Total Submitted Abstracts: 209


Submitted for: Postdoc Presentation Okay to Post to Web: Yes

 

  EFFECT OF DIRECT MYOFASCIAL RELEASE AND KINESIO TAPE ON AXILLARY WEB SYNDROME
  Presenter: Rehan Abdelaziz
  All Authors:Rehan Abdelaziz, Rehan Gamal Abd El Nasser Ibrahim,Mohamed Mahmoud; Abd El Khalek Khalaf,Yasser Moustafa Elkermand Mohamed M. El Safwany
  pharos university in alexanderia
   
 

Enter your abstract's text hBackground and Objective: Axillary web syndrome is a complication that can arise in patients following axillary dissection. It is considered as taut, stretched band underneath the skin, functional deficits and pain may be associated with AWS development so the aim of this study was to detect the effect of direct myofacial release, kinesio tape & combination between them on axillary web syndrome. Materials and Methods: Sixty female patients after 3 months post axillary dissection, their age between 40 to 50 years old, were defined as having AWS (palpable or visible) cord in the axilla. The study was conducted from November 2015 to April 2017 .The subjects recruited and underwent physical exam at oncology clinic in Medical Research Institute, Alexandria University , Alexandria ,Egypt and the patients received the physical therapy in outpatient clinic of physical therapy faculty, Pharos University, Alexandria ,Egypt. The patients were randomly divided into three groups (A, B, C). Each group consisted of 20 patients. Group (A) received a direct myofascial release and kinesio tape, group (B) received direct myofascial release, group (C) received kinesio tape, all three groups received their treatment for 4 weeks / 2 sessions per week, all patients were assessed pre and post treatment for these variables: VAS for the pain and ultrasound for assessed (the thickness of the cord and cord disorganization). Results: Statistical analysis revealed that there was a significant improvement in each groups in VAS scores, thickness of the cord and cord disorganization for all groups pre and post values but group (A) which was combination of kinesio tape and myofascial release had a superior effect on disorganization, compared to the other groups. There was no significant improvement in the VAS scores and thickness of the cord between groups except the cord disorganization variable which is significant (P<0.05). Conclusion: group (A) had a superior effect in axillary web syndrome treatment when compared to the other groups. Key words: direct myofascial release, kinesio tape, breast cancer, axillary cording and axillary web syndrome. ere.

   



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  Alpha(v) integrins engage autophagy components to regulate immune receptor trafficking and signaling
  Presenter: Mridu Acharya
  All Authors:Mridu Acharya, Anna Sokolovska, Jenny Tam, Caroline Stefani, Fiona Raso, Kara Conway, Raminik Xavier, Jatin Vyas, Lynda M. Stuart, Richard O. Hynes and Adam Lacy-Hulbert
  Benaroya Research Institute
   
 

Integrin-ligand interactions trigger an array of intracellular signaling events and cytoskeletal rearrangements, which include the rapid endocytosis and exocytosis of integrins and other membrane proteins. There is increasing appreciation that these intracellular trafficking events do more than just recycle and deliver integrins to sites of adhesion, but instead play a critical role in signaling. We have uncovered a novel role for alpha(v) integrins in regulating immune cell signaling by modulating trafficking of immune receptors. Specifically, we have found that alpha(v)beta3 integrin regulates Toll-like receptor (TLR) signaling by directing trafficking and maturation of TLR containing endosomes, through a mechanism involving src-family kinases and components of the autophagy pathway. We have defined this mechanism using a combination of biochemical and microscopic techniques in B lymphocytes. In B lymphocytes stimulated with TLR ligands, alpha(v) integrins direct endosomal trafficking of the TLRs from NF?B signaling endosomes to IRF7 signaling endosomes where the TLRs associate with autophagy components LC3 and this leads to down-regulation of TLR signaling. This regulation of TLR trafficking is initiated upon alpha(v) integrin internalization, which results in activation of src and syk kinases and leads to LC3 recruitment to these TLR containing endosomes. When B cells lack either alpha(v) integrins or autophagy components LC3 and atg5 this transition through signaling compartments is disrupted and leads to enhanced TLR signaling and cell proliferation. We have also used av conditional knockout mice to determine the consequences of loss of alpha(v)beta3-mediated immune regulation. Mice lacking B cell alpha(v) show increased antibody responses to antigens with TLR-ligands and develop increased levels of autoantibodies. We therefore propose that alpha(v)-mediated TLR regulation serves to maintain the balance between protective immunity to microbes and potential pathological autoimmune responses. We are currently studying the role of alpha(v) signaling and autophagy components in development of class switched, high affinity antibodies in response to antigens in the context of infection with a pathogen or in response to self-antigens.

   



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  HPV16 immunity induced by immune responses to mutations in E6 and E7 proteins
  Presenter: Maria Agarwal
  All Authors:Maria Agarwal, Ashley Saint-Fleur, Jie Fu, Hyam Levitsky,Cornelia L. Trimble
  Johns Hopkins University
   
 

Human papillomavirus (HPV) causes 30% of cancers attributable to infectious pathogens. HPV16 causes most cervical, anal, vaginal, vulvar, and oropharyngeal cancers. Although preventive vaccines targeting HPV16 and 18 are available, rates of vaccination are uneven. The incidence of HPV-associated cancers for which no screening algorithms have been validated continues to increase. Development of squamous cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN) 2/3 is associated with integration of HPV genome into the host genome, and expression of the HPV E6 and E7- gene products, which inactivate p53 and pRb respectively. Since E6 and E7 are constitutively expressed in cervical carcinomas and CIN, they present attractive immunotherapeutic targets. Development of immunotherapies has been limited, in part, by modest immunogenicity of vaccines tested to date. The purpose of this study is to augment the immunogenicity of the E6 and E7 antigens through mutational screening. A subset of CIN2/3 do undergo complete regression. Our lab has carried out a series of prospective clinical studies, including immunotherapeutic interventions, in subjects with CIN2/3. Peripheral blood T cell responses to E6 and E7 are marginal, and do not distinguish regressors in either vaccinated or unvaccinated subjects. However, in vaccinated subjects, the lesion microenvironment is comprised of new tertiary lymphoid structures, evidence of T cell proliferation elicited by cognate antigen, and clonally expanded TCRs. Tissue T cells in vaccinated subjects access dysplastic epithelium, in contrast to T cells in persistent lesions, which were confined to the stromal compartment. These findings suggest that T cells that could mediate regression can be elicited by vaccination. However, strategies to enhance immunogenicity would likely increase therapeutic effect. Here we present an approach designed to determine the effect of mutations in E6 and E7 on antigenicity. Mutations can enhance immune responses to antigens by several mechanisms, including altering antigen processing, trafficking, glycosylation, and affinity to major histocompatibility complexes. Using error prone PCR, we generated libraries containing random mutations in the coding sequence of E6 and E7. Clones from each library are currently being screened in vitro to identify immunogenic mutants, which could be potentially used for multi-epitope vaccines.

   



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  Dysregulated TLR7 signaling drives anemia via development of unique hemophagocytes
  Presenter: Holly Akilesh
  All Authors:Holly Akilesh, Matthew Buechler, Jeffrey Duggan, William Hahn, Xizhang Sun, Griffin Gessay, Elizabeth Whalen, Michael Mason, Keith Elkon, Adam Lacy-Hulbert, Marion Pepper, and Jessica Hamerman
  Benaroya Research Institute
   
 

Cytopenias are an important clinical problem associated with acute inflammatory disease and infection. We show that specialized phagocytes that internalize red blood cells develop in TLR7-driven inflammation and during mouse malaria infection, both associated with severe anemia. TLR7 signaling caused development of inflammatory hemophagocytic cells (iHPC) that resemble splenic red pulp macrophages (RPM), but are a distinct population derived from Ly6Chi monocytes independently of the RPM lineage-defining transcription factor Spi-C. These iHPC were responsible for anemia and thrombocytopenia in TLR7-overexpressing mice, which have a severe Macrophage Activation Syndrome (MAS)-like disease. Our findings uncover a novel mechanism by which TLR signaling specifies monocyte fate during inflammation, and identify a new population of phagocytes responsible for anemia associated with inflammation and infection.

