg cells, and B-1a cells proliferated even more than B-1b cells when stimulated with LPS. Ongoing studies using chimeras generated with purified B-1a cells will determine whether a similar conversion of B-1a to “B-1b” also occurs in vivo after influenza infection. Our data demonstrate that activated B-1a cells lose CD5 expression and become “B-1b like” IgM-secreting cells. The findings suggest that surface phenotype analysis alone is insufficient to conclude on the contributions of B-1a and B-1b cells to IgM production. Furthermore, our results indicate that CD19+ CD43+ CD5neg “B-1b”cells, currently regarded as a distinct subset of B-1 cells, and attributed to have memory-like functions, are composed, at least in part, of differentiated, IgM-secreting B-1a cells. (Funding: NIH/NIAID R01 AI051354, AI085568 and U19 AI109962, T-32 AI060555, 2T32OD010931-09, 5T35OD010956-14, UL1 TR000002 and linked award TL1 TR000133)

   



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  Subsets of Natural IgM-secreting cells differ in their requirement for Blimp-1 expression
  Presenter: Hannah Savage
  All Authors:Hannah Savage, Vanessa M. Yenson, Jacquelyn Dieter, Marc A. Morgan, Elizabeth K. Bikoff, Nicole Baumgarth
  University of California, Davis
   
 

The precise cellular origins and the signals that induce and regulate the continuous production of natural IgM, which has both immune regulatory and protective functions against pathogens, are largely unknown. Here we explored the need for terminal differentiation by the transcriptional master regulator of terminal differentiation, Blimp-1, for normal IgM production. Distinct populations of natural IgM antibody-secreting cells (ASC) were identified in spleen and bone marrow: Blimp-1-expressing CD138+ plasma cells (PC) and mostly CD138 negative B-1 cells, phenotypically indistinguishable from non-secreting B-1 cells. Using a neonatal chimera approach we demonstrate that both populations are reconstituted by adoptive transfer of FACS-purified peritoneal cavity B-1 cells. FACS-sorting of the various B-1 cells differing in Blimp-1 expression with help of Blimp-1 YFP reporter mice, followed by ELISPOT analysis, revealed a population of Blimp-1 independent B-1 cell IgM-ASC in bone marrow but not the spleen. This was confirmed with mice lacking Blimp-1 expression specifically in B cells, generated by deletion of exon 1A of prdm-1 (PRDM-1?Ex1A mice). These mice had significant reductions in the frequencies of IgM-ASC in the spleen but not the bone marrow. Consistent with these findings, natural IgM levels were reduced but not absent in PRDM-1?Ex1A mice compared to controls. Thus, our data reveal substantial heterogeneity within the pool of natural IgM-ASC. Of particular interest is the discovery of IgM-ASC among Blimp-1neg B-1 cells in the bone marrow that increase in frequency in the absence of Blimp-1 and are likely responsible for much of the remaining IgM in Blimp-1 deficiency. The data suggest that several distinct mechanisms regulate the pool of natural IgM-secreting cells.

   



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  PKM2 is a critical driver of pathologic immune responses during CNS demyelination
  Presenter: Scott Seki
  All Authors:Scott Seki, Max Stevenson, Lelisa Gemta, Vlad Serbulea, Norbert Leitinger, Timothy NJ Bullock, Alban Gaultier
  University of Virginia
   
 

Growing evidence suggests that inflammatory responses of T cells rely on metabolic adaptations, especially elevated glucose metabolism, for bioenergetic and biosynthetic support. While this has generated excitement about targeting metabolic pathways for inflammatory conditions, the potential side effects associated with systemic administration of glycolysis inhibitors has tempered the translational potential of this concept. An alternative approach, and the premise of this work, is that machinery specifically upregulated to facilitate metabolic adaptation, and thus inflammation, may represent valuable targets by which inflammatory responses could be selectively defused. One candidate for this approach is Pyruvate kinase isoform M2 (PKM2), an isoform of the pyruvate kinase enzyme that is weakly expressed under homeostatic conditions and robustly upregulated by activated immune cells. While commonly known as an enzyme in the glycolytic cascade, PKM2 can double as a transcription factor. Indeed, it has been shown in myeloid cells that PKM2 can translocate to the nucleus, where it is serves as an essential co-activator of inflammatory gene signatures. PKM2 is a superb example of how machinery induced during metabolic adaptation can drive downstream inflammation. My goal is to identify how metabolic adaptations, like elevated PKM2, support pathologic immune cells during relapsing remitting multiple sclerosis (RRMS), a disease driven by inflammatory responses in the central nervous system. Using experimental autoimmune encephalomyelitis, a mouse model of RRMS, I have found that immune cells of the demyelinating CNS upregulate glycolytic machinery, including PKM2, and that levels of glycolytic machinery expression correlate with expression of inflammatory genes. Blocking PKM2 nuclear translocation with the drug TEPP-46 inhibits inflammatory responses of a diverse array of immune cells isolated from the demyelinating CNS at peak disease severity. In addition, I found that TEPP-46 selectively blocks the differentiation of IL-17A-producing T cells, a T cell subset known to drive autoinflammatory responses including, but not limited to RRMS. Taken together, these data reveal PKM2 is a potent modulator of CNS inflammation during immune-mediated demyelination.

   



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  Structural dynamics of opioid-bound MD2 and the mechanisms of subsequent TLR4 modulation
  Presenter: Masaud Shah
  All Authors:Masaud Shah, Muhammad Ayaz Anwar, Dhanusha Yesudhas, Sangdun Choi*
  MST, Ajou University, Suwon Si, South Korea
   
 

The negative effect of opioids in analgesia and the initiation of central immune system, following TLR4 activation, have highlighted the underlying consequences for opioids pharmacodynamics. Successive studies have extended these findings and suggested that morphine and its opioid-receptors’ inactive metabolite M3G activate TLR4 pathway. However, detail structural insights of these ligands with TLR4/MD2 or MD2 alone have not yet been investigated. Here, we utilized extensive computational tools and highlighted the structural dynamics of the opioid-bound TLR4/MD2 and the underlying possible mechanism of their non-sterioselective activation of TLR4 pathway. Our docking results are supporting previous findings and further extend that the stability of morphine and naloxone, but not M3G, in MD2 cavity is TLR4 dependent. Further detail dynamics analysis suggests that morphine binds to the soluble MD2, but most likely this binding is reversible, which gains stability upon interacting with TLR4. Morphine establishes its interaction close to the Phe126 loop and facilitates the agonistic conformation of MD2C. M3G also confers MD2C and suggests the investment of H1 loop, in addition to Phe126 loop, in the ligand binding affinity and the overall stability of the TLR4/MD2-M3G complex. Naloxone remarkably switches the gating loop conformation from active (MD2C) to inactive (MD2O) form in MD2 and needs TLR4 for stabilization. Cumulatively, our findings suggest that ligand binding and receptor clustering occur subsequently in opioids-induced TLR4 signaling, and plasticity and pocket hydrophobicity in MD2 play key role in ligand recognition and accommodation.

   



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  Contribution of the transcription factor Nr4a1 to CD8+ T cell activation following vaccination
  Presenter: Lindsey Shallberg
  All Authors:Lindsey Shallberg, Anthony T. Phan, David A. Christian, Christopher A. Hunter
  University of Pennsylvania, Department of Pathobiology
   
 

CD8+ T cells are critical for resistance to a broad range of pathogens. Presentation of cognate antigen to naive CD8+ T cells results in their activation and expansion, and re-encounter of antigen induces cytokine production, cytolysis of infected cells, and can influence fate decisions. The transcription factor Nr4a1 is an immediate-early response gene to TCR stimulation. It has been reported that Nr4a1 expression is a driver of T cell exhaustion following LCMV-clone13 infection, however it is unclear what impact Nr4a1 expression has in CD8+ T cells that are stimulated by antigen but do not become exhausted. Utilizing vaccination with a non-replicating strain of the OVA expressing intracellular parasite Toxoplasma gondii, we find Nr4a1 expressing OT-I following vaccination. Using a Nr4a1 shRNA knockdown retrovirus, OT-I deficient in Nr4a1 fail to properly expand following vaccination. This is associated with decreased expression of the IL-2 receptor and effector markers such as CX3CR1, as well as a decrease in the proportion of CD62LlowCD127low effector cells. Further, loss of Nr4a1 also altered OT-I mitochondrial mass and function, indicating a role for Nr4a1 in coordinating the metabolic activity necessary to sustain T cell activation and expansion. These data suggest a role of Nr4a1 in generating the protective CD8+ T cell response following vaccination.

   



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  Id2 reinforces antiviral Th1 cell differentiation and inhibits Tfh cell differentiation
  Presenter: Laura Shaw
  All Authors:Laura Shaw, Simon Belanger, Kyla D. Omilusik, James P. Scott-Browne, J. Philip Nance, John Goulding, Anna Lasorella, Shane Crotty and Ananda Goldrath
  University of California, San Diego
   
 

Differentiation of CD4+ T cells into multiple effector subsets is critical for protection against various pathogens, and transcriptional regulation is central in these effector cell differentiation processes. Together, E protein transcription factors and the inhibitor of DNA binding (Id) proteins are known arbiters of development and differentiation of lymphocytes. As yet, the importance of these transcription factors in the differentiation of Th1 and Tfh cells has not been addressed. In this study, we show that the transcriptional repressor Id2 is robustly expressed in antiviral Th1 cells compared to Tfh cells. Complete loss of Id2 in CD4+ T cells (Id2fl/fl CD4-Cre) resulted in a severe loss of Th1 cells, which was found to be E protein dependent. Gene-expression changes between Id2-deficient and –sufficient CD4+ T cells revealed a loss of Th1-associated genes (78%), and an increase in the expression of Tfh-associated genes (65%). Finally, we find that Bcl6 binds to the Id2 locus and inhibited its expression, providing a mechanism by which bimodal expression of Id2 in Tfh and Th1 cells can be established. These data identify Id2 as a critical actor in enforcing the reciprocal development of Th1 and Tfh cell fates.

   



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  The role of Id proteins in CD4+ T cell memory
  Presenter: Laura Shaw
  All Authors:Laura Shaw, Kyla D Omilusik and Ananda W Goldrath
  UCSD
   
 

When activated by antigen in the context of MHC class II, CD4+ T cells initiate a specific transcriptional program that affords them the unique ability to differentiate into multiple helper (Th) populations, such as Tfh and Th1. Tfh cells are essential for germinal center responses and supporting long-lived antibody production, whereas Th1 cells are responsible for defending against intracellular pathogens. As the immune response wanes, the majority of the antigen-specific T cells will die but a few CD4+ T cells will survive indefinitely to establish memory populations, providing long-lived protection against reinfection. This ability of lymphocytes to ‘remember’ is the basis for protection following vaccination. Analogous to the CD8+ T cell response, we propose that E protein transcription factors and their natural inhibitors, Id proteins, play a major role in coordinating effector and memory CD4+ T cell differentiation. Using mice expressing knocked-in fluorescent reporters, we have identified specific expression of Id2 and Id3 in effector (Th1) and memory (Tfh) populations, respectively, following infection. Furthermore, with conditional knock-out models, we demonstrate an essential role for Id2 and Id3 in promoting the generation and survival of Tfh and Th1 populations. Understanding the transcriptional network that leads to the development and localization of these CD4+ T cell subsets will prove useful in designing vaccines that elicit an optimal cellular immune response.

   



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  Airway epithelial cells promote allergic airway inflammation to cockroach allergen through production of GM-CSF in a MyD88-dependent manner.
  Presenter: Alyssa Sheih
  All Authors:Alyssa Sheih, Steven F. Ziegler
  University of Washington
   
 

Asthma is a chronic lung disease that leads to airway inflammation, reversible airflow obstruction and airway hyperresponsiveness. The global prevalence of asthma has steadily increased over the past 50 years and is associated with a large socioeconomic burden. Environmental allergens are a major culprit in the development and exacerbation of asthma. Despite its public health importance, the pathways that lead to allergic sensitization of the airways have not been well defined. Airway epithelial cells play an important role during allergic sensitization. They serve as the first line of defense against foreign pathogens. In addition to providing a protective barrier, epithelial cells directly sense allergens through innate immune receptors and coordinate the immune response by releasing key cytokines. By studying airway epithelial cells at the air-liquid interface, we find that exposure to cockroach allergen leads to the production and release of GM-CSF by airway epithelial cells in a MyD88-dependent manner. To further understand how MyD88 and GM-CSF might contribute to allergic airway inflammation, we have characterized a mouse model that results in robust TH2 airway inflammation upon intranasal sensitization and challenge with cockroach allergen. This inflammatory response is significantly reduced in MyD88 knockout mice. However, bone marrow chimera experiments show that MyD88 is not required on hematopoietic cells for this response to cockroach allergen. In addition, we have shown that neutralization of GM-CSF at the time of sensitization lowers allergic airway inflammation to cockroach allergen. In this study, we have established a critical role for MyD88 signaling in airway epithelial cells and the subsequent production of GM-CSF in regulating airway inflammation to cockroach allergen.

   



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  Evaluation of TLR4 inflammatory pathway using TIRAP-derived peptides
  Presenter: Hyeon-Jun Shin
  All Authors:Hyeon-Jun Shin, Xiangai Gui and Sangdun Choi
  Department of Molecular Science and Technology, Ajou University, Suwon, Korea
   
 

Activation of Toll-like receptor 4 (TLR4) ensues the increased secretion of inflammatory cytokines, reactive oxygen species and NO production. TLR4-induced exaggerated immune responses are associated with the initiation of various diseases as well as aggravating the inflammatory diseases including rheumatoid arthritis, sepsis and acute lung injury. TIRAP (Toll/interleukin-1 receptor domain containing adaptor protein) is an important adaptor molecule that is involved in TLR4 signaling propagation. Its activation leads to the subsequent activation of NF-κB and mitogen activated protein kinases (MAPKs; extracellular regulated kinase, c-Jun N-terminal Kinase and p38), which then results in cytokine secretion and inflammatory responses. Herein, TIRAP-derived peptide has been evaluated to block TLR4 pathway. This peptide inhibited TLR4-mediated activation of NF-κB, MAPKs and other cytokines. The peptide hindered the induction of iNOS, COX2, NO and ROS. The peptide also diminished systemic cytokine responses elicited in vivo by lipopolysaccharide challenge. This peptide represents a TLR4 pathway inhibitor that blocks protein-protein interaction between Toll/interleukin 1 receptor domains and, thus, TLR4-mediated immune responses.

   



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  Defective NKT cell development in BRCA-1 mutant mice
  Presenter: Susannah C. Shissler
  All Authors:Susannah C. Shissler, Tonya J. Webb
  University of Maryland, Baltimore
   
 

Breast cancer accounts for 250,000 new cancer diagnoses annually – making it a significant health burden. Inherited mutations in Breast Cancer susceptibility (BRCA)-1 or 2 genes significantly increase the likelihood of cancer development. Upon diagnosis, women with BRCA-1 mutations are more likely to present at a younger age and with a more aggressive phenotype. Despite advances in screening and treatment, breast cancer incidence and mortality rates have remained constant over the past three decades, underscoring the need for innovative treatment strategies, particularly immunotherapy. In order for immunotherapy to be effective, one must have a functioning immune system. Preliminary studies indicate a profound reduction in natural killer T (NKT) cells in women with BRCA mutations. Similarly, in a BRCA-1 mutant mouse model, there is a 70-85% decrease in NKT cells in the periphery and in the thymus. NKT cells play a critical role in cancer immune surveillance, thus we hypothesize that mutations in BRCA lead to impaired NKT cell development and this loss contributes to cancer development. NKT cell development diverges from conventional T cells at the double positive stage where they divert into a developmental pathway characterized by four distinct stages. BRCA-1 mutant NKT cells are primarily restrained in stage 2 of development with very few progressing to stage 3. This study aims to determine why thymic NKT development is inhibited in BRCA-1 mutant mice, and the mechanism accounting for the paucity of NKT cells in the periphery. Importantly, this research identifies a major deficit in host anti-tumor immunity. This work will aid in understanding the mechanisms by which BRCA-1 regulates NKT cell development and maintenance. Collectively, these studies may lead to the development of novel immunotherapeutic strategies.