   



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  Chronic TLR7 and TLR9 signaling drives anemia via differentiation of unique hemophagocytes
  Presenter: Holly Akilesh
  All Authors:Holly Akilesh, Akilesh HM, Buechler MB, Duggan JM, Hahn WO, Matta B, Sun X, Gessay G, Whalen E, Mason M, Presnell SR, Elkon KB, Lacy-Hulbert A, Barnes BJ, Pepper M, and Hamerman JA
  Benaroya Research Institute at Virginia Mason
   
 

Cytopenias are an important clinical problem associated with inflammatory disease and infection. We show that specialized phagocytes that internalize red blood cells develop in TLR7-driven inflammation. TLR7 signaling caused development of inflammatory hemophagocytes (iHPC) that resemble splenic red pulp macrophages, but are a distinct population derived from Ly6Chi monocytes. iHPC were responsible for anemia and thrombocytopenia in TLR7-overexpressing mice, which have a macrophage activation syndrome (MAS)-like disease. IRF5, associated with MAS in human disease, participated in TLR7-driven iHPC differentiation. We also found iHPC during experimental malarial anemia, where they required endosomal TLR and MyD88 signaling for differentiation. Our findings uncover a mechanism by which TLR7 and TLR9 specify monocyte fate, and identify a unique population of phagocytes responsible for anemia and thrombocytopenia associated with inflammation and infection.

   



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  Toll-like receptor 9 signaling antagonizes the B cell receptor-dependent ability of B cells to process and present antigen to helper T cells.
  Presenter: Munir Akkaya
  All Authors:Munir Akkaya, Billur Akkaya, Ann S Kim, Pietro Miozzo, Haewon Sohn, Mirna Pena, Susan K. Pierce
  NIH/NIAID
   
 

B cells express both an adaptive antigen-specific B cell receptor (BCR) and innate Toll-like receptors (TLRs) allowing the functional outcome of the B cell’s engagement with antigen to be modulated in response to pathogen-derived TLR ligands. In T cell-dependent antibody (Ab) responses, the BCR both signals for B cell proliferation and differentiation and also internalizes bound antigen for processing and presentation to helper T cells (TH cells). Key events in T cell-dependent Ab responses in vivo are dependent on B cell presentation to TH cells. Ab responses are initiated in secondary lymphoid organs by the stable interaction of antigen-primed TH cells with activated antigen-specific B cells through MHC-class II peptide complexes presented on the B cell surface. Dependent, in part, on the quality of the B cell- TH cells interaction, B cells either enter germinal centers (GCs) or differentiate into either short-lived plasma cells (PCs) or GC-independent memory B cells (MBCs). Within the GC, B cells proliferate, somatically hypermutate and subsequently undergo antigen-dependent affinity selection. Selection is dependent on the ability of B cells to capture, process and present antigen to TH cells an event that ultimately results in the differentiation of GC B cells to long-lived MBCs and PCs. Here we show that although the TLR9 ligand, CpG, does not affect early antigen-driven BCR signaling, CpG alters the outcome of BCR signaling, resulting in a unique transcriptional profile, enhanced proliferation and differentiation to PCs. Remarkably, CpG dramatically limits the ability of B cells to process and present antigen to TH cells. In the presence of CpG, BCR-induced upregulation of the expression of CD86 and MHC class II is antagonized and antigen internalized by the BCR is not properly trafficked to antigen processing compartments resulting in reduced numbers of peptide-MHC class II complexes on the B cell surface. Indeed, CpG treated antigen-specific B cells show a reduced ability to maintain contact with antigen-specific T cells and to activate antigen-specific helper T cell proliferation in vitro. These results indicate that TLR9 activation of B cells in T cell-dependent Ab responses would drive B cell toward proliferation and antibody secretion and away from events that are highly dependent on the ability of B cells to present antigen to helper T cells and produce long-lived MBCs and PCs.

   



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  Antigen-specific T regulatory cells (Tregs) inhibit dendritic cell (DC) function by capturing peptide-MHCII complexes from the DC surface
  Presenter: Billur Akkaya
  All Authors:Billur Akkaya, Munir Akkaya, Amanda H. Holstein, Olena Kamenyeva, Juraj Kabat, Ethan M. Shevach
  NIAID
   
 

Tregs are a small fraction of T cells that express the transcription factor Foxp3 and are critical mediators of peripheral immune tolerance. There have been several mechanisms described for Treg mediated suppression of T effector cell (Teff) responses; however, the exact mode of inhibition is largely unclear. A majority of the mechanisms utilized by antigen-specific Tregs to suppress immune responses, suggest an initial inhibition of dendritic cell (DC) function that results in diminished activation of T effector cells (Teff) specific for the homologous target as well as diminished activation of other antigen-specific Teff cells that recognize different antigens presented by the same DC (bystander suppression). In an adoptive transfer model in vivo, we demonstrated that OTII transgenic iTregs specific for Ovalbumin323-339 markedly inhibited the expansion of OTII Teff, but did not inhibit the expansion of co-transferred SMARTA Teff (specific for LCMV NP61-76) even though the transferred DCs displayed both peptides. Analysis of iTreg-DC co-cultures in vitro using flow cytometry and confocal microscopy demonstrated that specific peptide-MHCII complexes were captured and internalized by iTregs and nTregs leaving DCs with decreased levels of antigen. Polyclonal iTregs, naïve, and activated antigen-specific Teff did not capture peptide-MHCII complexes to the same extent with antigen specific iTregs. Intravital microscopy of the popliteal lymph node showed that adoptively transferred OTII iTregs made larger clusters around the DC for a longer period of time compared to activated Teff which resulted in a decreased synapse formation between the DCs and co-transferred naïve OTII Teff cells.Taken together this study suggests that antigen-specific Tregs inhibit immune responses locally in an antigen-dependent fashion by forming firm interactions with DCs leading to a stripping of peptide-MHCII complexes from the DC surface by a transendocytosis-based mechanism.

   



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  Engineering adoptive T cell therapy for efficacy in ovarian cancer
  Presenter: Kristin Anderson
  All Authors:Kristin Anderson, Breanna M. Bates, Edison Y. Chiu, Christopher B. Morse, Nicolas Garcia, Philip D. Greenberg
  Fred Hutchingson Cancer Research Center and University of Washington
   
 

Over 20,000 women are diagnosed with ovarian cancer annually, and more than 50% will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for innovative therapies. One promising new strategy has the potential to control tumor growth without toxicity to healthy tissues, by employing immune T cells engineered to target proteins uniquely overexpressed in tumors. Recently, mesothelin (MSLN) has been identified as a valid antigen target in ovarian cancer; MSLN contributes to malignant and invasive phenotypes and has limited expression in healthy cells. In preclinical studies using patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express a high-affinity MSLN-specific T cell receptor (TCR) can kill ovarian tumor cells in vitro. In vivo, adoptively transferred TCR-engineered T cells preferentially accumulate within disseminated ID8 tumors, delay tumor growth and prolong mouse survival, but our data also show the tumor microenvironment (TME) can limit engineered T cell persistence and function. Immunosuppressive cells, inhibitory ligands that reduce T cell function, and cell death-inducing ligands are abundant within ID8 tumors. Further, the ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Cellular and molecular analyses of human ovarian cancer specimens showed similar TME-mediated obstacles exist for human T cell therapy. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.