   



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  Allergic airway inflammation and airway hyperresponsiveness are independently controlled by diacylglycerol kinase zeta
  Presenter: Brenal Singh
  All Authors:Brenal Singh, Wen Lu, Amanda M. Schmidt Paustian, Moyar Q. Ge, Cynthia J. Koziol-White, Cameron H. Flayer, Sara S. Killingbeck, Nadan Wang, Xinzhong Dong, Matthew J. Riese, Deepak A. Deshpande, Reynold A. Panettieri, Jr., Angela Haczku, and Taku Kambayashi
  University of Pennsylvania
   
 

Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, leading to airway obstruction caused by airway hyperresponsiveness (AHR) to contractile agonists. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protects against allergic asthma by simultaneously blocking airway inflammation and AHR by independent mechanisms. Targeted deletion of DGKζ in T cells led to decreased type 2 inflammation in an ERK-dependent manner with no attenuation of AHR. In contrast, loss of DGKζ in airway smooth muscle cells led to decreased AHR in an ERK-independent manner despite no changes in airway inflammation. Importantly, pharmacological inhibition of DGK diminished airway inflammation and AHR in mice, and also reduced bronchoconstriction of human airways. These data suggest that DGK is a novel therapeutic target for asthma and reveals that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

   



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  Unique signaling requirements facilitate a dichotomous response of human MAIT cells to commensal and pathogenic bacteria
  Presenter: Chloe Slichter
  All Authors:Chloe Slichter, Andrew McDavid, Hannah W. Miller, Greg Finak, Brenda J. Seymour, John P. McNevin, Gabriela Diaz, Julie L. Czartoski, M. Juliana McElrath, Raphael Gottardo, and Martin Prlic
  Fred Hutchinson Cancer Research Center
   
 

Human Mucosal-associated invariant T (MAIT) cells recognize and are activated by bacterial metabolites presented by the non-classical MHC molecule MR1, have a memory T cell phenotype, and are abundant in human blood and mucosal tissues. Given that commensal and pathogenic bacteria share some of the same metabolites known to activate MAIT cells, it raises the question of how MAIT cell effector function is regulated to avoid responses against commensal-derived metabolites, while still responding to pathogen-derived metabolites. To answer this question we examined MAIT cells isolated from blood and mucosal tissues of healthy human donors and found that effector function is controlled in a stringent manner. A TCR plus costimulatory signal is not sufficient to elicit robust effector function, but instead induces a specific monocyte-recruiting chemokine response. Importantly, we demonstrate that inflammatory cytokines are necessary and synergize with the TCR signal to induce robust effector function including interferon-γ and granzyme B expression. To define tissue-based differences of human MAIT cells, we used a single-cell gene expression approach and found that MAIT cells in the mucosal tissue of healthy donors have even more pronounced pro-inflammatory/activating potential than MAIT cells in the blood, but show no direct ex vivo effector function. This further underlines that tight functional control of these effector functions is necessary and occurs in vivo. We propose a novel model in which unique signaling requirements allow MAIT cells to respond to the same TCR signal in a dichotomous and situation-specific manner. These data also have important implications for understanding MAIT cell function following damage to the gastrointestinal luminal integrity such as bone marrow transplant conditioning or HIV infection.

   



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  Inflammatory signals license human MAIT cell effector function
  Presenter: Chloe Slichter
  All Authors:Chloe Slichter, Andrew McDavid, Hannah W. Miller, Greg Finak, Brenda J. Seymour, John P. McNevin, Gabriela Diaz, Julie L. Czartoski, M. Juliana McElrath, Raphael Gottardo, and Martin Prlic
  University of Washington
   
 

Mucosal-associated invariant T (MAIT) cells recognize bacterial-derived metabolites presented on MR1 via their semi-invariant T cell receptor (TCR). MAIT cells are a potent effector population located in the periphery as well as in tissues that are constantly exposed to the microbiome. This raises the question how MAIT cell activation is regulated to avoid unwanted responses to microbiome-derived metabolites. We examined the ability of MAIT cells to respond to inflammatory cues in the absence of bacterial metabolites and found that inflammation is sufficient to induce MAIT cell activation, but stimulating the MAIT TCR in the absence of inflammatory cytokines was not sufficient to induce effector function such as granzyme B and IFN-γ expression. Importantly, MAIT cells are not generally hyporesponsive to TCR-mediated signals as TCR stimulation in the absence of inflammation elicited a qualitatively distinct chemokine response. Additionally, single cell gene analysis of MAIT cells circulating in the blood and residing in the mucosal tissue revealed unique gene expression profiles. When CD8+ and CD8- MAIT cell populations were compared we found a similar transcriptional profile within each tissue type, but differences became apparent when comparing MAIT cells between blood and mucosal tissue. Interestingly, we found that expression of genes associated with activating/pro-inflammatory functions was increased in mucosal MAIT cell populations, indicating that mucosal MAIT cells have greater potential to enhance immune responses compared to their counterparts in blood. Our data suggest that the strict dependence of TCR signals on pro-inflammatory cytokines to induce effector function represents a MAIT cell-intrinsic mechanism to prevent unwanted responses to commensal bacteria-derived metabolites. We propose a novel model in which MAIT cells strictly require inflammatory signals to license induction of effector function following TCR engagement.

   



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  Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy
  Presenter: Drake Smith
  All Authors:Drake Smith, Levina J. Lin, Heesung Moon, Alexander T. Pham, Xi Wang, Siyuan Liu, Sunjong Ji, Valerie Rezek, Saki Shimizu, Marlene Ruiz, Jennifer Lam, Deanna M. Janzen, Sanaz Memarzadeh, Donald B. Kohn, Jerome A. Zack, Scott G. Kitchen, Dong Sung An, Lili Yang
  UCLA
   
 

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2R?-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

   



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  Induction of necroptotic cell death within the tumor microenvironment confers tumor control
  Presenter: Annelise G. Snyder
  All Authors:Annelise G. Snyder, Michelle N. Messmer, Nicholas W. Hubbard, Susana L. Orozco, Brian P. Daniels, Kristy Chiang, Andrew Oberst
  University of Washington, Department of Immunology (Seattle, WA, USA)
   
 

Cytotoxic therapies such as irradiation or chemotherapy can promote beneficial anti-tumor immune responses dependent on tumor cell death. However, the specific mechanisms underlying how distinct lytic cell death programs differentially instruct anti-tumor immunity have not been thoroughly investigated. It has been proposed that pro-inflammatory forms of tumor cell death increase the availability of tumor antigen, in addition to providing a burst of inflammatory signals within the typically immunosuppressive tumor microenvironment (TME). Work in our lab and others has shown that a RIPK3-dependent lytic cell death program, termed “necroptosis”, stimulates stronger inflammatory immune responses compared to apoptosis; we therefore hypothesized that necroptosis could induce anti-tumor responses within the TME. To test this, we created cell lines in which specific cell death programs can be triggered using bioavailable drug ligands. Using these “Pure Death” systems in syngeneic flank tumor models, we show that introduction of necroptotic cells to the TME leads to CD8-dependent tumor control that is associated with increased tumor antigen loading among tumor-associated antigen-presenting cells, as well as a favorable CD8:Treg ratio. Furthermore, inducing necroptosis within a primary tumor results in the control of distal, untreated tumors, indicating that this therapy confers a systemic response. Further elucidation of the cell subsets and signaling axes responsible for these observations will yield novel insights concerning the contribution of specific tumor cell death programs in promoting anti-tumor immunity.

   



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  Rare circulating PD1-high/ TIM3+/TIGIT+ T cells are seen in cervical cancer patients
  Presenter: Fabiola Solorzano-Ibarra
  All Authors:Fabiola Solorzano-Ibarra, Alan Guillermo Alejandre-Gonzalez, Gloria Gutierrez-Silerio, Blanca Estela Bastidas-Ramirez,Pablo Cesar Ortiz-Lazareno, Abraham Zepeda-Moreno, Oscar Joel Carrillo-Garibaldi,Arturo Chavira-Alvarado, Miriam Ruth Bueno-Topete, Susana del Toro-Arreola, Jesse Haramati
  Universidad de Guadalajara, Guadalajara, Mexico, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara; Centro de Investigación Biomédica de Occidente; Instituto Jalisciense de Cancerología; Hospital Civil de Guadalajara, Juan I. Menchaca. Guadalajara, Mexico.
   
 

Recent reports have shown the importance of immune checkpoint molecules in cancer escape. Cervical cancer is characterized by the persistent presence of human papilloma virus (HPV) infection. Progression from clinically detectable infection to invasive cervical carcinoma occurs in less than 1% of HPV-infected women. Here, we analyzed the expression of checkpoint molecules PD-1, TIM3 and TIGIT in NK and T cell populations in the blood of patients with cervical cancer and healthy controls. Significant increases in PD-1 were found. While PD-1 was also observed in controls, unique populations of PD-1 high cells (with MFI about 10x-100x higher than the regular population) were found increased in cancer, and sub-gating on these cells that expressed the highest PD-1 showed them to be also enriched for TIGIT and TIM3. In controls, a DNAM low/TIGIT negative population of T cells was found; in patients, in contrast, a DNAM low/TIGIT+ population appeared. Additionally, a unique population of CD56bright, TIGIT+ NK cells was observed. Conclusions: we show new cellular populations with the potential to explain immune escape in cervical cancer; the functional effects of these unique, possibly exhausted/ex-cytotoxic, populations will be investigated further in followup experiments.

   



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  Manipulation of lymphocytes by sphingolipid analog AD-2900 for therapeutic purposes
  Presenter: JING SONG
  All Authors:JING SONG, O. Almogi-Hazan, S. Gatt , A. Dagan, R. Or
  The Hebrew University of Jerusalem
   
 

Sphingolipids have significant roles in the immune system. Sphingolipid and its derivatives are involved in common signalling pathways which control the main stages of immune cells development and function. The use of sphingolipid analogues, such as FTY-720, is currently under investigation as a therapy for different immune disorders. We hypothesize different sphingolipid analogues have different effect on immune cell function and therefore different analogues can be used for intervention in various diseases. A group of sphingolipid analogues have been synthesized in our laboratory. Based on their effect on lymphocyte cell proliferation, we have selected one of our synthesized sphingolipid analogues, AD-2900. In this research we have characterized the properties of AD-2900 and examined its influence on lymphocytes function. We found that AD-2900 is an antagonist for all the S1P receptors 1 to 5; Specially, AD-2900 shows high antagonism property for S1P5 (IC50 = 0.405µM). Moreover, AD-2900 treatment leads to a significant but reversible down-regulation of S1PR1 cell surface expression on hPBLs. However, AD-2900 is not phosphorylated either by SphK1 or SphK2. We have tested the effect of AD-2900 on immune activation. In hPBL, AD-2900 inhibited T cell proliferation and induced apoptosis dose-dependently. However, it had a unique effect on immune activation comparing to FTY-720 and S1P. The inhibitory effect of the analogue on hPBL proliferation was dependent on cAMP reduction and calcium signal transduction, but not on phospholipase C (PLC) activation (which participates in S1P’s effect). The effect of FTY-720 on hPBL proliferation was not associated with any of these pathways. In addition AD-2900 induced ERK phosphorylation in non-activated cells. However, the Phosphorylation patterns were different as compare to cells treated with the other analogues. In order to investigate whether AD-2900 plays a role in lymphocyte localization in vivo, C57BL/6 female mice were oral administrated AD-2900 in their drinking water in different dosages; FTY-720 serves as a positive control. The results show that, in contrast to FTY-720, AD-2900 had no impact on blood WBC counting after 48h administration. However, while FTY-720 treatment decreased spleenocyte counting, AD-2900 significantly increased the number of WBC in the spleen. Modulations in the surface expression levels of S1PR1 and the chemokine receptor CCR7 on lymphocytes are involved in the circulation of these cells between the lymphatic organs and the blood. We found that AD-2900 significantly down-regulates the surface expression S1PR1 on lymph node and blood WBC. The expression of CCR7 on mice spleen cells was significantly up-regulated by AD-2900. Notably, FTY-720 had no influence on CCR7 expression in the spleen. In blood cells, both AD-2900 and FTY-720 treatments caused significant down-regulation of CCR7. Currently, we are expanding our knowledge regarding the specific sub-populations of cells which are affected by AD-2900. Hopefully, the results of this study may allow us to use our innovative sphingolipid analogue AD-2900 to develop an effective immune-modulatory drug for immunotherapy of immune related disorders.

   



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  Membrane-targeting of oligoadenylate synthetase 1 primes antiviral activity
  Presenter: Frank W. Soveg
  All Authors:Frank W. Soveg, Johannes Schwerk, Katharina Esser-Nobis, Alison Kell, Adriana Forero, Julian Smith, Justin Roby, Tien-Ying Hsiang, Amy Stone, Chiraag Balu, Jonathan Clingan, Daniel B. Stetson, Michael Gale Jr., and Ram Savan
  The University of Washington
   
 

Positive-strand RNA viruses, such as flaviviruses and picornaviruses, replicate within a modified subcellular compartment known as the replication organelle (RO). Derived from organelle membranes, the RO sequesters viral RNA (vRNA) synthesis from immune surveillance. Although many important vRNA sensors are cytosolic and have limited access to the RO, we hypothesized membrane-targeting of host sensors is a mechanism to gain access to the RO. We tested this hypothesis in the context of oligoadenylate synthetase (OAS) proteins, a family of innate vRNA sensors critical for defense against RNA viruses through activation of the latent endoribonuclease RNase L. Human OAS1 is C-terminally spliced into several isoforms and previous genetic studies have implicated a splice acceptor site SNP (A/G, rs10774671) in the OAS1 gene with West Nile virus (WNV) resistance. The resistance allele (G) controls p46 expression while the susceptibility allele (A) controls p42 expression, but how this SNP promotes resistance to WNV is unknown. We show, compared to the p42 isoform, p46 has significantly stronger antiviral activity against not only WNV, but also other positive-strand RNA viruses such as encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB). Although both OAS1 isoforms require catalytic activity and RNase L for their antiviral activity, only the p46 isoform contains a C-terminal prenylation motif. The addition of a hydrophobic lipid through prenylation facilitates membrane targeting. We tested if membrane-targeting drives the enhanced antiviral activity of p46 using confocal microscopy, biochemical, and genetic approaches. We found prenylation-dependent localization of p46 to the Golgi, while p42 is cytosolic. Through microscopy and RNA immunoprecipitation experiments, we revealed the p46 isoform has enhanced access to vRNA. Collectively, our study shows membrane-targeting of p46 through prenylation localizes this protein in proximity to pools of vRNA generated by positive-strand RNA viruses and provides a mechanistic explanation for the resistance to WNV conferred by the G allele of rs10774671. Broadly, these data show the subcellular positioning of innate immune sensors is important for proper detection of viral nucleic acids.

   



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  Kupffer cells resist irradiation through cell cycle arrest
  Presenter: Dilini Radika Soysa
  All Authors:Dilini Radika Soysa , Sarah Margit, Sebastien Yuen, Ian N. Crispe
  University of Washington
   
 

upffer cells (KCs) are liver resident macrophages that reside in hepatic sinusoidal lumen. Aside from being excellent phagocytes KCs play a key role in liver immune tolerance and induction of both inflammation and resolution during liver injury. Using irradiation born marrow chimera models our lab and others have reported that a subset of KCs resist irradiation and survive long term. The factors that determine radioresistance of a subset of KCs and whether the host and donor derived KCs have different immune functions in the chimeric liver are currently unknown. To answer this question we utilized an in situ cell specific mRNA isolation method based on “RiboTag approach” in combination with the bone marrow chimera. Here we report that the tissue resident macrophage specific Cre driver Emr1 combined with RiboTag allows KC labeling and isolation of KC specific mRNA. Gene expression analysis of KCs in Emr1Cre::RiboTag mice immediately after irradiation showed down regulation of cell cycle progression genes, cell death associated genes while cell cycle arrest genes are upregulated suggesting immediate cell cycle arrest as a mechanism of radioresistance. Furthermore in irradiation chimera mice host and donor derived KCs indicate that while most of the genes are expressed at a similar level, few genes maintain significant differences at least up to 13 weeks post irradiation. This initial result indicates that a subset of KCs resist irradiation through cell cycle arrest and maintain functional differences in the chimeric liver.