   



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  Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in solid tumors
  Presenter: Kristin Anderson
  All Authors:Kristin Anderson, Kristin G. Anderson*, Shannon K. Oda*, Breanna M. Bates, Madison G. Burnett, Edison Y. Chiu, Magdalia L. Suarez Gutierrez, Nicolas Garcia, Andrew W. Daman, Philip D. Greenberg
  Fred Hutchinson Cancer Research Center and the University of Washington
   
 

Background: Over 20,000 women are diagnosed with ovarian cancer annually, and over half will die within 5 years. This rate has changed little in the last 20 years, highlighting the need for therapy innovation. One especially promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors, with the potential to limit tumor growth without toxicity to healthy tissues. Mesothelin (Msln) is a rational target for ovarian cancer immunotherapy - it contributes to the malignant and invasive phenotype in these tumors and has limited expression in healthy cells. Methods: Deep transcriptome profiling of whole tumor tissue was used to confirm the expression of similar gene signatures in human cancers and in the preclinical ID8 mouse model, including comparable expression of immunosuppressive pathways. For example, RNA sequencing, flow cytometry and immunohistochemistry analysis revealed consistently high expression of the immunomodulatory protein Fas ligand (FasL). Human/mouse T cells were engineered to express a human/mouse Msln-specific high-affinity T cell receptor (TCRMsln) and tested for cytotoxic activity against human patient-derived or ID8 mouse ovarian cancer cell lines in vitro and in vivo. Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth, and significantly prolonged mouse survival. However, our data also revealed that elements in the tumor microenvironment (TME) limit engineered T cell persistence and anti-cancer activity. We and others previously detected FasL in the tumor vasculature and TME of human and murine ovarian cancers. FasL can induce apoptosis in infiltrating lymphocytes expressing Fas receptor (Fas). To overcome this potential T cell evasion mechanism, we generated a panel of immunomodulatory fusion proteins (IFP) containing the Fas extracellular binding domain fused to a CD28 or 4-1BB co-stimulatory domain, rather than the natural death domain. Relative to T cells modified with only TCRMsln, T cells engineered to express both TCRMsln and a Fas IFP preferentially infiltrate tumors, expand/persist and retain function in the TME of tumor-bearing mice. Moreover, adoptive immunotherapy with IFP+ T cells significantly prolonged survival in tumor-bearing mice, relative to TCRMsln T cells lacking an IFP. Conclusions: Fas/FasL signaling can mediate T cell death, including activation-induced cell death, an apoptotic mechanism responsible for regulating T cell expansion. Thus, tumor cells may upregulate FasL for protection from tumor-infiltrating lymphocytes. As many solid tumors overexpress FasL, IFPs may provide an opportunity to enhance engineered adoptive T cell therapy against many malignancies.

   



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  Failure to suppress CYLD-mediated cell death causes immune dysregulation.
  Presenter: Rosalind Ang
  All Authors:Rosalind Ang, Author Names separated by commas
  The Precision Immunology Institute at the Icahn School of Medicine at Mount Sinai
   
 

SHARPIN together with RNF31/HOIP and RBCK1/HOIL-1 form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked poly-ubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL-1 in humans and Sharpin in mice lead to auto-inflammation and immunodeficiency but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpin-/- mice is due to the failure to inactivate CYLD, the deubiquitinase that removes K63-linked poly-ubiquitin chains. The dermatitis, disrupted splenic architecture, and loss of Peyer's patches in the Sharpin-/- mice were fully reversed in Sharpin-/-Cyld-/- mice. In Sharpin-/- cells, there is impaired phosphorylation of CYLD, which physiologically inhibits its deubiquitinating activity. Without this suppressive modification, CYLD removes poly-ubiquitin from RIPK1 in response to TNF, favoring the association of RIPK1 with death-signaling molecules to initiate death in Sharpin-/- cells, which was reversed in Sharpin-/-Cyld-/- cells. The dermatitis in the Sharpin-/- mice was also ameliorated by the conditional deletion of Cyld using LysMcre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological condition, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent phosphorylation and inactivation of CYLD. The Sharpin-/- phenotype illustrates the pathological consequences when CYLD inhibition fails.

   



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  Eosinophils suppress Th1 responses and restrict bacterially induced gastrointestinal inflammation
  Presenter: Isabelle C. Arnold
  All Authors:Isabelle C. Arnold, Isabelle C. Arnold, Mariela Artola-Boran, Paulino Tallón de Lara, Andreas Kyburz, Christian Taube, Karen Ottemann, Maries van den Broek, Anne Müller
  University of Zurich
   
 

Eosinophils are predominantly known for their contribution to allergy. Here, we have examined the function and regulation of gastrointestinal eosinophils in the steady state and during infection with Helicobacter pylori or Citrobacter rodentium. We find that eosinophils are recruited to sites of infection, directly encounter live bacteria, and activate a signature transcriptional program; this applies also to human gastrointestinal eosinophils in humanized mice. The genetic or anti-IL-5-mediated depletion of eosinophils results in improved control of the infection, increased inflammation and more pronounced Th1 responses. Eosinophils control Th1 responses via the IFN-gamma-dependent upregulation of PD-L1. Furthermore, we find that the conditional loss of IFN-gammaR, but not of Myd88 signaling, phenocopies the effects of eosinophil depletion. Eosinophils further possess bactericidal properties that require their degranulation and the deployment of extracellular traps. Our results highlight two novel functions of this elusive cell type and link it to gastrointestinal homeostasis and anti-bacterial defense.

   



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  Mechanism of cytokine bias in natural killer T cell responses to diverse glycolipid antigens
  Presenter: Pooja Arora
  All Authors:Pooja Arora, Besra GS, Porcelli SA
  7184303226
   
 

CD1d-restricted Natural Killer T cells (NKT cells) are a subset of T lymphocytes that recognize glycolipid antigens, and respond by secreting both the pro-inflammatory Th1- or anti-inflammatory Th2-cytokines. Several variants of prototypical glycolipid antigen, alpha-galactosylceramide (αGC), have been described that induce responses in which either the Th1 or Th2 cytokines predominate. It has been proposed that the variation in cytokine responses with different glycolipid antigens could reflect their presentation by different cell types. By using a monoclonal antibody that selectively recognizes CD1d/αGC complexes, we have shown that a single type of cell, the CD8αPos DEC-205Pos dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of a-galactosylceramide that stimulate Th1 or Th2-biased cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and co-inhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8αPos DCs and iNKT cells, providing a mechanism for biasing towards either pro-inflammatory or anti-inflammatory responses.

   



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  Thresholds for Zap70 activity in T cell development and proliferation
  Presenter: Byron Au-Yeung
  All Authors:Byron Au-Yeung, Heather Melichar, Julie Zikherman, Jenny Ross, James Mueller, Debra Cheng, Yiling Chen, Kevan Shokat, Ellen Robey, and Arthur Weiss
  University of California, San Francisco
   
 

Upon TCR stimulation, it is generally accepted that Zap70 promotes the activation of downstream signal pathways associated with T cell activation. Less clear is the “amount” of Zap70-dependent signaling required to drive cellular responses. To quantitatively perturb Zap70 activity, we developed a chemical-genetic system in which a catalytically competent mutant of Zap70 can be selectively inhibited by a small molecule inhibitor. We also use a Nur77-GFP reporter transgene, which is expressed in relative proportion to TCR signal magnitude or duration. Stimulation of thymocytes or mature T cells in the presence of graded concentrations of Zap70 inhibitor resulted in similarly graded reductions in reporter expression. Using a model system of synchronized thymocyte positive selection, we showed that DP cells accumulate Zap70-dependent signals over 36 continuous hours to complete positive selection. Zap70 inhibitor titration resulted in graded decreases in positively selected cells. However, positively selected cells accumulated a relatively invariant “amount” of TCR signaling (Nur77-GFP expression), regardless of the concentration of Zap70 inhibitor. These results suggested a level of heterogeneity within a population of thymocytes, even if they all express the OT-I TCR. In mature CD4 and CD8 T cells, we also showed that titration of Zap70 inhibitor concentration quantitatively reduced the TCR signaling accumulated by activated T cells, and decreased the proportion of cells that enter the proliferative response. However, cells that divided also integrated an invariant amount of TCR signaling (Nur77-GFP) regardless of inhibitor concentration. Our studies indicate the existence of TCR signaling thresholds for development and activation, and that such thresholds remain invariant despite tuning of TCR signal magnitude and duration.

   



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  Obesity Potentiates TH2 Immunopathology via Dysregulation of PPAR??
  Presenter: Sagar Bapat
  All Authors:Sagar Bapat, Mark Ansel, Alexander Marson, Richard L. Gallo, Ronald M. Evans, Ye Zheng
  University of California, San Francisco
   
 

How obesity affects immune function is not well understood. Clinically, obesity is strongly associated with severe TH2 immunopathology, though the physiological, cellular, and molecular underpinnings of this association remain obscure. Here, we demonstrate that obese mice are susceptible to severe atopic dermatitis (AD), a major manifestation of TH2 immunopathology and disease burden in humans. Mechanistically, we show that dysregulation of the nuclear hormone receptor (NHR) PPARγ (peroxisome proliferator-activated receptor gamma) in T cells is a causal link between obesity and the increased TH2 immunopathology. We find that PPARγ oversees a cellular metabolic transcriptional program that restrains nuclear gene expression of the chief TH2 priming and effector cytokine interleukin-4 (IL-4). Accordingly, thiazolidinediones (TZDs), potent PPARγ agonists, robustly protect obese mice from TH2 immunopathology. Collectively, these findings establish PPARγ as a molecular link between obesity and TH2 immune homeostasis and identify TZDs as novel therapeutic candidates for TH2 immunopathology. Fundamentally, these findings demonstrate that shifting physiologic metabolic states can shape the tone of adaptive immune responses to modulate differential disease susceptibility.

   



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  IL-6 and IL-27 exert unique and overlapping effects of CD4+ T cells during chronic viral infection
  Presenter: Burton Barnett
  All Authors:Burton Barnett, James Harker, Phuc Bao, Aleksandr Dolgoter, Elina Zuniga
  University of California, San Diego
   
 

Persistent viruses such as human immunodeficiency virus and hepatitis C and B viruses are associated with diminished cytotoxic and antibody responses, which in spite of their delayed and weakened activity, exert a limited level of viral control. It is understood that CD4+ helper T cells are central to establishing effective cytotoxic CD8+ T cell and antibody mediated immunity. Therefore, understanding the molecular factors that control CD4+ T cells during chronic viral infection is of utmost importance. By using lymphocytic choriomeningitis virus (LCMV) clone 13 infection in mice as a model system, we set out to investigate the role of interleukin-6 (IL-6) and IL-27 in regulating CD4+ T cell responses during chronic infection. To overcome potential redundancies between these two cytokines we examined IL6R and IL27R double knockout mice infected with LCMV clone 13. We observed reduction in the number of virus-specific CD4+ T cells and compromised TFH responses and IL-21 production, which become evident by day 30 after infection. These CD4+ T cell deficiencies were cell-intrinsic, as shown by mixed bone marrow chimera experiments, and were accompanied by cell-extrinsic defects in CD8+ T cell responses, GC B cell responses, lower virus specific antibody titers, and impaired viral control. In IL-6R and IL-27R single knockout mice, TFH and CD4+ T cell survival (but not IL-21 production) were affected, respectively. Together, these data support a model in which IL-6 and IL-27 exert unique (i.e. TFH and survival) and redundant (IL-21 induction) effects in virus specific CD4+ T cells at late stages during chronic viral infection.

   



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  Characterization of the molecular and cellular mechanisms controlling protective myeloid cells in cancer
  Presenter: Kevin C. Barry
  All Authors:Kevin C. Barry, Joy Hsu, Miranda L. Broz, Francisco J. Cueto, Mikhail Binnewies, Alexis J. Combes, Amanda E. Nelson, Kimberly Loo, Raj Kumar, Michael D. Rosenblum, Michael D. Alvarado, Denise Wolf, Dusan Bogunovic, Nina Bhardwaj, Adil Daud, Joshua L. Pollack, Bushra Samad, Saurabh Asthana, Vincent Chan, Matthew F. Krummel
  University of California, San Francisco
   
 

Intratumoral stimulatory dendritic cells (SDCs) play an important role in locally restimulating cytotoxic T cells and driving immune responses against cancer. Here, we show that the frequency of SDCs in the tumor microenvironment (TME) of human melanoma patients predicts better overall patient survival and responsiveness to PD-1 blockade. However, the mechanisms that control SDC numbers in tumors remain poorly understood. We find that in human melanoma SDC numbers correlate with intratumoral expression of the gene encoding the cytokine FLT3LG. Given the correlation between protective SDCs and FLT3LG expression in the tumor, we generated a novel reporter mouse to identify the cells responsible for producing this cytokine. Here we present live-imaging and functional studies of the interactions that lead to delivery of Flt3L to SDCs in the tumor. Flt3l expressing cells formed stable conjugates with SDCs in the mouse TME and genetic and cellular ablation of Flt3l expressing cells in mice demonstrated their role in regulation of SDC numbers through production of Flt3L. We further identify FLT3LG producing cells in the TME of human melanoma samples and correlate these cells with the levels of protective SDCs in human cancers, patient responsiveness to anti-PD-1 immunotherapy, and better overall survival. Our studies reveal that innate immune SDCs and FLT3LG producing cells in the TME cluster together as the best prognostic tool for patient responsiveness to T cell directed immunotherapy and that these cells are necessary for enhanced T cell tumor responses, suggesting this axis for novel therapies.

   



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  A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.
  Presenter: Kevin Barry
  All Authors:Kevin Barry, Hsu J, Broz ML, Cueto FJ, Binnewies M, Combes AJ, Nelson AE, Loo K, Kumar R, Rosenblum MD, Alvarado MD, Wolf DM, Bogunovic D, Bhardwaj N, Daud AI, Ha PK, Ryan WR, Pollack JL, Samad B, Asthana S, Chan V, Krummel MF
  University of California, San Francisco
   
 

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 'checkpoint' immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell-directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.Enter your abstract's text here.