   



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  Turn Down For What: Loss of CD4 expression Facilitates Antigen Exchange Between 33D1+ Dendritic Cells
  Presenter: J. Michael Stolley
  All Authors:J. Michael Stolley, Daniel J. Campbell
  University of Washington
   
 

MHC class-restriction, the concept that helper T cells expressing the co-receptor CD4 encounter antigen in the context of MHC-class II while cytotoxic T cells expressing the co-receptor CD8 receive TCR activation through MHC-class I, is immunological dogma. Paradoxically, dendritic cells (DCs) which express high levels of MHC molecules differentially express the T cell co-receptors CD4 and CD8a, and these are in-fact used to subset distinct populations of DCs within secondary lymphoid tissues. Why DCs concurrently express MHC molecules and T cell co-receptors has puzzled immunologists since the discovery that DCs expressed CD4 or CD8a in the 1990’s. However, studying the function of these molecules on DCs is confounded by the fact that their T cell counterparts are absent in CD4 or CD8a-deficient mice. Here we provide a glimpse into the potential function of CD4 on DCs. CD4 expression on 33D1+ DCs is markedly diminished following TLR agonist exposure in vivo in a cell-intrinsic manner. Using chimeric mice in which CD4-deficiency can be restricted within the DC compartment, we provide evidence suggesting loss of CD4 expression on 33D1+ DCs facilitates the exchange of antigens between 33D1+ DCs. Consistent with these findings, peptide-loaded CD4-deficient DCs relinquished more antigen to congenically marked DCs in vitro than CD4-sufficient DCs. Therefore, we suggest a model by which CD4 down-regulation succeeding phagocytosis (and subsequent TLR activation) of pathogenic material expedites the exchange of microbial peptides from activated to non-activated DCs; rapidly increasing the overall number of DCs which can initiate CD4+ T cells responses.

   



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  Non-typhoidal Salmonella that causes invasive disease evades reactive oxygen species production by neutrophils
  Presenter: Annica Stull-Lane
  All Authors:Annica Stull-Lane, Hirotaka Hiyoshi, Vladimir Diaz-Ochoa, Renée Tsolis
  University of California, Davis
   
 

Neutrophils are important phagocytic innate immune cells that help combat non-typhoidal Salmonella (NTS) disease, from gastroenteritis to systemic infection. ST19 NTS isolates are associated with classic gastroenteritis, whereas ST313 NTS isolates are more likely to cause invasive disease globally. Invasive NTS disease has a case fatality rate of 20-25% and disproportionately affects the immunologically vulnerable. As part of their antimicrobial arsenal, neutrophils can eliminate microbial pathogens with a potent oxidative burst of reactive oxygen species (ROS). In this study we characterize induction of ROS by ST19 and ST313 strains in both mouse neutrophils ex vivo and in human HL-60 cells differentiated into neutrophil-like cells in vitro. ROS is detected by luminol-based chemiluminescence. Further, we assess the potential for intracellular survival of these strains within the neutrophil with a gentamicin protection assay. Interestingly, ST313 strains induce significantly less ROS than ST19 strains in both mouse and human neutrophils. Without potent ROS induction, levels of intracellular ST313 increase over the course of 2 hours, whereas levels of ST19 do not. These results suggest that invasive disease isolates may use neutrophils as a temporary intracellular niche. Unraveling these host-microbe mechanisms will deliver important fundamental knowledge to the pathogenesis of virulent Salmonella strains, informing future research and therapeutics to combat invasive disease.

   



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  What does the fox say? Regulation of Foxo1 in T cell homeostasis
  Presenter: Jenna M. Sullivan
  All Authors:Jenna M. Sullivan, Kerri Thomas and Daniel J. Campbell
  University of Washington
   
 

The Foxo (forkhead box O) family of transcription factors is important for maintaining homeostasis of both effector and regulatory T cells (TR). Interestingly, Foxo1 is necessary for proper development and function of TR and absence of Foxo1 results in fatal inflammation due to TR dysfunction. During T cell activation, phosphorylation of Foxo1 by Akt results in inactivation its and translocation from the nucleus. We have bred mice that contain a floxed non-phosphorylatable Foxo1 allele under control of CD4-cre (CD4cre-Foxo1CA), resulting in constitutive activation of Foxo1 in both CD4 and CD8 T cells. Mice which are unable to properly regulate Foxo1 have reduced body weight, chronic inflammation and develop spontaneous autoimmunity. CD4cre-Foxo1CA displaying symptoms of autoimmunity have tissue damage in the lung along with increases in eosinophils, alveolar macrophages and mucus production. In the periphery CD4cre-Foxo1CA mice have reduced TR number, increased activated effector T cells and an altered B cell compartment. Together this data demonstrates that proper regulation of Foxo1 is essential for T cell homeostasis and prevention of autoimmunity.

   



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  T cell specific dysregulation of Foxo1 results in spontaneous inflammatory disease
  Presenter: Jenna Sullivan
  All Authors:Jenna Sullivan, Kerri Thomas, Jeffrey Duggan and Daniel J. Campbell
  University of Washington
   
 

The Foxo (forkhead box O) family of transcription factors is important for maintaining homeostasis of both effector and regulatory T cells. In conventional T cells, Foxo1 is phosphorylated downstream of the PI3K/Akt/mTOR signaling pathway resulting in its inactivation and translocation from the nucleus and subsequent altered transcription of downstream targets. While many studies have examined the effects of Foxo1 deficiency, few have addressed the role of proper downregulation of Foxo1. To address this question we have obtained mice that carry a non-phosphorylatable Foxo1 allele (Foxo1CA) knocked into the ROSA26 locus, along with a ‘stop-flox’ cassette that prevents expression. We have crossed Foxo1CA mice to CD4-Cre mice (CD4cre Foxo1CA), resulting in deletion of the ‘stop-flox’ cassette and constitutive activation of Foxo1 in both CD4 and CD8 T cells beginning at the double positive stage of thymic development. Interestingly neonate thymi contain normal numbers of double positive thymocytes by have reduced mature single positive CD4 T cells with decreased cortex area and increased cell death. Mice which are unable to properly regulate Foxo1 in T cells are characterized grossly by runting, dermatitis and splenomegaly. In the periphery CD4cre Foxo1CA mice have reduced CD4 T cell populations comprised mainly of activated effector T cells and a dramatically reduced Treg population. Together this data demonstrates that proper regulation of Foxo1 is essential for thymic development and prevention of autoimmunity.

   



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  Novel Mechanism for YY1-mediated Control of Dynamic X-chromosome Inactivation Maintenance in Female B Lymphocytes
  Presenter: Camille Syrett
  All Authors:Camille Syrett, Camille M Syrett, Arpita Myles, Anna Martin, Michael Cancro, Montserrat C Anguera
  University of Pennsylvania
   
 

The X chromosome is enriched for genes with important functions in immunity. XX females are also more prone to autoimmune disorders such as systemic lupus erythematosus (SLE) where X-linked immune-related genes are overexpressed in lymphocytes. The mechanism underlying this sex bias is unclear, and hypotheses suggest an important role for X-chromosome dosage. Interestingly, female lymphocytes, unlike other somatic cells, uniquely regulate X-chromosome inactivation (XCI), where the inactive X chromosome (Xi) lacks canonical features including heterochromatin modifications and Xist RNA localization. Epigenetic profiling of this unusual Xi reveals that Xist RNA is lost from the Xi in pro-B cells, and that heterochromatic modifications are gradually lost from the Xi during B cell development. Xist RNA and Xi-heterochromatin return back to the Xi upon mature B cell activation in a YY1-dependent fashion. We show that the method of B cell stimulation, involving the B cell receptor or toll-like receptors, influences the timing and patterns of Xist RNA localization to the Xi. To determine if deletion of Xist RNA in lymphocytes causes reactivation of X-linked immune genes, we conditionally deleted Xist in female murine B cells (mb1CRE XistCKO/+). Remarkably, female XistCKO/+ animals develop serum ds-DNA autoantibodies at similar levels to classical lupus animal models, as well as proteinuria and glomerulonephritis. We also observed alterations in mature B cell subsets, as XistCKO/+ animals have elevated numbers of activated, memory, germinal center, and Tbet+ B cells, compared to age-matched controls. We propose that Xist RNA is essential for maintaining the dosage of X-linked immune genes in female B cells, and that perturbations to XCI maintenance contribute to autoimmunity.

   



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  Expression of DNAM-1 (CD226) on inflammatory monocytes
  Presenter: Eri Takenaka
  All Authors:Eri Takenaka, Anh Van Vo, Akira Shibuya, Kazuko Shibuya
  University of Tsukuba
   
 

DNAM-1 is an activating receptor expressed on NK cells and T cells and plays an important role in cytotoxicity of these cells against target cells. Although the role of DNAM-1 in the function of T cells and NK cells has been well studied, the expression and function of DNAM-1 on myeloid cells have been incompletely understood. In this study, we investigated expression of DNAM-1 on monocyte subsets in mouse peripheral blood and found that only inflammatory monocytes (iMos), but not patrolling monocytes (pMos), expressed high levels of DNAM-1. In addition, we found that DNAM-1 was highly expressed on iMos, rather than pMos, also in human. Furthermore, we found that DNAM-1 on inflammatory monocytes was involved in cell adhesion to CD155-expressing cells. Therefore, we propose that expression of DNAM-1 on inflammatory monocytes are evolutionally conserved and act as an adhesion molecule on blood inflammatory monocytes.

   



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  CX3CR1+ Bronchus-Associated Macrophages are Positioned for Surveillance of the Airways
  Presenter: Xin-Zi Tang
  All Authors:Xin-Zi Tang, Christopher Allen
  UCSF
   
 

In allergic asthma, allergen exposure leads to inflammation around the bronchial airways. However antigen uptake around the airways has never been detected. By two-photon laser scanning microscopy, we found that myeloid cells were present around the airways in the steady state and that majority of the antigen was captured from the airway lumen by a CX3CR1-GFP+ myeloid population, which was distinct from CD11c-YFP+ dendritic cells (DCs). In particular, the CX3CR1-GFP+ cells that took up antigen at the bronchial airways are dendritic in morphology but had a surface marker profile characteristic of macrophages. We therefore termed them Bronchus-Associated Macrophages (BAMs). In contrast to DCs, BAMs showed minimal displacement over time and did not migrate to the draining lymph nodes upon antigen uptake. Thus BAMs are unlikely to be necessary for T cell priming and sensitization. However, BAMs expressed high levels of MHC-II, were capable of antigen processing and presentation, and formed extended interactions with T cells that were recruited to the airway proximal regions during inflammation. In mouse models of asthma, we demonstrated that DCs were not necessary during the re-challenge phase for allergic inflammation, suggesting that memory T cells can be activated by BAMs upon allergen re-exposure. We have therefore identified a resident population of macrophages near the bronchial airways that capture antigen from the airway lumen and contribute to local T cell activation in the context of allergic airway disease.

   



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  Naïve Helper T Cells with high CD5 expression have increased calcium signaling
  Presenter: Claudia M Tellez Freitas
  All Authors:Claudia M Tellez Freitas, Garrett J Hamblin; Carlee M Larsen; Scott Weber
  Brigham Young University
   
 

The adaptive immune response is orchestrated by T helper cells and interactions between the T cell receptor (TCR), peptide MHC (pMHC) and co-receptors. These TCR-pMHC interactions initiate calcium signaling cascades which determine T cell activation and function. CD5 is a co-receptor that plays an important role in regulating T cell signaling and fate during thymocyte education. CD5 surface expression on mature single positive thymocytes correlates with the TCR signal strength for positive selecting self-ligands. CD5 also plays a role in T cell function after thymic development is complete. Peripheral T cells with higher CD5 expression respond better to foreign antigen than those with lower CD5 expression and are CD5 high T cells are enriched in memory populations. In our study, we examined the role of CD5 expression and calcium mobilization in the primary response of T cells using two Listeria monocytogenes specific T helper cells (LLO118 and LLO56). These T cells recognize the same immunodominant epitope (LLO190-205) of L. monocytogenes and have divergent primary and secondary responses as well as different levels of CD5 expression. We characterized the role of CD5 expression and calcium influx in these CD5 high and CD5 low T cells over the course of 8 days. We found significantly different calcium signaling levels in naïve and day 3 post-stimulation LLO56 and LLO118 T helper cells. To further investigate the role CD5 expression plays in calcium mobilization, we measured the calcium influx in T cells from LLO118-CD5 knockout mice versus those from LLO56-CD5 knockout mice. We found that CD5 expression is important in regulating calcium mobilization in the CD5 high naïve LLO56 T cells during the initial response to antigen and as CD5 levels decrease over time its role in regulating calcium also decreases.

   



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  CD5 co-receptor plays a role in T cell metabolism
  Presenter: Claudia M. Tellez Freitas
  All Authors:Claudia M. Tellez Freitas, Tyler D. Cox, Anne Dunn, and K. Scott Weber
  Department of Microbiology & Molecular Biology, Brigham Young University
   
 

During activation, T cells undergo metabolic reprogramming, which helps determine their distinct functional fates. CD5 is a co-receptor found on T cells and plays a significant role in regulating T cell thymic development, signaling, and cytokine production. Although CD5 is best known as a regulatory coreceptor during selection in T cell development, it has been reported to play a regulatory role similar to PD-1 and CTLA-4 upon T cell activation. We have found that CD5 levels influence calcium mobilization and T cell activation. Differential calcium mobilization, calcineurin function, and NFAT are known to affect glycolysis and mitochondrial respiration. Previous studies have shown that CD5 knockout mice (CD5KO) have increased T cell activation, leading to elevated levels of cytokine production and T cell proliferation. These functional changes suggest that CD5 may be affecting metabolic reprogramming. We hypothesized that CD5 deficient T cells have different bioenergetic demands that affect metabolic pathways key to T cell activation and function. We evaluated the effects of the CD5 co-receptor on metabolism by measuring the metabolic profiles of CD5KO and wild type T cells. Our preliminary data suggests that CD5KO T cells have higher mitochondrial respiration than wild type T cells and we are examining the mitochondrial mass in CD5KO naïve T cells. Thus, CD5 may play an important role in metabolic programing in T cells and could potentially be useful used in modulating the T cell response in the tumor microenvironment.

   



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  Calcium Signaling in T helper cell Primary and Secondary Responses
  Presenter: Claudia Tellez Freitas
  All Authors:Claudia Tellez Freitas, Kurt R. Williams, K. Scott Weber
  Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602
   
 

CD4+ helper T cells play a critical role in the immune response to infectious disease as they interact with macrophages and dendritic cells and activate B cells and cytotoxic CD8+ T cells. CD4+ T cells are essential for the pathogen specific responses of B cells and CD8+ T cells and are essential for the proliferation, differentiation, and memory cell formation of CD8+ T cells. Since helper T cells have such a central role in the adaptive response many studies have examined how they are activated and calcium (Ca2+) signaling is an important way to measure activation. Calcium is a universal second messenger and plays an important role in the regulation of cell differentiation, gene transcription and other effector functions of the cell. Calcium signaling has been well characterized in lymphocytes and the characteristics of the calcium signal for specific helper T cell subsets such as TH1, TH2 and TH17 have been identified. However, the unique calcium signal of helper T cells involved in primary and secondary responses is not yet well elucidated. To address this question, we are using two helper T cells from TCR transgenic mice specific for Listeriolysin O (LLO) of Listeria monocytogenes called LLO118 and LLO56. These helper T cells differ by only 15 amino acids and are specific for the same Listeria monocytogenes epitope, yet they have dramatically different primary and secondary responses. After preparing and isolating our desired helper T cell populations we are using ratiometric live cell calcium imaging to measure calcium profiles of T cells after primary and secondary stimulation. This project will help to elucidate the role that calcium signaling plays in helper T cell activation in primary and secondary responses and should produce additional understanding into activation of helper T cells in response to infectious disease.

   



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  Towards an understanding of NAIP/NLRC4 inflammasome assembly
  Presenter: Jeannette Tenthorey
  All Authors:Jeannette Tenthorey, Author Names separated by commas
  University of California, Berkeley
   
 

NAIP/NLRC4 inflammasomes assemble upon detection of conserved bacterial proteins in the host cell cytosol. Once assembled, inflammasomes serve as a scaffold that activates the protease CASPASE-1. Active CASPASE-1 initiates inflammation by cleaving pro-IL-1ß and IL-18 to their active forms, and by inducing a lytic form of cell death known as pyroptosis. However, the mechanism by which inflammasomes bind their ligands, assemble a signaling-competent oligomer, and coordinate CASPASE-1 activation remains elusive. Mice express multiple paralogs of NAIP that respond to distinct bacterial ligands. By generating chimeras between different NAIP paralogs, we found that ligand discrimination is not mediated by the leucine-rich-repeat (LRR) domain, as expected, but rather by several internal helical domains of NAIP. Furthermore, we found that each NAIP protomer must bind its cognate ligand to assemble into inflammasomes. To further refine the ligand binding site on NAIPs and to elucidate the mechanism by which ligand binding activates NAIPs, we have initiated structural studies of the NAIP/NLRC4 inflammasome. We expect these structural studies will be critical in clarifying how inflammasome assembly is initiated in response to cytosol-accessing bacterial pathogens.

   



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  Recognition of multiple motifs on bacterial ligands by NAIP receptors constrains bacterial immune evasion
  Presenter: Jeannette Tenthorey
  All Authors:Jeannette Tenthorey, Ella Hartenian, Elise Adamson, Russell Vance
  University of California, Berkeley
   
 

The innate immune system faces the challenging task of using a small number of receptors to recognize diverse classes of microbes based on features that distinguish microbes from host cells. Because microbes can rapidly evolve to evade detection, it might be beneficial for innate immune receptors to bind promiscuously to variable microbial ligands. On the other hand, promiscuous binding might also allow for aberrant recognition of self ligands to trigger auto-immune responses. We investigated how the innate immune system balances the specificity of ligand recognition with robustness to mutagenesis in the context of NAIP inflammasomes. Mouse NAIP paralogs bind to conserved bacterial proteins, including flagellin and structural components of the type III secretion system, that are translocated by the pathogen into the host cell cytosol. To determine how NAIPs recognize their target ligands, we screened a library of mutants in two of these ligands for their ability to activate their cognate NAIPs. In each case, we found that NAIP activation requires at least two ligand motifs that are segregated in primary sequence. Furthermore, both motifs bound to their cognate NAIP, suggesting that each is directly recognized. Importantly, single point mutations in either recognition motif are not sufficient to evade NAIP recognition. Instead, both sites must be mutated to evade immune recognition. Mutants that escape NAIP recognition are correspondingly more likely to be non-functional. We suggest that a bipartite mode of ligand recognition is a generalizable strategy for constraining bacterial immune evasion while simultaneously increasing the stringency of off-target recognition.

   



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  Presenter: Test Test
  All Authors:Test Test, Test
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  The Role of IL-25 in the Initiation of a Type-2 Inflammatory Response
  Presenter: Tennille Thelen
  All Authors:Tennille Thelen, Steven F. Ziegler
  University of Washington
   
 

We are interested in elucidating the precise role of IL-25 in the development of type-2 inflammation. Specifically, we would like to determine which cells are the main producers of IL-25, the cells it is capable of acting upon, and what effect it has on those cells. At mucosal sites, epithelial cells respond to environmental contaminants as well as infections by producing inflammatory cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP). IL-25 (IL-17E) is well known for its ability to induce TH2 cytokines (IL-4, IL-5, and IL-13) thereby enhancing the TH2 response, while correspondingly suppressing TH1 and TH17 responses. This leads to increased IgE production and eosinophilia, demonstrating its importance in both host defense to helminth infections as well as pathogenesis in allergic disorders. Cells capable of producing IL-25 include CD4+ and CD8+ T cells, mast cells, eosinophils, epithelial and endothelial cells. The IL-25 receptor is heterodimeric, consisting of IL17RA and IL17RB, and is expressed on T cells, macrophages, epithelial cells, and type 2 innate lymphocytes (ILC2s). In a lung tissue specific IL-25 over-expression mouse model, we found that turning on IL-25 expression results in dramatic increases IL-25 protein in lung and bronchial alveolar lavage (BAL) fluid, eosinophilia, increased mucus production, and increased serum IgE levels. When WT mice are given intranasal injections of IL-25 every other day for two weeks, we found that IL-25 alone is capable of inducing Type-2 inflammation, and this response is enhanced in the presence of antigen. When an a-CD4 antibody is used to deplete CD4+ cells the Type-2 response is diminished but not completely abolished. These results suggest IL-25 is capable of activating both innate and adaptive cell types. We are currently crossing a conditional knockout in which the IL-25 receptor subunit specific to IL-25, IL-17RB, is flanked with LoxP sites to CD4- and Lysm-CRE mice. This will allow us to eliminate IL-25 signaling within specific cell types to more precisely determine which cells are responding to IL-25 and generating a TH2 response. Another important question we would like to answer is what the interdependence is between the three epithelial cytokines; IL-25, IL-33, and TSLP. Using the same model of intranasal injections every other day for two weeks, we found that the Type-2 response was severely diminished in both TSLP and IL-33 receptor knock-out mice. These results are also seen when using blocking antibodies against the TSLP or IL-33 receptors. Interestingly, the abrogated response was only observed when IL-25 was given alone. When IL-25 is administered along with Ova, the response was similar to WT. This indicates there is cross-talk between these cytokines and we are currently investigating the mechanism behind this response.

   



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  Tumor-derived CSF-1 induces the NKG2D ligand RAE-1[d] on tumor-infiltrating myeloid cells
  Presenter: Thornton W. Thompson
  All Authors:Thornton W. Thompson, Benjamin T. Jackson, Po-Yi J. Li, David H. Raulet
  University of California - Berkeley
   
 

Expressed by NK cells and some T-cells, the cell-surface receptor NKG2D binds a set of host-encoded ligands typically absent from healthy cells but upregulated upon viral infection and on tumor cells, which targets them for destruction. Unexpectedly, we recently showed that in tumor-bearing mice, persistent interactions between NKG2D and its ligand(s) expressed on non-tumor cells reduce the cytotoxic activity of NK cells against cancer cells and facilitate tumor growth. Using spontaneous and transplanted cancer models, we show here that intra-tumoral macrophages and monocytes are induced to express the NKG2D ligand RAE-1δ. Conditioned media from tumor cells was sufficient to induce RAE-1δ on macrophages ex vivo, so we screened a panel of soluble factors and found that the cytokine CSF-1 was a robust inducer of RAE-1δ. Tumor cells secrete abundant CSF-1, and antibody blockade of the CSF-1 receptor (CD115) prevented macrophage RAE-1δ induction by tumor cell supernatants in vitro. Tumor-bearing mice injected with blocking antibodies against CD115 or CSF-1 rapidly downregulate myeloid RAE-1δ expression in tumors without substantial reduction in macrophage/monocyte numbers. We then used CRISPR/Cas9 to knock out Csf1 from tumor cells, which abrogated the induction of intra-tumoral myeloid RAE-1δ in vivo. Furthermore, we show that induction of RAE-1δ by CSF-1 requires signals from PI3K, and we identify PI3K p110α as the relevant isoform for RAE-1δ induction. Thus, cancer cell secretion of CSF-1 utilizes PI3K to induce the NKG2D ligand RAE-1δ on the cell surface of macrophage/monocyte cells that infiltrate the tumor.

   



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  Hemolytic activity of adenylate cyclase toxin is not required for colonization capacity but contributes to virulence of Bordetella pertussis
  Presenter: Barbora Tomalova
  All Authors:Barbora Tomalova, Karolina Skopova, Pavel Rossmann, Marek Kovar, Jiri Masin, Ilona Bibova, Nicole Guiso, Radim Osicka and Peter Sebo
  Institute of Microbiology ASCR, Videnska 1083, Prague 4, Czech Republic
   
 

The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a crucial role in virulence of the whooping cough agent Bordetella pertussis. The 1706 residue-long bi-functional leukotoxin targets cells expressing the complement receptor 3 (CR3, known as aMß2 integrin, CD11b/CD18, or Mac-1) and delivers into their cytosol an N-terminal adenylyl cyclase (AC) enzyme domain. This ablates bactericidal functions of host phagocytes by unregulated conversion of cytosolic ATP into second messenger molecule cAMP. In parallel, the hemolysin moiety of CyaA forms cation-selective pores that permeabilize target cell membranes and account for the hemolytic (Hly) activity of B. pertussis on blood agar. While importance of the cAMP-elevating activity of CyaA for colonization and immunomodulatory capacities of B. pertussis has previously been established, the role of the pore-forming (hemolytic) activity could not yet be addressed. Here we constructed the first non-hemolytic B. pertussis mutant (AC+Hly-) that produces a CyaA toxin exhibiting an intact capacity to elevate cAMP in host phagocytes. Upon intranasal infection, the AC+Hly- strain colonized mouse lungs to equal levels as the parental AC+Hly+ strain, showing that the pore-forming (hemolytic) activity of CyaA is per se not required for mouse lung colonization by B. pertussis. Compared to AC+Hly+, however, the AC+Hly- strain induced much milder lung pathology, caused lethal infections only at significantly increased bacterial doses and provoked much lower levels of neutrophil, macrophages and dendritic cell infiltration into infected lungs. While infection by the AC+Hly+ strain provoked significant decrease of mean MHC II molecule expression levels on the lung populations of macrophages and dendritic cells, infections by the AC+Hly- strain did not result in reduced MHC II expression levels. Hence, the pore-forming (hemolytic) activity of CyaA synergizes with the cAMP-elevating activity of CyaA in promoting immune evasion of the pathogen. Acknowledgement: This work was supported by Czech Science Foundation grant 13-12885S and Institutional Research Concept RVO 61388971.

   



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  Comparative study on efficacy of HPMA copolymer-based drug conjugates in treatment of solid and disseminated tumors
  Presenter: Barbora Tomalova
  All Authors:Barbora Tomalova, Milada Sirova, Pavel Rossmann, Petr Chytil, Eva Koziolova, Viktor Cerny, Jakub Tomala, Blanka Rihova, Karel Ulbrich, Tomas Etrych, Marek Kovar
  Institute of Microbiology ASCR v.v.i, Vídenská 1083, 142 20, Prague 4, Czech Republic, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic
   
 

In this study, we compared in vivo biological activity of linear (Mw ~ 27 000) and star-like (Mw ~ 250 000) N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymer conjugates bearing anthracycline antibiotic doxorubicin (DOX) bound via pH-sensitive hydrazon bond. We evaluated toxicity of both conjugates and compared their anti-tumor activity in murine B-cell leukemia (BCL1) and mouse T-cell lymphoma (EL4) model. The investigation of intestinal barrier integrity via FITC-dextran assay revealed no pathology caused by neither conjugate compared to free DOX. Histological analysis, however, showed yet to be explained accumulating process in spleen positively correlating with dosage of conjugates which was also accompanied with occurrence of mitotic hepatocytes in liver parenchym. Nevertheless, no additional abnormalities were seen in other tested organs (i.e. kidney, vertebral bone marrow). In vivo anti-tumor activity showed differences in treatment of various tumors. Therapy of BCL1 leukemia by either conjugate seemed to have quite similar outcome with slightly better effects of star-like conjugate. However, it appears that long persistence of star-like conjugate could cause side toxicity. Compared to that, treatment of EL4 lymphoma seems to be more efficient when linear conjugate is used. Star-like conjugate, on the other hand, seems to promote development of anti-tumor resistance in cured mice. In conclusion, we suppose that the ideal drug-bearing conjugate should have HMW structure which could be rapidly disintegrated into small polymer chains during first few days after administration. Acknowledgement: This work was supported by Czech Science Foundation grant P301/11/0325, by the Ministry of Education, Youth and Sports grant CZ.1.07/2.3.00/20.0055 and by Institutional Research Concept RVO 61388971.

   



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  Macrophage Polarization in the Colon Cancer Tumor Microenvironment
  Presenter: M.H. Townsend
  All Authors:M.H. Townsend, E.G. Weagel, W. Meng, R.A. Brog, E.J. Valezquez, R.A. Robison, K.L. O’Neill
  Brigham Young University
   
 

As an integral component of the innate immune response, macrophages are one of the most efficient and skilled killers of foreign pathogens and apoptotic bodies. While extremely effective against external invaders, their role in relation to cancer cells is very different. In certain tumors, tumor associated macrophages (TAMs) make up 50% of the tumor mass, and play an essential role in the survival and propagation of cancer cells. Macrophages in the tumor microenvironment largely switch from an M1 classically activated phenotype towards an M2 phenotype. While M1 macrophages are pro-inflammatory and specialized in phagocytosis, M2 macrophages are characterized by their role in tissue repair. Cellular signals released from the tumor cells influence surrounding macrophages to switch to an M2 profile. These M2 macrophages release anti-inflammatory cytokines, which will stimulate tissue repair and trigger angiogenesis. This will provide the tumor with the necessary nutrients during tumor growth. In this project we evaluated the effects of SW620 and HT29 colon cancer cells on the polarization of macrophages. U937 cell lines were stimulated with phorbol 12-myristate 13-acetate (PMA) over the course of 24 hours. Cells were first evaluated for correct morphology and differentiation changes characteristic of macrophages, then incubated with HT29 or SW620 cells at a 1:1 cellular concentration. Meanwhile, differentiated U937 cells were also incubated in 500µL of spent media from HT29 or SW620’s to evaluate signals that may be released from the tumor. Cells were incubated for 48 hours under these experimental conditions. Macrophages were exposed to fluorescent microspheres and incubated for an hour. The U937-derived macrophages were then analyzed through flow cytometry to measure the bead engulfment. We found that there was a 63% decrease in the three bead engulfment and a 47% decrease in the two bead engulfment when exposed to HT29 colon cancer cells. Macrophages exposed to HT29 spent media had reductions of 32% and 18% in the three bead and two bead engulfment population respectively. We discovered that macrophages exposed to HT29 cells were more affected in their bead engulfment than macrophages exposed to SW620 cells. Reductions of 11% in the three bead population and 15% in the two bead population were observed when cells were exposed to SW620 cells. Macrophages incubated in SW620 media decreased by 26% and 35% in the three bead and two bead engulfment populations respectively. These data suggest that the difference in engulfment between the two cancer cell lines may be an indicator of tumor aggressiveness. To further evaluate the polarization of macrophages towards an M2 phenotype in the tumor microenvironment, we evaluated the gene expression of IL-10, IL-1B, and IL-12. While exposed to cancer cells there was a significant increase in the IL-10 production and a decrease in both IL-1B and IL-12 production. This change in gene regulation stimulates the switch from a pro-inflammatory M1 phenotype towards an M2 anti-inflammatory phenotype in the tumor microenvironment. These results provide evidence of a direct relationship between tumor cells and the resulting polarization of macrophages towards a less aggressive, M2 phenotype.