   



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  Identification of a developmentally restricted hematopoietic stem cell that gives rise to innate-like B and T cells
  Presenter: Anna Beaudin
  All Authors:Anna Beaudin, Scott W. Boyer, Gloria E. Hernandez, Jessica Perez-Cunningham, E. Camilla Forsberg
  UC Santa Cruz
   
 

The generation of distinct hematopoietic cell types, such as tissue-resident immune cells, distinguishes fetal from adult hematopoiesis, but the mechanisms responsible for differential cell production during prenatal hematopoietic development remain to be established. Using an irreversible lineage tracing model, we have identified a novel, definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients, but does not persist into adulthood in situ. Despite their multilineage potential, these novel HSCs display higher lymphoid cell production, lymphoid lineage priming, and greater capacity to generate tissue-resident innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Our lineage tracing identifies a developmentally restricted HSC that contributes to the formation of a layered immune system and reveals the mechanism underlying developmentally regulated hematopoietic waves. As early lymphoid cells play essential roles in establishing self-recognition and tolerance, defining their origin and generation has critical implications for understanding the development of autoimmune disease, allergy, and tolerance induction upon organ transplantation

   



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  Proinflammatory microenvironments within the intestine regulate the differentiation of CD8 T cells responding to infection
  Presenter: Tessa Bergsbaken
  All Authors:Tessa Bergsbaken, Michael J. Bevan
  University of Washington
   
 

CD8 T cells in mucosal tissues play an important role in controlling infection by a variety of pathogens. We report that oral infection with the enteric pathogen Yersinia pseudotuberculosis (Yptb) results in two distinct populations of pathogen-specific CD8 TRM cells in the lamina propria (LP). Surprisingly, one population did not require TGFß signaling and did not express CD103. Despite these unusual features, CD103neg T cells were true resident memory cells, as they were long lived and resistant to systemic depletion. Unlike the CD103+ CD8 T cells, which were TGFß-dependent and scattered in the tissue, CD103neg T cells were clustered with CD4 T cells and CX3CR1+ macrophages/dendritic cells around areas of bacterial infection. CXCR3-dependent recruitment to areas of inflammation was critical for development of the CD103neg population and for pathogen clearance. These studies have identified the preferential development of CD103neg LP TRM cells in inflammatory microenvironments within the LP and suggest that this subset plays a critical role in controlling infection.

   



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  GM-CSF plays a pathogenic role in autoimmunity to the neuroretina in the absence of Th1 and Th17 lineage cytokines
  Presenter: So Jin Bing
  All Authors:So Jin Bing, Phyllis B. Silver, Reiko Horai, Yingyos Jittayasothorn, Rachel R. Caspi
  Laboratory of Immunology, NEI, National Institutes of Health, Bethesda, MD, USA
   
 

Autoimmune uveitis is a complex group of sight-threatening diseases caused by activated retina-specific Th1 or Th17 cells that acquired the ability to cross the blood-retinal barrier, but their respective contribution to autoimmune uveitis is not fully understood. In this study, we used mice deficient in IL-17A or IFN-? (GKO), or both IL-17 and IFN-? (DKO) to dissect the role of Th1 and Th17 lineage-specific cytokines in pathogenesis of uveitis. Experimental autoimmune uveitis (EAU) was induced by active immunization with the retinal autoantigen IRBP in complete Freund’s adjuvant. IL-17A-/- mice were protected from EAU, whereas GKO mice had exacerbated disease. Surprisingly, DKO mice were fully susceptible to EAU, with scores similar to WT controls. To test whether other lineage-specific proinflammatory cytokines compensate for lack of IL-17A and IFN-?, EAU-challenged DKO mice were treated with blocking antibodies to IL-17F, IL-22, TNF-a, or GM-CSF. Blockade of IL-17F, IL-22, or TNF-a in DKO mice did not affect the severity of EAU. By contrast, treatment of DKO mice with anti-GM-CSF antibody, either as prevention or as a reversal paradigm, significantly suppressed EAU. These results suggest that IL-17F, IL-22 or TNF-a individually are dispensable, but GM-CSF appears to play a major and nonredundant role in development of EAU when IFN-? and IL-17A are both absent. To examine whether GM-CSF-secreting T cells (ThGM) are pathogenic effectors, IRBP-specific T cells (from R161H mice) were polarized in vitro to ThGM, Th1 or Th17 phenotypes, and were adoptively transferred to naïve WT mice. A high proportion of ThGM-polarized cells produced GM-CSF, and many co-produced IFN-?, Notably, these cells induced a more severe disease than did Th17 cells, (but less severe than Th1 cells), supporting the notion that ThGM cells are also uveitogenic. Together, these results indicate that ‘classic’ Th1 and Th17 cells are not the only pathogenic effectors in uveitis, and that other inflammatory cytokine(s), such as GM-CSF, can also drive disease.

   



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  Effect of normal microbial exposure on the immune response to airway allergens
  Presenter: Katharine Block
  All Authors:Katharine Block, Koji Iijima, Sara E Hamilton Hart, David Masopust, Hirohito Kita, Stephen C Jameson
  University of Minnesota
   
 

Allergy and asthma are chronic conditions that can result in acute, life-threatening episodes. Our understanding of what makes individuals susceptible to these pathological immune responses is incomplete. Improvements in sanitation, vaccination programs, and other changes to individuals’ microbial exposure are hypothesized to contribute to the rise in atopy in recent decades. This “hygiene hypothesis” posits that an immune system that has not been properly trained through fighting off natural infections is at risk of responding improperly to innocuous allergens, resulting in allergic asthma. We set out to experimentally test how this microbial training of the immune system influences responses to airway allergens using a murine model of normal microbial exposure. Laboratory mice are generally housed under specific pathogen free (SPF) conditions and their immune systems have a phenotype similar to a neonatal human with limited microbial exposure. Recent work has demonstrated that physiological animal-to-animal transmission of natural mouse pathogens alters the immune system and results in “dirty” mice with a phenotype that more closely resembles adults that have experienced a higher degree of microbial exposure. We used SPF mice to model children that have not been exposed to many infections or microbial products and “dirty” mice to model children with increased microbial experience and that appear to be protected from the development of allergy and asthma. We found that normal microbial exposure reduced recruitment of eosinophils into the lungs upon airway allergen exposure. Bulk CD4+ T cells, Th2 cells and allergen-specific CD4+ T cells were also decreased in dirty mice compared to SPF mice after allergen exposure. Future work will test whether dirty mice are resistant to lung inflammation and pathology in response to airway allergens and will investigate the parameters of allergen-specific CD4+ T cells responding in the lungs. The results generated from these studies will increase our understanding of how microbial exposures influence susceptibility to asthma and allergy. By addressing these questions, potential mechanisms underlying the development of allergy will be revealed, which could lead to intervention strategies.

   



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  The role of neutrophils and extracellular adenosine in shaping host resistance to Streptococcus pneumoniae lung infection
  Presenter: Elsa Bou Ghanem
  All Authors:Elsa Bou Ghanem, Stacie Clark, Sara E. Roggensack, Sally R. McIver, Pilar Alcaide, Phil G. Haydon and John M. Leong
  Tufts University
   
 

Streptococcus pneumoniae (pneumococcus) invasive infections, such as pneumonia, bacteremia and meningitis, remain a leading cause of mortality and morbidity worldwide despite the availability of a vaccine. An important determinant of the course of disease following S. pneumoniae infection is polymorphonuclear leukocyte- (PMN-) mediated pulmonary inflammation. However, different studies report that, during pneumococcal infection, these innate immune cells may promote either immune protection or severe disease. By careful monitoring of the influx of PMNs throughout the course of infection, we found that upon intratracheal challenge of mice, early PMN influx coincided with a decrease in pneumococcal burdens in the lungs, but continued inflammation correlated with increased pulmonary and blood-borne pneumococci. Depletion of PMNs prior to lung challenge significantly increased the susceptibility of mice to S. pneumoniae, but PMN depletion at peak infiltration 18 hours post-infection lowered bacterial numbers a 100-fold in both the lungs and blood and enhanced survival. Thus, our findings demonstrate that optimal host defense against pneumococcal disease requires an initial robust PMN response followed by modulation of this response later in infection. To better understand the signals that regulate the influx of PMNs into the lungs during pneumococcus infection, we investigated a crucial regulator of pulmonary inflammation, extracellular adenosine (EAD), whose production requires the cell surface enzyme, 5'-nucleotidase CD73. We found that diminishing EAD production by pharmacological inhibition or genetic ablation of CD73, or by chemically blocking EAD signaling receptors led to dramatically increased accumulation of PMNs in the lungs upon S. pneumoniae infection of mice. Importantly, blocking EAD production or signaling also increased the susceptibility of mice to systemic pneumococcal infection. This susceptibility of CD73-deficient mice was reversed by PMN depletion following infection, suggesting that control of PMN influx is a central factor in EAD-mediated resistance. Our findings identify EAD as a negative regulator of pulmonary PMN influx, an activity that is crucial for innate host resistance to S. pneumoniae.