   



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  Is Interleukin-10 a key factor in colon cancer metastasis?
  Presenter: Michelle Townsend
  All Authors:Michelle Townsend, Abi Felsted, Edwin Velazquez, Evita Weagel, Richard Robison, and Kim O’Neill
  Brigham Young University
   
 

Interleukin 10 (IL-10) is a crucial cytokine synthesis inhibitory factor involved in cancer development. As a potent anti-inflammatory molecule, IL-10 is commonly elevated within the tumor microenvironment. IL-10 is primarily produced by helper T-cells along with regulatory T-cells and functions by inhibiting Th1-mediated responses. We examined the abundance of IL-10 expression in patients with colon adenocarcinoma and lymph node metastatic adenocarcinoma from the colon in order to evaluate whether IL-10 expression was increased within metastatic cancers. Tissue from 100 patients ranging in age from 30 to 79 with either colon adenocarcinoma, lymph node metastatic adenocarcinoma (from the colon), adenoma, normal adjacent tissue, and normal colon tissue were obtained and stained for IL-10 expression using standard Immunohistochemistry techniques. Briefly, tissues were treated with a monoclonal anti-IL-10 antibody along with a GADPH positive control and an isotype negative control. Tissues were incubated with an HRP-polymer conjugated antibody, followed by a DAB substrate which reacts with antibody-antigen binding sites to produce a colorimetric change within the tissue. Tissues were further quantified using Image J software and an IHC toolbox plugin in order to measure DAB staining intensity within samples and data is reported in gray value. We found a significant difference between the levels of IL-10 between metastatic tissue and malignant adenocarcinoma. While only 23% of the Adenocarcinoma samples showed a positive level of IL-10 binding, over 55% of the metastatic samples had significant IL-10 expression. These data indicate a significant elevation in IL-10 expression in tumor tissue derived from a metastatic site. In addition, we also found differences in IL-10 presence between patients. While some patient tissue showed a significant up-regulation of IL-10 (p =0.0018), other patients had no apparent up-regulation in comparison to normal controls. This indicates that IL-10 production is variable between patients and may have implications on recurrence and outcome. This study shows that the production of IL-10 from tumor cells is not only variable between patients, but is also increased within metastatic tumor tissue.

   



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  Features of CD8+ T cell tolerance to the self-antigen Trp2
  Presenter: Emily Truckenbrod
  All Authors:Emily Truckenbrod, Kristin Renkema, Stephen Jameson
  Center for Immunology, University of Minnesota
   
 

Immunological tolerance to self is critical for avoiding autoimmune pathology, but the nature and enforcement of this tolerance are incompletely understood. Defining these mechanisms is critical for effectively treating autoimmune disease and for safely targeting tumor-associated self-antigens. We are studying the CD8+ T cell response to the melanocyte/melanoma-associated enzyme tyrosinase-related protein 2 (Trp2) in wild-type (WT) C57BL/6 and Trp2-deficient mice to clarify the characteristics of self-tolerance. Deletion of self-reactive T cells during development is a known tolerance mechanism, but we find a relatively large number of Trp2-specific CD8+ T cells in the lymphoid tissues of both WT and Trp2 knock-out (Trp2 KO) mice. Mixed bone marrow chimeras established in these mice using donor cells from Trp2-specific T cell receptor transgenics confirmed that clonal deletion plays a negligible role. We detected a slightly higher avidity for Trp2 in Trp2 KO CD8+ T cells relative to WT cells as measured by Trp2 tetramer median fluorescence intensity. No consistent differences in the expression of activation (CD69, CD44, CD122) or anergy markers (PD-1, CTLA-4, LAG3, 2B4) were identified between Trp2 KO and WT Trp2-specific CD8+ T cells. Despite these relatively modest differences in the Trp2-specific populations from preimmune WT and Trp2 KO mice, we observed striking differences in the performance of Trp2-specific cells from these mice when challenged with Trp2 in an immunogenic context (recombinant Listeria monocytogenes expressing Trp2 or TriVax immunization with Trp2 peptide, poly(I:C), and agonist anti-CD40 antibody). WT mice were poorly responsive to Trp2, while we were able to elicit a functional response to this antigen in Trp2 KO mice. Trp2 KO mice produced a significantly larger number and proportion of Trp2-specific CD8+ T cells at effector and memory time points. Following Listeria infection, the avidity of responding Trp2 KO cells was significantly higher, and cytokine production in response to ex vivo Trp2 stimulation was greater in cells from Trp2 KO mice. Transfers of bulk polyclonal CD8+ T cells suggest a cell-intrinsic tolerance mechanism, as cells from Trp2 KO donors mount a response to Trp2 in both KO and WT recipients whereas cells from WT mice do not respond in either KO or WT recipients. Ongoing work includes assessment of differences in gene expression between tolerant (WT Trp2-specific cells) and non-tolerant cells to clarify the profile of self-tolerant CD8+ T cells and more precisely delineate the intrinsic mechanism restraining tolerant cells in this model. To date, the majority of our work has focused on the Trp2 (180-188) epitope shared between C57BL/6 mice and humans (HLA-A*0201-restricted). However, we have identified similar populations of CD8+ T cells specific for an alternative Trp2 epitope and an epitope from another melanocyte-associated enzyme, tyrosinase-related protein 1. CD8+ T cells specific for these epitopes also escape thymic deletion and the majority have a naïve, not overly anergic phenotype in preimmune WT mice. Our studies of polyclonal CD8+ T cells responding to the same antigen as self vs. foreign represent a physiologically relevant approach likely to reveal insights generalizable to other melanocyte antigens and applicable to recently described non-deletional CD8+ T cell anergy in humans.

   



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  The role of caspase-8 in regulating dendritic cell activation during both homeostasis and chronic viral infection
  Presenter: Jennifer Tsau
  All Authors:Jennifer Tsau, Stephen Hedrick
  UCSD
   
 

Dendritic cells (DCs) link innate and adaptive immunity by alerting the host to the presence of pathogens. However, DCs must also avoid initiating T cell responses to harmless self-antigens. Caspase-8, required for apoptosis, is also a negative regulator of Rig-I signaling, which is activated upon detection of RNA viruses such as lymphocytic choriomeningitis virus (LCMV). We found that mice with caspase-8-deficient DCs (dcCasp8-/- mice) develop an age-dependent autoimmunity, yet mount an enhanced response to a chronic viral infection. Aged dcCasp8-/- mice develop hyperactivated DCs and T cells, display organ immunopathology, and have CD4 T cells that skew towards a Th1 phenotype when stimulated ex vivo. When we infected young adult dcCasp8-/- mice with the chronic strain of LCMV, we found that these mice had enhanced antigen-specific T cell responses. A month after infection, dcCasp8-/- mice had fewer proportions of exhausted antigen-specific T cells, more LCMV-specific IgG2a antibodies, and lower viral loads. In order to link our observations to a heightened sensitivity of caspase-8-deficient DCs to Rig-I signaling, we transfected DCs lacking caspase-8 with a Rig-I ligand. A larger proportion of CD11b+ Casp8-/- DCs became highly activated upon Rig-I stimulation. Our findings suggest that caspase-8 is important for regulating DC activation, which must be tightly controlled: excessive DC activation aids in viral clearance, but also leads to the development of autoimmunity in the steady state.

   



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  Determining how Toxoplasma strains drive strain-specific CNS immune response
  Presenter: Shraddha Tuladhar
  All Authors:Shraddha Tuladhar, Yarah Ghotmi, Apoorva Bhaskara, Joseph S. Lagas, and Anita Koshy
  Department of Immunobiology, Bio5 Institute, University of Arizona
   
 

Toxoplasma gondii is an obligate intracellular parasite that infects up to 1/3 of the world’s population. Though this persistent infection is asymptomatic in most, in immunocompromised individuals, this infection can give rise to symptoms ranging from fever, focal neuro-inflammation to death. While the determinants of disease variability are poorly understood, recent human data suggest that the genotype of the infecting Toxoplasma strain may influence disease outcomes. Additionally, prior work in the mouse model has shown that different Toxoplasma strains provoke different central nervous system (CNS) immune responses and recent in vivo data have revealed that strain-specific polymorphic effector proteins injected into host cells can lead to different innate immune responses. Thus, we hypothesize that different Toxoplasma strains provoke distinct, strain-specific immune responses, which in turn would affect disease severity. To test this hypothesis, we used quantitative immunohistochemistry to compare CNS immune responses in mice infected with either type II or type III parasites. At 3 weeks post infection (wpi), we found that despite having similar parasite burden, the CNS of type III-infected mice had a more pro-inflammatory CNS immune response (macrophages and T-cell response, and cytokines/chemokines) as compared to type II-infected mice. Consistent with these findings, our flow cytometry analysis of immune cells isolated from the spleen and brain of 3 wpi mice showed that type III-infected mice had a significantly lower numbers of alternatively activated macrophages (AAMs) and regulatory T cells as compared to type II-infected mice. However, flow cytometry analysis on splenocytes during acute infection (0.5 and 1.5 wpi), showed that type III-infected mice had higher numbers of AAMs. Taking these data together, we propose that by inducing a robust pro-inflammatory response early on, type II-infection provokes a compensatory anti-inflammatory response that will ultimately hinder the host from eliminating CNS parasites. Conversely, early in infection type III parasites elicit a less inflammatory response, allowing for a subtle increase in the peripheral parasite burden that then drives a more pro-inflammatory response, resulting in an immune response that will effectively clear CNS parasites. This model predicts that despite having equivalent parasite burdens early in CNS infection, type III-infected mice should have a lower CNS parasite burden chronically. We are currently testing this possibility by evaluating the CNS parasite burden at 16 wpi. We also hypothesize that polymorphic effector proteins drive the differences in the initial immune response. In particular, we hypothesize that ROP16 and GRA15, two effector proteins that differentially modulate major immune signaling pathways in a Toxoplasma strain-specific manner, underlie these immune response differences. Currently we are engineering parasites to test this hypothesis.

   



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  Malaria Induced Inflammation by Xanthine Oxidase
  Presenter: Maureen Ty
  All Authors:Maureen Ty, Anton Goetz, Ana Rodriguez
  NYU School of Medicine
   
 

Malaria blood-stage infection induces high fever and a strong inflammatory response that peaks at the time of rupture of infected erythrocytes. Despite the crucial importance of this inflammatory response in the pathology of malaria, the mechanism of activation by the parasite is not completely understood. An important paradox still unresolved in malaria research, is that in vitro co-cultures of macrophages with infected red blood cells (iRBCs) do not induce strong secretion of inflammatory cytokines. Patients with malaria exhibit elevated levels of oxidative stress markers in the blood, especially of Xanthine Oxidase (XO), an enzyme that converts hypoxanthine to uric acid, releasing reactive oxygen species that cause oxidative stress. When in vitro cultures of macrophages and iRBCs are incubated with XO, a strong secretion of inflammatory cytokines is detected, suggesting that oxidative stress functions as a trigger for the inflammatory activation. The cytokine response observed is comparable to LPS-induced response in magnitude and includes typical inflammatory cytokines that are a signature of malaria: TNF, IL1-beta, IL-10, IL-6 and IL-8. This secretion can be inhibited with the addition of the XO specific inhibitor, Febuxostat. These results suggest that oxidative stress through XO may play a role in the inflammatory response to malaria.

   



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  Distinct Phenotype of Pancreatic Regulatory T cells during Autoimmune Diabetes
  Presenter: Tan Tze Guan
  All Authors:Tan Tze Guan, Author Names separated by commas
  Harvard University
   
 

Regulatory T cells (Treg) are known to play a critical function in restraining inflammation in a variety of autoimmune diseases, including type 1 diabetes. However, the precise phenotype and role of Treg cells in the insulitic lesion during diabetes development remain poorly characterized. We thus sought to describe the phenotype and function of Treg cells in the pancreas in the NOD mouse over the course of insulitis, and found a striking enrichment of T-bet-dependent, CXCR3+ Treg cells in the pancreas relative to the spleen and lymph nodes. Pancreatic CXCR3+ Treg cells are induced by IFN-γ and manifest an activated phenotype, with elevated expression of a panoply of immunoregulatory molecules, and their frequency correlates negatively with the size of the insulitic infiltrate. Thus the Th1 inflammation in the insulitic lesion induces and recruits CXCR3+ Treg cells that may help to control eventual progression to overt disease.

   



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  Determining the optimal TCR:pMHC avidity for CD4+ T cell memory generation
  Presenter: Kiara Vaden
  All Authors:Kiara Vaden, K. Scott Weber
  Brigham Young University
   
 

The development of more effective vaccines requires an understanding of the requirements for strong short and long-term immune responses. Memory cell formation is a hallmark of the adaptive immune system and CD4+ T cells play a central role in the generation of productive recall responses and protective immunity. CD4+ T cells coordinate the adaptive and innate immune responses to infection, but their role as memory cells is not well understood. An unresolved question is what CD4+ TCR:pMHC affinity leads to optimal CD4+ memory cell formation. To address this, we have generated two CD4+ TCR transgenic mouse lines that are specific for the same naturally occurring epitope from Listeria monocytogenes. The TCRs from these mice lines (LLO118 and LLO56) differ by only 15 amino acids and while LLO118 and LLO56 T cells have a similar in vitro response to antigen, their in vivo responses to infection are strikingly different. LLO118 has a stronger primary response whereas LLO56 has a much stronger memory response. Understanding why these TCRs cause such different T cell responses provides a novel system to understand how to improve vaccines and immunotherapies. We have L. monocytogenes mutants with different capacities to stimulate LLO118 and LLO56 T cells to test the role of TCR:pMHC binding affinity in CD4+ primary and memory responses. Our studies are designed to identify the characteristics of CD4+ T cells responsible for differential immune responses and could be highly relevant for improving therapeutic and protective vaccine design and immunotherapies.

   



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  Determining the Optimal TCR:pepMHC Affinity for CD4+ T cell Primary and Memory Response?
  Presenter: Kiara Vaden
  All Authors:Kiara Vaden, John C. Hancock III, K. Scott Weber
  BYU
   
 

The strength of TCR:pepMHC interaction (affinity) helps determine T cell activation and differentiation, which influences the outcome of the immune response to infection. A predominant hypothesis has been that stronger TCR:pepMHC affinity produces better T cell activation and proliferation than weaker interactions. Recent research, however, suggests that low or intermediate affinity may promote strong T cell responses. This elicits the need to determine the affinity conditions necessary for the best CD4+ T cell responses. We have used two T cells from transgenic Rag1 -/- mouse lines, called LLO118 and LLO56, to better understand this question. The TCRs on these CD4+ T cells recognize the same epitope from Listeria monocytogenes (listeriolysin O (LLO190-205), however, they differ by fifteen amino acids and respond differently in vivo. Using three L. monocytogenes APL mutants, we have studied LLO118 and LLO56 T cell response in vitro and in vivo by measuring their proliferation upon exposure to the epitope. We have found that for LLO118, the mutants that act as partial agonists in vitro generate the strongest primary response in vivo. Understanding how TCR:pepMHC affinity affects T cell function is crucial for the development of cancer immunotherapies, vaccines, and understanding immune-mediated diseases such as autoimmunity and graft rejection.

   



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  Determining IL-7/IL-7R function in fetal macrophage development
  Presenter: Clint Valencia
  All Authors:Clint Valencia, Gabriel Leung, Anna Beaudin
  University of California, Merced
   
 

Little is known regarding the mechanisms or factors that play a part in the development of specialized macrophages during fetal development. Our recent work has revealed the interleukin-7 receptor (IL-7R) as an important regulator of fetal macrophage development. Germline deletion of IL-7R or pharmacological blockade during development impairs macrophage development in the liver, lung, and epidermis. We now aim to delineate how IL-7R regulates tissue resident macrophage development together with its cognate cytokine, IL-7. In adult hematopoiesis, IL-7 is a critical cytokine responsible for the proliferation and survival of lymphocytes, including T cells. While the role of IL-7/IL-7R signaling in promoting the lymphoid lineage is well-established, little is known regarding the role of IL-7/IL-7R signaling in myeloid cell development. To gain insight into how IL-7/IL-7R signaling is regulating myeloid development, we investigated IL-7 expression in fetal tissues and the spatial relationship between IL-7R-expressing macrophages and IL-7-expressing cells in distinct fetal tissues. Preliminary analysis suggests that clusters of IL-7R expressing macrophages lie adjacent to cells that express IL-7 within fetal tissues. We also investigated tissue macrophage cellularity in IL-7 KO mice, and found that germline deletion of IL-7 also significantly impaired tissue macrophage development in the brain, liver, and epidermis of neonates. Together with ongoing experiments to define how IL-7 regulates tissue macrophage development, these findings help elucidate developmental mechanisms underlying the establishment of unique populations of tissue-resident macrophages across ontogeny.

   



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  Interferon gamma independent control of Mycobacterium tuberculosis by CD4 T cells
  Presenter: Erik Van Dis
  All Authors:Erik Van Dis, Kimberly M Sogi, Chris S Rae, Kelsey E Sivick, Janet Peace Babirye, Lily H McCann, Sarah M McWhirter, Daniel A Portnoy, and Sarah A Stanley
  Univ. of California, Berkeley
   
 

IFN-γ produced by CD4 T cells is crucial for controlling Mycobacterium tuberculosis (Mtb). However, results from both human and mouse studies suggest that production of IFN-γ is not sufficient for control of infection. Here, we show that a protein subunit vaccine formulated with STING-activating cyclic dinucleotides elicits durable protective immunity to Mtb in the mouse model, and that this protection correlates not with IFN-γ-producing Th1 T cells but with a robust Mtb-specific Th17 response. This vaccine provides protection that is superior and more durable than that of the live attenuated vaccine strain BCG, and full protective efficacy is IL-17 dependent. These results and other published mouse studies suggest that IFN-γ-independent CD4 T cell mechanisms contribute to control of Mtb, but how CD4 T cells exert a protective effect independent of IFN-γ remains unknown. Using an ex vivo co-culture system we show that CD4 T cells isolated from the lungs of Mtb-infected mice activate macrophages to control Mtb replication in an IFN-γ independent manner. Supernatant from cultured lung-derived CD4 T cells is sufficient for IFN-γ-independent control of Mtb. Roles for known CD4 T cell effectors including TNF-α, CD40, CD153 and Type I IFN have been excluded, suggesting that a novel factor produced by CD4 T cells can activate macrophages to control Mtb infection. RNAseq on infected macrophages cultured with IFN-γ deficient CD4 T cells revealed a unique pattern of activation when compared with conventional IFN-γ-dependent activation. These findings further elucidate the role of CD4 T cells during Mtb infection and may help inform vaccine development which has focused primarily on exploiting classic IFN-γ-centric Th1 immunity.

   



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  Steady-state interferon-stimulated gene expression in the intestinal epithelium depends on interferon lambda and the bacterial microbiome
  Presenter: Jacob Van Winkle
  All Authors:Jacob Van Winkle, Stefan Peterson, Michael Wheadon, Sanghyun Lee, Megan Baldridge, and Timothy Nice
  Oregon Health & Science University
   
 

Distinct cellular responses have evolved that are specialized for eliminating pathogens in mucosal surfaces, such as the gastrointestinal tract. In particular, intestinal epithelial cells (IECs) are uniquely responsive to interferon lambda (IFN-λ) and depend on this cytokine for antiviral defense to a greater extent than other types of interferon. IFN-λ signaling protects IECs by inducing expression of antiviral IFN-stimulated genes (ISGs), but it is unclear how this signaling pathway interacts with the intestinal microbiome. We find that depletion of the intestinal microbiome with broad spectrum antibiotics significantly reduces steady-state ISGs in the gastrointestinal tract when assessed by whole-tissue RNA sequencing. Steady-state ISG expression is also significantly reduced in mice with conditional knockout of IFN-λ receptor (IFNLR) in IECs. These data suggest that enteric bacteria stimulate expression of IFN-λ and, subsequently, epithelial ISGs at homeostasis. Strikingly, imaging data reveals distinct pockets of IFNLR-dependent ISGs throughout the small intestinal epithelium. These highly localized ISGs are also dependent on the presence of bacteria and IFNLR expression by IECs. Notably, in addition to localized ISG expression along the length of the gastrointestinal tract, ISG expression is concentrated in the most mature IECs at the tips of individual villi. These novel observations indicate that ISGs are robustly expressed in localized regions of the intestinal epithelium at homeostasis. This robust ISG expression depends on the bacterial microbiome and may preemptively activate antiviral defenses in vulnerable IECs to improve host fitness against enteric viruses.

   



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  Burkitt’s lymphoma cells affects macrophage polarization.
  Presenter: Edwin J. Velazquez
  All Authors:Edwin J. Velazquez, E. G. Weagel, W. Meng, M. H. Townsend, R. E. Brog, R.A. Robison, and K.L. O’Neill
  Brigham Young University
   
 

The immune system plays an important role in the development and progression of cancer. Macrophages are a heterogeneous population of innate myeloid cells and undergo specific differentiation depending on the local tissue environment. Macrophage activation may exhibit two functional states, which represent the extremes of a continuum of activation states. M1, or classically-activated macrophages, promote inflammation, activate a Th1 immune response, and tumoricidal activities. Whereas M2, or alternatively-activated macrophages, regulate tissue repair, promote a Th2 response, and allow tumor progression. Cells exposed to tumor microenvironments including cancer cells, behave differently. By secreting a number of diverse chemoattractants, primary tumors recruit blood circulating monocytes. Once in the tumor site, these monocytes differentiate to Tumor-Associated Macrophages (TAMs). TAMs have shown to display an M2-like phenotype in most cancers and are thought to be able to maintain their M2-like phenotype by receiving polarization signals from malignant cells. The engulfment activity and cytokine profile expression levels of macrophages are good indicators of macrophage polarization and may help us to understand how tumors are able to evade the immune system. Here, we investigated the influence of cancer cells on the behavior of macrophages. We assessed the engulfment capacity of macrophages using an engulfment assay with fluorescent beads in conjunction with flow cytometry. Using Q-PCR we measured the expression levels of key cytokines in macrophages co-cultured with Burkitt’s lymphoma cancer cells. During these experiments, we exposed U937 cells to phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow differentiation. Then we co-cultured these macrophages with Raji cancer cells for 48 hours. Subsequently, we allowed the macrophages to phagocytose fluorescent microbeads for 1 hour and we measured their engulfment using flow cytometry. In parallel, we measured the expression levels of IL-1B, IL-10 and IL-12 in PMA-stimulated U937 cells before and after exposure to a Raji spent media, Raji cells, and normal media. We found a significant decrease of the total engulfment and engulfment capacity of the U937 cells after being exposed to Raji spent media and Raji cells. The U937 cells showed a decrease of almost the half their engulfment capacity. 12% and 19 % decrease of the total engulfment was observed in U937 cells exposed to Raji spent media and Raji cells respectively. We observed a 5 fold increase in the IL-10 expression for those cells exposed to Raji spent media and Raji cells, whereas a down regulation of IL-B and IL-12 was observed for the same treatments. These results show that PMA-stimulated U937 cells undergo macrophage polarization from M1 to M2 phenotype after exposure to cancer cells and their microenvironment through immunomodulatory cytokine signals. This suggests that cancer cells greatly affect the immune system by downregulating macrophage aggressiveness, allowing for further tumor development. Currently, we are performing additional experiments to better understand the complexity of macrophage polarization exposed to various types of cancer microenvironments.

   



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  Role of CXCR5+ CD8+ T cells in Regulating Autoimmunity
  Presenter: Christi Waer
  All Authors:Christi Waer, Kristen Valentine, Genevieve Mullins, Kirk Jensen, Katrina K. Hoyer
  University of California Merced
   
 

CXCR5+ CD8+ T cells are an emerging population of immune cells that arise during chronic disease. Depending on the disease setting the cells have been termed CD8+ T follicular cells or stem-like progenitor memory cells. How CXCR5+ CD8+ T cells interact to promote or protect during autoimmunity and cancer remains largely unknown. Our lab has previously discovered CXCR5+ CD8+ T cells within autoimmune germinal centers that promote B cell class switching and plasma cell differentiation. Other labs have observed an expansion of CXCR5+ CD8+ T cells in pancreatic, colon, and lung cancers. This population has also been seen in models of infection, such as LCMV, SIV and HIV. CXCR5+ CD8+ T cells are known to express inhibitory markers that may be suggestive of exhaustion phenotypes or helper responses, such as Tim3 and PD-1. Here we propose to explore CXCR5+ CD8+ T cell interactions within tumor and autoimmune settings to delineate the functional state and impact on disease outcomes.

   



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  Influence of myelin-specific CD8+ T cells on CD4+ T cell-initiated experimental autoimmune encephalomyelitis.
  Presenter: Catriona Wagner
  All Authors:Catriona Wagner, Joan Goverman
  University of Washington
   
 

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although the pathogenic pathways of MS are not fully understood, it is hypothesized that self-reactive T cells specific for myelin proteins initiate CNS inflammation, demyelination, and axonal damage. Myelin-specific CD4+ T cells have been the main focus of MS research but recent studies have suggested that CD8+ T cells are also involved in MS pathogenesis. We hypothesize that simultaneous recruitment of myelin-specific CD8+ T cells to the site of inflammation initiated by CD4+ T cells could influence the pathology, clinical signs, and severity of CNS autoimmune disease. To test our hypothesis, we use a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) in which adoptive transfer of CD4+ T cells specific for the myelin protein myelin oligodendrocyte glycoprotein (MOG) induces CNS autoimmunity characterized by both brain and spinal cord inflammation. To determine the influence of CD8+ cells, we transfer CD8+ T cells from a TCR-transgenic mouse model expressing a TCR specific for a MHC class I-restricted epitope of myelin basic protein (MBP) into the periphery of mice just after disease is initiated by CD4+ T cells. We found that the recruitment of the MBP-specific but not control CD8+ T cells exacerbated CD4+ T cell-initiated EAE. Intriguingly, preliminary data suggest that recruitment of myelin-specific CD8+ T cells increases the number of activated CD4+ T cells, but not CD8+ T cells, within the CNS. These data suggest that the interplay between CD4+ and CD8+ T cells specific for two different myelin proteins influences the manifestation of CNS autoimmune disease.

   



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  Myelin-Specific CD8+ T cells Exacerbate CNS Autoimmunity and Increase Clinical Signs of Brain Inflammation.
  Presenter: Catriona Wagner
  All Authors:Catriona Wagner, Joan Goverman
  University of Washington
   
 

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). Although the pathogenic pathways of MS are not fully understood, myelin-specific T cells are hypothesized to initiate disease. Myelin-specific CD4+ T cells have been the main focus of MS research and animal models of MS have relied exclusively on the activity of CD4+ T cells. However, substantial data from MS patients indicate that CD8+ T cells also contribute to MS pathogenesis. We hypothesized that recruitment of myelin-specific CD8+ T cells to the site of inflammation initiated by CD4+ T cells will influence disease. To test our hypothesis, we use a mouse model of MS, experimental autoimmune encephalomyelitis (EAE) in which we can investigate the interplay between myelin-specific CD4+ and CD8+ T cells when disease is initiated by adoptive transfer of CD4+ T cells specific for myelin oligodendrocyte glycoprotein (MOG). To determine the influence of CD8+ T cells, we transferred MOG-specific CD4+ T cells into wild-type mice that also received an injection of naïve CD8+ T cells from TCR-transgenic mice that express a TCR specific for a MHC class I-restricted epitope of myelin basic protein (MBP). We found that the recruitment of the MBP-specific but not control CD8+ T cells increased the incidence and severity of symptoms associated with brain inflammation. Increased disease severity was associated with a higher number of activated MOG-specific CD4+ T cells within the brain at peak disease. Furthermore, an increased number of GM-CSF- and TNFa-producing MOG-specific CD4+ T cells were detected in the brain of these mice. MBP-specific CD8+ T cells produced more TNFa in the brain and spinal cord as compared to WT CD8+ T cells, suggesting a potential mechanism by which MBP-specific CD8+ T cells exacerbate disease. These data suggest that the interplay between CD4+ and CD8+ T cells specific for two different myelin proteins is critical for determining the manifestation of CNS autoimmune disease.

   



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  Diet-induced obesity promotes tumor growth but greater responsiveness to PD-1 blockade
  Presenter: Ziming Wang
  All Authors:Ziming Wang, Ziming Wang, Jesus I. Luna, Ethan G. Aguilar, Lam T. Khuat, Arta M. Monjazeb, William J. Murphy
  University of California Davis
   
 

T cell exhaustion has been observed in the tumor microenvironment. Exhaustion involves increased expression of inhibitory receptors, decreased cytokine production and impaired cytolytic activity, which lead to the inability to eliminate cancer. PD-1 is a critical marker of T cell exhaustion. The blockade of the PD-1/PD-L1 axis can augment T cell responses and result in significant responses across a wide spectrum of cancers, which has dramatically altered the landscape of immunotherapy in cancer. Despite the success of checkpoint blockade, a significant percentage of patients still do not respond to therapy. The goal of this study is to determine the impact of obesity on cancer outcome with a focus on T cells and the anti-tumor effects of PD-1 blockade in tumor models. We generated lean and diet-induced obese (DIO) C57BL/6 mice by feeding mice 60% vs 10% fat diets starting at age 6 weeks. 6-month DIO mice had an average body weight of 50 g compared to the 35 g average of lean mice. DIO and lean mice were implanted with 106 B16 cells subcutaneously. The B16 tumors grew significantly faster in the DIO mice compared to the lean mice. There was increased fluorodeoxyglucose (FDG) uptake in the tumors of DIO mice as determined by PET/CT indicating increased metabolic activity. Importantly, a significantly higher proportion of tumor-infiltrating CD8 and CD4 T cells in DIO mice expressed PD-1, which reflects the exhaustion of T cells in DIO tumor bearing mice. This exhaustion phenotype was also demonstrated using RNAseq analysis on sorted CD44hi T cells (purity >95%) from the spleen and lymph nodes. Histological assessment of B16 tumors excised at 21 days post-inoculation showed intact epidermis in the lean mice, while the tumors in DIO mice caused epidermis erosion and necrosis with the tumor invading the subcutaneous fat. The administration of the antagonist monoclonal antibody to PD-1 led to marked reduction of tumor burden only in the DIO mice, not the lean mice, with no toxicity observed. This decrease in tumor burden inversely correlated with tumor infiltrating CD3+ T cells. These results have implications for the efficacy of PD-1 blockade in different metabolic environments.

   



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  Lin28b alters the neonatal CD8+ T cell response to infection by reprogramming cellular metabolism
  Presenter: Jocelyn (Jie) Wang
  All Authors:Jocelyn (Jie) Wang, Erin Wissink, Neva Waston, Norah L. Smith, Andrew Grimson, and Brian D. Rudd
  Cornell University
   
 

Neonates often generate incomplete immunity against intracellular pathogens. As CD8+ T cells are essential for clearing intracellular pathogens, it is crucial to understand why they behave differently than adults during infection. We previously showed that neonatal CD8+ T cells fail to form memory because they rapidly differentiate into short-lived effectors. However, the underlying basis for these age-related differences is unclear. To gain insight into these age-related differences, we first examined gene expression profiles in CD8+ T cells isolated from the thymus and periphery of neonatal and adult mice. Interestingly, we observed distinct transcriptomes in neonatal CD8+ T cells from the time they were initially created in the thymus, indicating that neonatal CD8+ T cells are made differently than their adult counterparts. To examine whether different hematopoietic stem cells (fetal liver vs. adult bone marrow) underlie these age-related differences, we performed intrathymic transfers with fetal and adult progenitors and compared how their progeny responded to infection with listeria monocytogenes. Whereas fetal-derived CD8+ T cells selectively formed short-lived effectors, the adult-derived cells preferentially gave rise to memory precursors. Given that Lin28b has been shown to play an important role in fetal lymphopoiesis and is selectively expressed in neonatal CD8+ T cells, we next asked whether ectopic expression of Lin28b in adult CD8+ T cells enables them to behave more like neonatal CD8+ T cells. Strikingly, adult CD8+ T cells with overexpressed Lin28b showed higher OXPHOS and glycolytic activities upon activation and preferentially formed short-lived effector cells compared to wild-type cells. However, when mTOR activity was limited with rapamycin, the defective formation of Lin28b-induced memory precursor cells could be restored. This data is consistent with the metabolic and phenotypic differences observed between adult and neonatal CD8+ T cells. Collectively, our data suggests that Lin28b regulates the neonatal CD8+ T cell response through the reprogramming of cellular metabolism.