   



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  Transposon Mutagenesis Screen to Identify Host-Encoded Anti-Viral Factors
  Presenter: Anna Bruchez*
  All Authors:Anna Bruchez*, Ky Sha*, Li Chen, Adam Lacy-Hulbert, Lynda Stuart
  Benaroya Research Institute, Massachusetts General Hospital
   
 

Ebola and Lassa virus cause sporadic outbreaks of severe hemorrhagic fever. Owing to their high mortality rates, they pose significant health risks and are potential bioterrorism agents. However, our knowledge of how they usurp host cells to complete their life cycle, and more importantly, how this can be combated, is limited. Here we describe a new genome-wide approach to identify genes and pathways that confer resistance to infection. This work will provide insights into host anti-viral defense mechanisms and may be used to identify new therapeutic targets that can be manipulated to promote host-encoded anti-viral immunity. We are using a forward genetics approach that our lab previously developed to identify host genes, known as restriction factors, which confer resistance to viral infection. In this system cells are mutagenized with a transposon that activates genes close to its insertion site. Libraries of mutagenized cells are screened with a cytotoxic virus and resistant cell populations are selected. Illumina sequencing is then used to characterize transposon insertion sites and identify candidate resistance genes. Using this method we have performed a screen in the human cell lines A549, U-2 OS, HEC-1-B, HELA and 293T for genes conferring resistance to either vesicular stomatitis virus (VSV) or recombinant VSV bearing either the Ebola virus (Ebo-VSV) or Lassa fever virus surface glycoprotein (LFV-VSV). We have identified genes associated with resistance to only one type of virus, or to multiple types, demonstrating that resistance can occur through targeting of either viral entry or viral replication. One of the genes our screen identified is NPC1, a known cofactor for Ebola virus entry; thus, confirming that this is a valid method of identifying virus-host interactions. We are currently characterizing the novel candidates to confirm resistance genes, and extending the screen to additional cell lines and viruses. Supported by NIAID Grant R21AI102266.

   



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  Peritoneal podoplanin-expressing cells control peritoneal macrophage identity and localization
  Presenter: Matthew B Buechler
  All Authors:Matthew B Buechler, Shannon J Turley
  Genentech
   
 

Peritoneal macrophages are important for tissue repair and pathogen clearance. The transcription factor GATA6 restricts their localization and expression of this gene is regulated by environmental factors, such as retinoic acid. Yet, the accessory cells that metabolize retinol and program peritoneal macrophages have not been identified. Here, we show that GATA6 and other lineage-defining genes are imprinted by non-hematopoietic podoplanin (PDPN)-expressing cells in the omentum. Surprisingly, we reveal that PDPN+ mesothelial cells, which ensheath all peritoneal tissues, exhibit a high Wt1-Raldh2 index that imbues GATA6 expression in peritoneal macrophages to restrict their localization to the peritoneal space in the steady state. During the macrophage disappearance reaction, peritoneal macrophage re-localized to the omentum via CCR1 ligand release by omental PDPN+ cells. Collectively, these results demonstrate that PDPN+ cells regulate gene expression and localization of peritoneal macrophages under homeostasis and LPS-induced inflammation.

   



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  Microvilli enable efficient T cell antigen search and ligand detection
  Presenter: En Cai
  All Authors:En Cai, Kyle Marchuk, Peter Beemiller, Casey Beppler, Matthew G. Rubashkin, Valerie M. Weaver, Audrey Gérard ,Tsung-Li Liu, Bi-Chang Chen, Eric Betzig, Frederic Bartumeus, Matthew F. Krummel
  University of California-San Francisco
   
 

During antigen detection, T cells survey the surface of antigen-presenting cells (APCs), which typically display mainly nonstimulatory peptide-loaded major histocompatibility complexes (pMHCs) mixed with more rare cognate antigen in a process involving close (nanometer-scale) membrane apposition. T cells must thus solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet microscopy and quantum dot-enabled synaptic contact mapping microscopy to show how microvilli on the surface of T cells search opposing cells and surfaces before and during antigen recognition. We uncovered that microvilli on T cell surfaces dynamically survey the majority of opposing surfaces within one minute through anomalous diffusion. T cell receptor (TCR) recognition resulted in selective stabilization of receptor-occupied protrusions, which was independent of tyrosine kinase signaling and the actin cytoskeleton. We now reveal that TCRs on activated T cells are non-uniformly distributed on cell membrane: some TCRs were concentrated on microvilli, while other TCRs formed microclusters on flatter membrane patches. Most of microvilli are TCR-enriched, but a small population of microvilli do not contain appreciable densities of TCR. A second feature of recognition is revealed by examining the opposing surface. We found that during T cell-APC interaction, T cell microvilli project deep into 3D pockets formed by veil structures on the surface of dendritic cells (DC); and DC membrane also conformed to accommodate T cell microvilli, which increased the effective close-contact area between T cell and APC. Such scanning patterns enable T cells to efficiently scan more APC surface in given time. This work defines the efficient cellular search process against which ligand detection takes place in T cells.

   



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  Host-pathogen lipid interactions influence mycobacterial pathogenesis
  Presenter: CJ Cambier
  All Authors:CJ Cambier, Steven Banik, Joseph A. Buonomo, Carolyn Bertozzi
  Stanford
   
 

Several lipids of the pathogen Mycobacterium tuberculosis are known to promote virulence at various stages of disease. However, the inability to probe these lipids during in vivo infection makes elucidation of their pathogenic mechanisms difficult. Using chemical extraction and reconstitution methods, we were able to define the lipid composition of the outer mycomembrane of Mycobacterium marinum prior to infection. Combining this approach with the synthesis of clickable, semi-synthetic lipids, we introduced a chemically tractable, biologically active variant of the virulence lipid phthiocerol dimycocerosate (PDIM) into the mycomembrane. We find that following infection of zebrafish larvae, PDIM spreads away from bacterial surfaces into the membranes of host cells. Importantly, we found that PDIM spreading into host epithelial cells prior to the arrival of macrophages to the site of infection was required for the bacteria to avoid toll-like receptor (TLR)-dependent antimicrobial immune responses. Structure-function analysis found that PDIM’s biophysical properties promoting occupation of host membranes and subsequent virulence were dependent on PDIM’s fatty acid tails containing methyl branches. In the absence of methyl branched lipid tails, PDIM exhibited decreased fluidity, the inability to spread into epithelial cells, and the inability to prevent TLR-dependent immune responses. Finally, we found that the concentration of cholesterol in host membranes directly correlated with the amount of PDIM spreading. Treating zebrafish with the statin atorvastatin lead to a decrease in host cholesterol, a decrease in PDIM spreading, and the fish were resistant to mycobacterial infection in a TLR-dependent manner. While it has long been appreciated that dyslipidemia promotes Tuberculosis (TB) pathogenesis, and that statins are associated with a decrease in TB incidence, our data finally suggest a potential molecular mechanism underlying these observations.

   



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  Tumors Reprogram Early Myeloid Differentiation in the Bone Marrow to Generate Immunosuppressive Neutrophils
  Presenter: Amy-Jo Casbon
  All Authors:Amy-Jo Casbon, Amy-Jo Casbon, Damien Reynaud, Chanhyuk Park, Emily Khuc, Dennis D. Gan, Koen Schepers, Emmanuelle Passegué and Zena Werb.
  University of California San Francisco
   
 

The growing interest in myeloid cells that suppress T cell function has led to several findings regarding the contribution of these myeloid cells in immune suppression in cancer and more recently, their role in promoting lung metastasis. However, the markers utilized to study these T cell-suppressive myeloid cells (also called myeloid-derived suppressor cells or MDSCs) are shared with myeloid cells found during normal homeostasis and controversy exists as to whether these cells are a unique cell population, alternatively activated myeloid cells, or immature myeloid cells blocked during differentiation. Although these cells differ from naïve myeloid cells in terms of different functional properties, differences in activity do not necessarily define these cells as a unique population. Using a mouse model of breast cancer that closely mimics the stages the of tumor progression observed in human patients, we took a novel, comprehensive approach to determine the molecular and cellular mechanisms which regulate production of these suppressive cells in tumor-bearing mice by evaluating changes in both mature and immature myeloid cell populations in multiple tissues in a longitudinal study. We found that tumor reprogramming of hematopoiesis in the bone marrow skewed myeloid differentiation at the level of the primitive multipotent progenitor to generate activated, T cell-suppressive, and Retinoblastoma-low-expressing neutrophils, which predominantly accumulated in peripheral sites, not the primary tumor. These data reveal a long-range crosstalk between the developing tumor and hematopoietic tissue that contributes to development of an immunosuppressive environment and to metastasis.

   



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  DAP12 promotes resolution of allergic airway inflammation
  Presenter: Cierra Casson
  All Authors:Cierra Casson, Jessica Hamerman
  Benaroya Research Institute
   
 

Asthma is a chronic respiratory disease that affects over 300 million people and is increasing in prevalence worldwide. Allergic asthma, the most prevalent form of asthma, is driven by chronic, antigen-dependent airway inflammation that develops following an aberrant response to common environmental stimuli. This allergic airway inflammation causes the wheezing, coughing, and shortness of breath that are typical symptoms of asthma. Though many of the mechanisms that contribute to the initiation of allergic airway inflammation have been defined, strikingly little is known about how airway inflammation is resolved to limit disease. Our data show that the signaling adaptor DAP12 is required for resolution of airway inflammation in response to cockroach antigen (CRA), one of the most common human airway allergens. In the absence of DAP12, mice had increased eosinophil numbers in the airway space following CRA sensitization. Additionally, mRNA encoding the type-2 cytokines IL-5 and IL-13 were increased in the lungs of Dap12-/- mice compared to wild-type controls. Though innate lymphoid cells (ILC2) are thought to be the major producers of IL-5 and IL-13 during allergic inflammation, ILC2 do not express DAP12. Instead, DAP12 is highly expressed by myeloid cells and natural killer cells. Early during sensitization with CRA, we observed an expansion of CD11b+ myeloid cells in Dap12-/- mice. The presence of these CD11b+ myeloid cells corresponded with an increase in IL-33 mRNA in Dap12-/- lungs, a cytokine known to induce IL-5 and IL-13 production. We propose that DAP12 expression in CD11b+ myeloid cells limits IL-33 production, either directly from myeloid cells or indirectly via interactions with the epithelium. This dampening of IL-33 production through DAP12 likely restricts IL-5 and IL-13 production to promote resolution of airway inflammation. In addition to having increased type-2 inflammation after sensitization with CRA, Dap12-/- mice also had increased eosinophil numbers in the BALF in response to challenge with CRA 17 days-post sensitization. These data suggest that DAP12 is not only required to resolve airway inflammation after acute sensitization with allergens, but that an inability to resolve initial inflammation in response to sensitization may lead to exacerbated allergic inflammation upon re-exposure to these allergens. Thus, we have identified DAP12 as a novel factor important for the resolution of allergic airway inflammation.

   



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  In lymphoid progenitors, a novel lncRNA, termed AJAX, repositions the EBF1 locus from the heterochromatic to the euchromatic compartment, to orchestrate early B cell development
  Presenter: Vivek Chandra
  All Authors:Vivek Chandra, Marty Flores and Cornelis Murre
  University of California, San Diego, USA
   
 

B cell development relies on a highly regulated and hierarchical gene expression program involving at least four transcription factors. The E-protein E2A induces the expression of early B cell factor 1 (Ebf1), which in turn activates the transcription factors Pax5 and Foxo1. Together, these factors orchestrate the B cell fate. Recent genome-wide studies have suggested that transcription also occurs in non-coding regions of the genome; however, the role of this activity in B-cell development is not known. Here, we describe a B cell specific antisense lncRNA coded by the immunoglobulin heavy chain locus (IgH) that we have named AJAX. AJAX expression occurs early during B cell development most highly at the common lymphoid progenitor (CLP) LY6D+ and pro-B cell stages. Using silencing and overexpression approaches, we found that AJAX is critical not only for early B cell development but also IgH locus contraction. Furthermore, we found that E2A regulates AJAX expression and that the vast majority of AJAX transcripts localize to pro-B cell nuclei. Global gene expression profiling suggests that AJAX regulates the expression of Ebf1 along with several other genes responsible for B cell development. Previous studies in our laboratory found that in progenitor cells, the transcriptionally inactive locus encoding Ebf1 was sequestered at the nuclear lamina to preserve multipotency. During development into pro-B cells, Ebf1 switched compartments and established a B lineage–specific transcription signature. We show that AJAX regulates this movement of the Ebf1 locus away from the nuclear lamina to the transcriptionally permissive compartment. Taken together, these data indicate that lcRNAs is critical for early B-cell development and have the ability to regulate the localization of loci during developmental progression.

   



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  A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation
  Presenter: Shilpi Chandra
  All Authors:Shilpi Chandra, Archana Khurana, James Gray, Sonia Sharma, Bjoern Peters, Mitchell Kronenberg
  La Jolla Institute for Allergy and Immunology, San Diego
   
 

Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid, and their absence does not have any effect on MHC class I or class II antigen presentation pathways. Therefore our data indicate that the CD1d and MHC class II antigen presentation pathways are highly divergent, although both depend on antigen loading in endolysosomal compartments.

   



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  A pooled RNA interference screen in vivo identifies regulators of antiviral CD8+ T cell differentiation
  Presenter: Runqiang Chen
  All Authors:Runqiang Chen, Simon Bélanger, Megan A. Frederick, Bin Li, Sonia Sharma, Bjoern Peters, Anjana Rao, Shane Crotty, Matthew E. Pipkin
  La Jolla Institute
   
 

Memory CD8+ T cells play an essential role in long-term immunity against intracellular infections, but our understanding of the cell-intrinsic molecules that control their differentiation is limited. To identify previously unrecognized transcriptional regulators that control effector and memory CD8 T cell development in vivo, we developed a pooled approach to screen 30–100+ genes individually in antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). In vivo screen using T cell receptor (TCR)-transgenic CD8+ T cells responding to lymphocytic choriomeningitis virus (LCMV) identified both known and new genes that regulated short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). The screen revealed the role for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1) in effector and memory CTLs differentiation in vivo. Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of protective short-lived effector CTL and enhanced memory precursor CTL formation in vivo. This pooled shRNA screening approach should accelerate the understanding of T cell differentiation in vivo.