   



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  Localization of iNKT Subsets Determines Their Cytokine Response
  Presenter: Haiguang Wang
  All Authors:Haiguang Wang, You Jeong Lee, Kristin Hogquist
  Center for Immunology, University of Minnesota
   
 

Invariant natural killer T (iNKT) cells are a specialized subset of aß T cells recognizing lipid antigens presented by CD1d molecule. They can be rapidly activated by a-galactosylceramide (a-GalCer) without priming, a-GalCer and analogues of it are being actively tested for clinical use as immunological adjuvants or therapeutics for cancers and autoimmune diseases. iNKT cells modulate immunity through cytokines production at steady state or during infection to influence the development, activation and recruitment of other immune cells. Instead of being an homogenous population, iNKT cells were comprised of three effector subsets, NKT1, NKT2, and NKT17, which produce distinct cytokines at steady state or after stimulation, but little is known about their distribution and localization. Here, we report the systemic distribution and anatomic localization of these subsets and measured their cytokine production at steady state or after stimulation. NKT1 cells were found to be mainly microvasculature associated while NKT2 cells preferentially localized in tissue parenchyma. Furthermore, intravenous injection of a-GalCer robustly activated microvasculature associated NKT1 cells in spleen and liver, but not lymph node (LN) or thymic iNKT cells, leading to a systemic production of interferon-? and IL-4. In contrast, oral administration of a-GalCer solely activated NKT2 cells in the mesenteric LN, resulting in localized IL-4 effect. These findings indicate that the localization of iNKT subsets determines their cytokine response. Given most iNKT cells are resident rather than circulating, these results demonstrate how the steady-state localization of iNKT subsets governs their systemic or localized cytokine effect, providing critical considerations to the design of iNKT based therapeutic applications.

   



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  Bortezomib sensitizes cancer stem cells from solid human tumors to natural killer cell-mediated killing
  Presenter: Ziming Wang
  All Authors:Ziming Wang, Steven K. Grossenbacher, Erik. Ames, Jesus I. Luna, Ian R. Sturgill, Robert J. Canter, William J. Murphy
  University of California Davis
   
 

Cancer stem cells (CSCs) from solid and hematopoietic tumors resist conventional cytotoxic therapies that target rapidly proliferating cells. Thus, residual CSCs can hide within the tumor niche and seed relapse and metastasis. Due to their relapse potential there is an urgent need to identify ways to therapeutically target CSCs. We previously found that cells expressing high amounts of the stem cell associated protein aldehyde dehydrogenase (ALDH) are effectively killed by activated natural killer (NK) lymphocytes. NK cells are known to kill malignant cells though apoptotic processes inherent to the target cell, such as TRAIL-DR5 or Fas-FasL binding, without prior immunization. We and others have also found that the FDA approved proteasome inhibitor, bortezomib, sensitizes tumor cells to NK cell killing by upregulating DR5 and intracellular machinery associated with apoptosis. Based on these, we investigated the effects of bortezomib to promote NK cell killing of ALDHbright CSCs. We evaluated CSCs derived from glioblastoma (GBM), in vitro and in vivo, for the induction of receptors associated with NK cell mediated killing, and for their susceptibility to NK killing after treatment. In this study, bortezomib led to a 3-fold increase in the percentage of viable ALDHbright GBM, in vitro, compared to untreated controls. Moreover, it enhanced the median fluoresence intensity (MFI) of Fas, DR5, and MICA/B in either U87 and or T98G cell lines, on both ALDHbright and ALDHdim cells. Bortezomib had also increased the percentage of viable ALDHbright in patient-derived primary GBM in vitro compared to untreated controls. Additionally, the pretreatment of bortezomib leads to a 98% decrease in viable ALDHbright cells following NK cytotoxicity assays in vitro. In vivo, it improved the efficacy of adoptive NK cell therapy in GBM xenograft models. These data indicate that the combined use of bortezomib with activated natural killer cells could act as a potential anti-CSC strategy to improve outcomes for patients with GBM.

   



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  Thymic epithelial miR-155 promotes regulatory T cell development by safeguarding medullary thymic epithelial cell maturation
  Presenter: Lindsey Warner
  All Authors:Lindsey Warner, Jiayi Dong, Ling-Li Lin, Mei-Chi Chen, Ryan O'Connell, and Li-Fan Lu
  Division of Biological Sciences, University of California, San Diego; Moores Cancer Center, University of California, San Diego; Center for Microbiome Innovation, University of California, San Diego; Department of Immunology, University of Washington; Department of Pathology, University of Utah.
   
 

During thymocyte development, medullary thymic epithelial cells (mTECs) provide an instructive cellular cross-talk important for shaping discrete thymic microenvironments for not only negative selection, but also the generation of regulatory T (Treg) cells. Here, we identify that miR-155, a microRNA whose expression in Tregs is crucial for their development and homeostasis, also contributes to thymic Treg (tTreg) differentiation by promoting mTEC maturation. Mechanistically, we show that RANK signaling induces miR-155 in order to safeguard the thymic medulla via targeting multiple known and previously uncharacterized molecules within the TGFß signaling cascade, a pathway known for its role in restricting mTEC development and differentiation. Our work locates a miR-155-TGFß axis in the thymic medulla to determine mTEC maturity and, consequently, the quantity of tTregs, underscoring that miR-155 ensures proper tTreg cell development in both cell-intrinsic and -extrinsic manners.

   



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  Prostate cancer exosomes affect macrophage polarization and phagocytosis
  Presenter: E. G. Weagel
  All Authors:E. G. Weagel, W. Meng, R. A. Robison, and K. L. O'Neill
  Brigham Young University
   
 

Prostate cancer is the second most common cancer in men in the United States after skin cancer and the fifth most common cancer-related death in the world. This study explores the effects of prostate cancer-derived exosomes on macrophages. Exosomes are small vesicles secreted by cells and found in virtually found in all bodily fluids. Exosomes contain parts of the genome of the cell of origin, some protein, and large amounts of RNA, including miRNAs. Exosome secretion is thought to be upregulated in cancer patients, as it is found abundantly in cancer patients’ secretions (urine, serum, etc.). One of the functions of exosomes is to mediate cellular communication. This happens as they are secreted from one cell and they distribute their contents into another cell. Some exosomes specifically target immune cells. The miRNA the exosomes carry can silence genes involved in the activation of these immune cells, leading to immune suppression and tumor growth. Macrophages are often found surrounding the tumor microenvironment, as they are recruited by tumor signals to promote angiogenesis and metastasis. These macrophages, which are often called tumor associated macrophages (TAMs), are exposed to various signals deriving from the tumor microenvironment, including exosomes. Our lab has previously generated data that suggests that the co-culture of macrophages with PC3 and DU145 cell lines results in a decrease in macrophage phagocytosis and an M2-like polarization state. For this study, we exposed U937 cells to phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow for differentiation. Then, we co-cultured these macrophages with isolated exosomes from spent media from both PC3 and DU145 cell lines at various time intervals (1, 2, 3, 4, 5, 6, 12, and 24 hours). We then allowed the macrophages to phagocytose fluorescent microbeads for 1 hour. Total bead phagocytosis decreases at 3 hours of exosome co-culture compared to control. Interestingly, total bead phagocytosis resumes to normal after 12 hours exosome co-culture. Gene expression analysis of these macrophages suggests an M2 phenotype from hours 2-24. These results suggest that prostate cancer-derived exosomes can target macrophages and affect their phagocytosis. Further research is required to elucidate the specific genes these exosomes target.

   



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  Epigenetic modifications modulate CD4+ T cell responses and autoimmunity
  Presenter: Lindsay Webb
  All Authors:Lindsay Webb, Himanshu Savardekar, Kyle Jablonski, Stephanie A. Amici, Amanda Panfil, Linsen Li, Patrick Green, Chenglong Li, Robert Baiocchi, Mireia Guerau-de-Arellano
  Ohio State University
   
 

In the autoimmune disease Multiple Sclerosis (MS), pro-inflammatory T helper (Th) 1 and Th17 cell responses overwhelm regulatory Th2 and Treg responses. MS treatments that suppress inflammatory T cell responses and promote regulatory T cell responses would be beneficial to the field. Epigenetic modifications are essential to T cell expansion and effector functions. Interestingly, it is known that pan-methylation reaction inhibitors suppress T cell proliferation and experimental autoimmune encephalomyelitis (EAE), but the enzyme responsible for these effects remains to be determined. Protein arginine methyltransferase 5 (PRMT5), the major Type II methyltransferase enzyme, mediates symmetric dimethylation of arginine residues of histones. Although PRMT5 has been shown to be up-regulated in many lymphoid malignancies, currently there is no known role for PRMT5 in T cells. In this study, we use memory Th cells and animal models to study the role of PRMT5 in T cell responses and autoimmunity. PRMT5 was up-regulated in memory Th1 and Th2 cells upon activation and treatment with novel PRMT5-selective inhibitors differentially impacted pathogenic Th1 and benign Th2 cell proliferation. Finally, we show that PRMT5-selective inhibitors ameliorate disease severity in EAE, the mouse model of MS. These results indicate that PRMT5 may be a promising therapeutic target in T cell-mediated autoimmune diseases such as Multiple Sclerosis.

   



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  Novel immunotherapy based on IL-2-poly(HPMA)nanoconjugate
  Presenter: Petra Weberova
  All Authors:Petra Weberova, Jakub Tomala, Vladimir Subr, Jiri Strohalm, Karel Ulbrich, Blanka Rihova and Marek Kovar
  Complex of Biomedical Institutes at Krc Prague, Czech Republic
   
 

IL-2 is important for the proliferation, differentiation, and survival of lymphocytes that display antitumor activity, thus stimulating considerable interest for the use of cytokines in cancer immunotherapy. More than 20 years ago, IL-2 was approved by the FDA for treatment of metastatic renal cell carcinoma and malignant melanoma [1,2]. However, although standard high-dose IL-2 therapy results in a modest clinical response rate of 14% [3], the treatment is limited by substantial toxicities [3,4], vascular leak syndrome (VLS) being the most serious one [5–7]. Alternatively, low-dose IL-2 treatment has shown activity in renal cell cancer (response rates of 18–23%), without the toxicity of high dose IL-2 [8,9]. Yet, IL-2 cancer therapy is of continued interest in part because about one-third of the clinical responses are complete, durable remissions. So far, IL-2 immunotherapy has been shown to be beneficial in a variety of clinical trials [10–14]. For example, studies to identify antigen-nonspecific strategies for enhancing immune reconstitution in individuals with HIV infection include those using IL-2 [15]. In order to increase IL-2 half-life in vivo, we covalently conjugated synthetic semitelechelic polymeric carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) to IL-2. Thus, we synthesized IL-2-poly(HPMA) conjugate containing 2-3 polymer chains per IL-2 molecule in average. Such conjugate exerts much higher activity than IL-2 in vivo as shown by expansion of memory CD8+ T, NK, NKT, ?dT and Treg cells. IL-2-poly(HPMA) shows also much longer half-time in circulation than IL-2 (~4h versus ~ 5min). Collectively, modification of IL-2 with poly(HPMA) chains dramatically improves its potency and pharmacologic features in vivo, which have implications for immunotherapy.

   



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  Novel IL-2-poly(HPMA)nanoconjugate based immunotherapy
  Presenter: Petra Weberova
  All Authors:Petra Weberova, Jakub Tomala, Vladimir Subr, Jiri Strohalm, Karel Ulbrich, Blanka Rihova, Marek Kovar
  Institute of Microbiology ASCR, v.v.i., Videnska 1083, Prague 14220, Czech Republic, Institute of Macromolecular Chemistry ASCR, v.v.i., Heyrovskeho sq. 2, Prague 16202, Czech Republic
   
 

Interleukin-2 (IL-2) possesses a strong stimulatory activity for activated T and NK cells and it is an attractive molecule for immunotherapy. Nevertheless, extremely short half-life and severe toxicities associated with high-dose IL-2 treatment are serious and limiting drawbacks. In order to increase IL-2 half-life in vivo, we covalently conjugated synthetic semitelechelic polymeric carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) to IL-2. Thus, we synthesized IL-2-poly(HPMA) conjugate containing 2-3 polymer chains per IL-2 molecule in average. Such conjugate has lower biologic activity in comparison to IL-2 in vitro. However, it exerts much higher activity than IL-2 in vivo as shown by expansion of memory CD8+ T, NK, NKT, ?dT and Treg cells. Moreover, IL-2-poly(HPMA) extremely effectively potentiates CD8+ T cell peptide-based vaccination. IL-2-poly(HPMA) shows also much longer half-time in circulation than IL-2 (~4h versus ~5min). Collectively, modification of IL-2 with poly(HPMA) chains dramatically improves its potency and pharmacologic features in vivo, which have implications for immunotherapy. To our knowledge, this is the first proof-of-concept report of the use of polymer/protein modification of IL-2 to obtain more pronounced biological activity. Acknowledgement: This work was supported by Czech Science Foundation grant 301/11/0325, grant AS CR Praemium Academiae, by Institutional research concept RVO 61388971 and by by the project

   



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  Quantifying the tissue-resident immune system
  Presenter: Sathi Wijeyesinghe
  All Authors:Sathi Wijeyesinghe, Lalit K. Beura, Pamela C. Rosato, Mark Pierson, David Masopust
  University of Minnesota - Twin Cities
   
 

Parabiosis studies have demonstrated that nonlymphoid tissues (NLTs) are primarily surveyed by tissue-resident memory CD8 T cells (TRM), independently of circulating memory T cells. (TRM) are established in nonlymphoid tissues following infection and mediate protective immunity during pathogen re-exposures. However, the protective utility of TRM is dependent on their longevity within nonlymphoid tissues. To test this critical aspect of TRM biology, we sought to rigorously enumerate TRM at multiple timepoints following a murine infection. Consistent with previous findings, we observed that memory T cells within circulation exhibited no decay over 450 days following infection. In nonlymphoid tissues, we observed tissue-specific changes in the frequency of mucosal TRM over time, which could be approximated by exponential decay models. However, tissue residence is not unique to T cells. Parabiotic surgery of congenically distinct mice can be used to identify any immune population that exists in NLTs independently of replenishment from circulation. We used parabiosis to distinguish tissue-resident CD45+ leukocytes from bloodborne leukocytes that entered nonlymphoid tissues over the duration of 30-day parabiosis. In all nonlymphoid tissues surveyed, over 50% of immune cells were tissue-resident over the course of 30-day parabiosis. Moreover, in specific mucosal tissues including the intestinal tract and female reproductive tract, 70-90% of the immune system was tissue-resident. These studies highlight the discordance in the immune system between vasculature, lymphoid tissues and nonlymphoid tissues. TRM exhibit distinct population dynamics within nonlymphoid tissues, not reflected by peripheral blood sampling, and the majority of the immune system is self-renewing within nonlymphoid tissues.

   



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  Investigating the mechanism for type I interferon repression by the epigenetic reader Sp140
  Presenter: Kristen Witt
  All Authors:Kristen Witt, Daisy Ji
  University of California, Berkeley
   
 

Type I interferon (IFN-I) is an antiviral response that must be tightly regulated to prevent host pathology. How IFN-I is negatively regulated at a transcriptional level is unknown. Our lab has discovered a novel role for IFN-I repression by the epigenetic reader and nuclear body protein Sp140. Sp140 is part of a larger Sp family that play a role in antiviral and antibacterial responses, but the mechanisms of action and function of these proteins is largely unknown. I am investigating the target genes of Sp140 and how Sp140 regulates transcription at these target genes, which domains of Sp140 promote IFN-I repression, and which components of Sp140 nuclear bodies contribute to IFN-I repression.

   



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  B cell receptor affinity may instruct the long-lived plasma cell fate
  Presenter: Rachel Wong
  All Authors:Rachel Wong, Haiyan Zhao, Justin Richner, Michael S. Diamond, Daved H. Fremont, and Deepta Bhattacharya
  Washington University
   
 

Germinal center B cells are selected to enter either the memory B cell or the long-lived plasma cell compartment, yet the underlying mechanisms that control this decision are unknown. Our recent work utilizing a West Nile Virus (WNV) infection system demonstrated that the antigen specificity between the memory B cell and long-lived plasma cell compartments differed. Long-lived plasma cells primarily recognized the dominantly neutralizing epitope, the lateral ridge, of West Nile Virus envelope protein domain III (DIII). In contrast, memory B cells could recognize the lateral ridge epitope and non-lateral ridge epitopes. We hypothesized that non-lateral ridge-specific B cell receptors fail to reach an affinity threshold that promotes the long-lived plasma cell fate. To assess this, we utilized DIII-tetramers that are or are not mutated at the lateral ridge to identify antigen-specific memory B cells and long-lived plasma cells. V(D)J sequences from these cells were cloned into expression vectors for antibody production. The binding patterns of these monoclonal antibodies, at the affinity matured and germline-reverted states, were assessed by ELISA and Bio-Layer Interferometry (BLI). Preliminary data indicates that non-lateral ridge-specific antibodies bind DIII more poorly than lateral ridge-specific antibodies. This difference is more striking when germline forms of these antibodies are compared. Non-lateral ridge-specific antibodies are present during early germinal center reactions when memory B cells are formed, but are progressively lost at later timepoints when long-lived plasma cells emerge. Our preliminary data suggest the existence of an antibody affinity threshold that promotes germinal center B cell retention and long-lived plasma cell selection.

   



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  Monoallelic and monogenic Tcrb assembly enforced by Vb recombination signal sequences and physiological roles for Tcrb repertoire and allelic exclusion
  Presenter: Glendon Wu
  All Authors:Glendon Wu, Author Names separated by commas
  University of Pennsylvania
   
 

Monoallelic expression of antigen receptor (AgR) genes is assumed to be critical for the proper selection and function of T and B lymphocytes. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that most cells assemble and express functional AgR genes from one allele (allelic exclusion). The V gene segment recombination signal sequences (RSSs) of Tcrb and Igh loci are weak and have been proposed to mediate monoallelic V-to-DJ recombination. To test the role of Vb RSSs in promoting Tcrb allelic exclusion, we created and studied mice harboring replacements of the DJCb-distal V2 and -proximal V31 RSSs with the stronger 3’Db1 RSS. Here, we show a substantial role for weak Vb RSSs in limiting: Vb recombination frequency, biallelic functional Vb-to-DJb recombination and biallelic TCRb expression, and dual TCRb chain expression encoded by a single Tcrb allele. Our data indicate that weak Vb RSSs limit Vb recombination to promote monogenic Tcrb assembly in the time window before feedback inhibition halts Vb recombination. Given that AgR allelic exclusion is most stringently applied to Tcrb and Igh, whose assembly and expression drives cellular proliferation, we hypothesized that mechanisms directing monoallelic induction of RAG double strand breaks (DSBs) evolved at Tcrb and Igh loci in part to suppress DSBs from entering S phase and forming oncogenic AgR translocations. We find that p53-/- mice with V2 and V31 RSS replacements succumb more rapidly to thymic lymphomas than their p53-/- counterparts, which is consistent with our model. As Vb RSS replacement mice exhibit dramatic shifts in the Vb repertoire, we also have begun using them to test relationships between Vb repertoire and host defense.

   



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  Dual TCR co-expression promotes immune response and memory formation during viral infection
  Presenter: Letitia Yang
  All Authors:Letitia Yang, Burhan Jama, Huawei Wang, Gerald P Morris
  Primary University of California San Diego
   
 

The normal repertoire of T cells includes a subpopulation co-expressing 2 T cell receptor (TCR) clonotypes. We have shown that dual TCR expression promotes thymic development and homeostatic proliferation, potentially via increased recognition of self-ligands. We hypothesize this could affect T cell repertoires and immune responses, as reactivity against self-antigen influences development and persistence of foreign antigen-specific memory. Despite evidence for the effects of dual TCR cells, they have been understudied due to an inability to definitively identify and test cells expressing 2 TCRs. To address this, we developed a novel mouse model linking eGFP (GFP) or tdTomato (RFP) reporters to the TCR? constant region to generate mice with definitive labeling of single- and dual-TCR cells. Flow cytometry identifies ~16% of peripheral T cells in immunologically naive adult mice as expressing dual TCRs, significantly higher than previous estimates of < 10%. Confocal microscopy demonstrates GFP and RFP signals localized to the cell membrane, indicating functional co-expression. We examined dual receptor T cells during immune responses using a model infection with LCMV-Armstrong. Dual TCR expression did not influence recognition of LCMV antigens in mice pre-infection. However, examination of I-Ab:GP66-77 and H2-Db:GP33-41 reactive cells on d28 post-infection revealed significant increases in LCMV-specific dual TCR cells. Furthermore, dual TCRs cells were specifically and significantly increased among antigen-specific CD8+ central memory cells. These data identify a novel potential role for dual TCR-expressing cells in protective immune responses and formation of immune memory.

   



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  Understanding how Helicobacter pylori Uses Chemoreceptor TlpB to Modulate Host Inflammation
  Presenter: Christina Yang
  All Authors:Christina Yang, Kevin Johnson, Karen Ottemann
  UCSC
   
 

Helicobacter pylori is a chronic gastric pathogen that infects roughly half the world’s population. A subset of those infected develop chronic and unresolved inflammation, which can promote the development of gastric ulcers and cancer. H. pylori can colonize the gastric glands, which are invaginations of the stomach epithelium, and it is proposed that these glands are a reservoir that allow the bacterial populations to persist. Factors that regulate gland colonization are poorly understood, but we have observed that bacteria use chemotaxis to colonize the glands. Previously, our lab has reported that H. pylori utilizes chemotaxis to promote gland colonization in the corpus and antrum regions of the stomach. We report here that TlpB is required to regulate gland colonization in the stomach. We performed an 8-month time course examining the gland colonization phenotype of WT and tlpB mutants in the corpus and antrum. We observed that tlpB mutants do not have gross defects colonizing the antrum and corpus regions of the stomach. Interestingly, tlpB mutants demonstrate advantages in gland colonization characterized by an increase in the number of bacteria per gland but not in the percentage of glands occupied in the corpus and antrum when analyzed by Bacterial Localization in Isolated Glands (BLIG), a method developed in our lab using fluorescently labeled bacteria. This increase in gland colonization occurs during a heightened state of inflammation when measured by histology. These data suggest that H. pylori uses chemotaxis to restrict the number of bacteria in the glands to limit recognition by the immune system to maintain chronic colonization.

   



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  Enhancer landscapes revealing CD8+ T cell fate decision during bacterial infection
  Presenter: Bingfei Yu
  All Authors:Bingfei Yu, Ananda Goldrath
  University of California, San Diego
   
 

In response to acute infections, CD8+ T cells undergo expansion and differentiation to effector and memory precursor populations, mediating the clearance of pathogens and long-term protection against re-infection. CD8+ T cell fate commitment occurs at effector stage and cell surface markers KLRG1 and CD127 are used to distinguish two CD8+ effector T cell subsets with distinct memory potential: terminally differentiated-effector (KLRG1hiCD127lo) and memory precursor-effector (KLRG1loCD127hi) CD8+ T cells. Transcriptional regulation of CD8+ T cell fate decision has been extensively studied. However, the epigenetic mechanism underlying this cell fate decision is largely unknown. Here, we first generate genome-wide chromatin state maps revealing putative enhancers of terminally differentiated- and memory precursor-effector CD8+ T cell subsets during Listeria monocytogenes infection. We have identified and classified enhancers into active, intermediate and inactive enhancers. Functional annotation of enhancers of both subsets suggest that apoptosis-related genes are required to be activated and repressed in terminally differentiated- and memory precursor-effector CD8+ T cells respectively. Further analysis of de novo motifs enriched in enhancers recovers potential transcription factors that might regulate CD8+ T cells differentiation by binding to enhancers.

   



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  Radiation Therapy Permits CD8+ T Cell-Mediated Control of Tumors through Tumor Cell Phenotype Alterations
  Presenter: Lauren Zebertavage
  All Authors:Lauren Zebertavage, Alejandro Alice, Marka Crittenden, Michael Gough
  Oregon Health and Science University; Earle A Chiles Research Institute
   
 

Background: Radiation therapy is traditionally used to induce catastrophic DNA damage leading to the death of rapidly-proliferating cancer cells and reduction of local tumor burden. While this basic premise remains true, it is now recognized that radiotherapy (RT) can trigger potent and effective anti-tumor immunity, primarily mediated by CD8+ T cells (CD8s), although the mechanisms by which this happens are unclear. Previously, we demonstrated that RT required pre-existing CD8 immunity to tumor antigens to drive clearance, and only moderately increased tumor reactive CD8s in circulation, indicating that RT functions poorly as an in situ vaccination. Interestingly, we observed that cytotoxic T cells were unable to control tumor cell growth in vitro unless the targets had been pretreated with RT or IFNγ. Hence, instead of directly altering T cell effector function, we questioned whether RT might render tumor cells more susceptible to CD8-mediated cytotoxicity via direct changes in tumor cells. Hypothesis: We hypothesized that RT altered tumor cell phenotypes to increase susceptibility to CD8-mediated tumor regression in vivo. Methods: For these studies, we used a murine model of pancreatic adenocarcinoma expressing the model antigen SIYRYYGL (SIY) and purified for high expression of the antigen (Panc02SIY100). Treatment of Panc02SIY100 with single-dose radiation (12 Gy) with aPD-L1 blockade results in tumor regression and clearance greater than 50% of mice. CD8+ T cells used for adoptive cell transfer (ACT) and in vitro assays were derived from 2C (SIY-specific) or OT-I (SIINFEKL-specific) transgenic TCR splenocytes activated in vitro with αCD3, αCD28 and IL-2 and purified with magnetic beads prior to administration. Irradiation was performed in vitro by timed exposure to radioactive Cs-137 and in vivo using the Xstrahl Small Animal Radiation Research Platform (SARRP). To generate MHC-I(hi) tumor cell lines, cells were transfected with a plasmid harboring the MHC Class I TransActivator (CITA, or NLRC5) under the constitutive EF-1α promoter and purified for cell-surface MHC-I by FACS and clonal expansion. Results: Using a Rag1-/- dual flank tumor model with adoptive transfer of CD8+ T cells, we found that RT-mediated tumor suppression was dependent on the presence of SIY-specific CD8s (d29 vs OT-I, p=0.023), while these cells had no effect on the size of unirradiated tumors in the same mouse (d29 vs OT-I, p=0.1938). We compared 2C vs OT-I infiltration into irradiated vs untreated tumors and found that RT did not draw more SIY-specific CD8s into the tumor (d7, per mg tumor, p=0.0637) and did not retain more 2C vs OT-I CD8s intratumorally (p=0.5435). We demonstrated in vitro that IFNγ (p=0.0023) and RT (10 Gy, p=0.0005, 20 Gy, p<0.0001) increased cell-surface expression of MHC-I by flow cytometry. Significantly, we observed that Panc02SIY100 was poorly controlled by 2C cells in vitro (p=0.6057) unless pretreated by IFNγ (p=0.0426) or constitutively expressing high levels of MHC-I (p=0.0074). Conclusion: Taken together, these data support the conclusion that, in this model, tumor cell phenotype modifications driven by RT are essential to CD8-mediated tumor regression and support MHC-I upregulation as a potential mechanism driving this improved anti-tumor response by pre-existing tumor-specific T cells.

   



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  Radiation Therapy as an Endogenous Vaccine
  Presenter: Lauren Zebertavage
  All Authors:Lauren Zebertavage, Alejandro Alice, Marka Crittenden, Michael Gough
  Oregon Health and Science University
   
 

Radiotherapy (RT) is one of the three main arms of traditional cancer therapy, but its role as an immunotherapeutic agent has not been fully characterized. In our murine model of pancreatic adenocarcinoma expressing model antigen SIY (Panc02-SIY), we have observed CD8-dependent clearance of tumors given single, high-dose radiation (20Gy) and anti-PD1 checkpoint blockade. Notably, we have observed inefficacy of radiation + checkpoint blockade to induce tumor clearance when T cell priming is blocked during tumor challenge (by αCD40L antibody, FTY720 or CD8 depletion). We therefore hypothesized that the role of RT was to boost existing CD8 T cell responses against tumor antigens and drive an influx of tumor- reactive cytotoxic CD8 T cells into the microenvironment, as opposed to priming new responses against tumor antigens. We found that boosting tumor-reactive T cell numbers in the tumor with a live-attenuated Listeria monocytogenes (Lm) vaccine against a major tumor antigen (SIYRYYGL, or SIY) was not sufficient to replicate the efficacy of RT when combined with checkpoint blockade. Mice treated with LmSIY and αPD1 showed no tumor growth control advantage when compared to controls, despite having up to two-thirds of CD8 T cells in circulation responding to the tumor antigen. Immunohistochemistry of embedded Panc02-SIY tumors indicate that Lm vaccination does drive T cell infiltration into the tumor, and in vitro studies show that LmSIY-induced T cells are capable of responding and secreting cytokines in response to Panc02-SIY. Additionally, in vivo specific lysis experiments show Lm-peptide vaccines are capable of inducing CD8 T cell responses capable of rapidly killing peptide-pulsed cell targets in circulation, indicating that failure of this therapy is tumor-specific. Significantly, in vitro T cell killing assays indicate that SIY-expressing Panc02 requires pretreatment (i.e. with IFNγ) in order for Listeria-SIY-primed CD8s to effectively kill. Our results indicate that the efficacy of RT in the context of checkpoint blockade cannot be replicated by boosting the number of tumor-reactive CD8 T cells within the tumor, and that the role of radiation in effective CD8-mediated therapy of immunogenic tumors may be modification of the tumor cells themselves to increase susceptibility to killing by pre-existing tumor-specific T cells.

   



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  Mechanism of lymph node barrier immunity
  Presenter: Yang Zhang
  All Authors:Yang Zhang, Yang Zhang, Elizabeth Gray, Jinping An, Jason Cyster
  University of California, San Francisco
   
 

Our ability to mount adaptive immune responses against skin-invading pathogens depends on the delivery of antigens to lymph nodes (LNs) for encounter by naïve lymphocytes. However, activation, clonal expansion and effector lymphocyte differentiation takes several days whereas pathogens can undergo marked replication in a matter of hours. The skin contains immune effector cells that help keep pathogen replication in check, in a process referred to as ‘barrier immunity’. Despite this, in many cases, intact pathogens travel within minutes via lymph fluid to draining LNs. Recently there has been evidence indicating the existence of barrier immunity within lymph nodes. Upon infection, the first LN cells exposed to lymph-borne antigens are the CD169+ sinus-associated macrophages. Crosstalk between sinus-assocaited macrophages and IFNg precommited CD8 T cells and NK cells are critical for mounting rapid Th1 like response against acute infection by Pseudomonas aeroguinosa and Toxo gondii. Recently, the Cyster lab and others identified populations of innate (pre-formed effector) lymphocytes that are closely associated with LN CD169+ macrophages and that rapidly produce cytokines (IL17) upon stimulation. In this study, we found those innate lymphocytes are responsible for rapid IL17 production in lymph nodes upon acute bacteria (Corynebacterium Accolens, Staphylococcus Aureus) and fungus (Candida Albicans) infection. In order to find guidance cues for innate lymphocytes positioning, we found CCL20 contributes to position innate lymphocytes in close proximity with CD169+ macrophages. S1P contributes to promote lymphocytes to access subcapsular and lymphatic sinus. LFA1 helps innate lymphocytes get into lymph node parenchyma from lymphatic sinus. We also found evidence for a novel mechanism employed by innate lymphocytes to resist the shear forces of lymph flow while interacting with sinus macrophages.