   



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  Toxoplasma and host interaction: the injection alone changes Macrophage Polarization
  Presenter: Longfei Chen
  All Authors:Longfei Chen, David Christian, Anita Koshy, Christopher A. Hunter
  University of Pennsylvania
   
 

The ability of IFN-gamma to activate macrophages is associated with an M1 phenotype that includes increased anti-microbial activity and is critical for the ability to inhibit replication of Toxoplasma gondii. Alternatively, when type I and III strains of T. gondii infect macrophages they engage an M2 transcriptional program and generate a population of cells that are typically characterized as having poor anti-microbial activity. Here, in vitro and in vivo studies using parasites that express Cre we show that a group of macrophages that have been solely injected but which lack the parasite express an M2 phenotype. Thus, injection of parasite derived rhoptry proteins alone has a significant impact on host cell function and raises new questions about the persistence of these M2 populations and their impact on virulence and the outcome of infection.

   



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  Effects of Eif4e Gene Dosage in Murine Primary B cells and Leukemia Cells
  Presenter: Honyin Chiu
  All Authors:Honyin Chiu, Leandra V. Jackson, Sharmila Mallya, David A. Fruman
  Benaroya Research Institute
   
 

The eukaryotic translation initiation factor 4E (eIF4E) protein binds to the 7-methylguanosine cap present in most mRNAs, recruits the scaffolding protein eIF4G and other proteins to form the translation initiation complex known as eIF4F. In cancer, eIF4F contributes to progression of the disease by preferentially translating mRNAs involved in sustained proliferative signaling, evasion of growth suppression, resistance to programmed cell death, replicative immortality, angiogenesis, invasion and metastasis. This makes cap-dependent translation an attractive target for cancer therapy, and efforts are underway to develop small molecule inhibitors of eIF4E, eIF4G and eIF4A for oncology. In lymphocytes, the 4E-binding protein (4E-BP)/eIF4E axis coordinates cell growth and proliferation during lymphocyte activation and our lab found that eIF4E activity is important for B cell differentiation and antibody class switching. This pathway is important in both normal B cell function and tumorigenesis; thus, we wanted to test the effects of reduced eIF4E protein levels in these parallel systems. Our findings show that reduced eIF4E protein slows tumorigenesis in a mouse model of pre-B cell leukemia, while normal mouse primary B cells can maintain B cell proliferation and antibody class switching. These findings establish that altering eIF4E dosage has significant yet selective biological consequences during tumor progression and supports studies showing that cancer cells are more sensitive than non-transformed cells to reductions in eIF4E protein.

   



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  The Transcriptional Cofactor Ski in Thymic Epithelial Cells Regulates Peripheral T Cell Responses
  Presenter: Honyin Chiu
  All Authors:Honyin Chiu, Steve Ziegler
  Benaroya Research Institute
   
 

The thymus is an important site for the establishment and maintenance of an appropriate immune response through positive and negative selection of developing T cells. The thymus contributes to thymocyte development through interactions with cortical and medullary thymic epithelial cells (TECs), termed cTECs and mTECs, respectively. Developing thymocytes also support the maturation and proliferation of mTECs by secreting transforming growth factor-beta (TGF-β) superfamily cytokines. Our lab has previously shown that deletion of TGF-β signaling in TECs enhanced negative selection and functional maturation of thymocytes, and increased the production of regulatory T cells. To investigate whether the proto-oncogene product Ski (Sloan-Kettering Institute), a negative regulator of TGF-β signaling, is associated with thymocyte development in homeostatic condition and inflammatory conditions, we generated mice that deleted Ski specifically in TECs (Foxn1CRESkifl/fl mice). We found that the mTEC population was decreased as predicted, however we did not find any differences in thymocyte development between the Foxn1CRESkifl/fl mice and the Skifl/fl control mice. Furthermore, antibody responses to NP-OVA were also unaffected. However, in an experimental autoimmune encephalitis (EAE) mouse model of multiple sclerosis, Ski deletion had a significantly greater protective effect than deletion of TGF-β. Overall, our finding suggests that Ski signaling in TECs regulates peripheral T cell self-reactivity responses.

   



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  miR-23~27~24 clusters restrict Th2 immunity and associated immunopathology during airway allergic reaction
  Presenter: Sunglim Cho
  All Authors:Sunglim Cho, Cheng-Jang Wu, Tomoharu Yasuda, Leilani Cruz, Ling-Li Lin, Marina Miller, Hyang-Mi Lee, David Broide, Klaus Rajewsky, Li-Fan Lu
  Division of Biological Sciences, University of California, San Diego. Immune Regulation and Cancer, Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany. Department of Medicine, University of California, San Diego. Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
   
 

MiRNAs (miRNAs) are important regulators in T cell differentiation and function. Here we show miR-23~27~24 clusters play a pivotal role in controlling type II immunity. Specifically, under Th2 polarizing condition, T cells with overexpression of miR-23~27~24 clusters exhibited reduced IL-4 secretion whereas T cells devoid of miR-23~27~24 clusters produced elevated amounts of IL-4. Further mechanistic studies revealed miR-24 and miR-27 could repress Gata3, a key regulator of Th2 cell differentiation through an indirect and a direct manner, respectively. Finally, by using an OVA-induced asthma model, we have shown that mice with T cell-specific ablation of miR-23~27~24 clusters developed a more severe airway inflammation characterized by increased IL-4 secretion, lung eosinophil infiltration, mucin production and serum IgE levels. Taken together, our studies identify a miRNA family with important biological function particularly in controlling Th2 immunity and suggest that a tight regulation of this miRNA family is required to maintain optimal effector T cell function and to prevent aberrant immune responses.

   



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  Parasite-induced Alternative Activation of Host Cells Regulates Immune Pathology During Infection
  Presenter: David Christian
  All Authors:David Christian, Lucas A. Dawson, Anita A. Koshy, Daniel P. Beiting, Jung Oh, David R. Roos, David Artis, Christopher A. Hunter
  University of Pennsylvania
   
 

In response to T. gondii infection, the host develops a robust Th1 response to produce IFN-g, which is required for control of this intracellular parasite. It is well established that during infections with Type II strains of T. gondii, the regulatory cytokines IL-10 and IL-27 are required to prevent lethal immune pathology mediated by CD4+ T cells. However, during infection with Type III strains, mice that lack IL-10 or IL-27 develop similar degrees of weight loss as Type II infections but survive this challenge. These data indicate the presence of a mechanism that is independent of IL-10 and IL-27 that limits immune pathology that is specific to Type III infections. One of the critical differences between these two strains is that the Type IIII strains express a kinase (rhoptry protein 16, ROP16) that is injected into host cells and which results in prolonged activation of the host transcription factor STAT6. These events lead to the induction of an alternatively-activated phenotype of macrophages and DCs that is STAT6-dependent but independent of IL-4 or IL-13 signals. Using transgenic parasites and reporter mice to identify injected host cell populations, these studies revealed that challenge with type III strains was characterized by the presence of large numbers of uninfected DCs and macrophages that were injected and expressed an alternatively-activated phenotype characterized by the expression of RELMa and CD206. Studies on the role of these injected cells to control immune pathology during infection with Type III strains of T. gondii will be discussed.

   



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  BATF3-dependent dendritic cells are required for the CD4+ T cell response to immunization against parasitic infection
  Presenter: David Christian
  All Authors:David Christian, Gretchen Harms Pritchard, Anita A. Koshy, Christopher A. Hunter
  University of Pennsylvania
   
 

BATF3 has been shown to be a critical transcription factor for the development of a distinct subset of dendritic cells (DCs) characterized by the expression of CD24 and CD103 in the periphery and CD8α in lymphoid tissues. Using mice deficient for BATF3, these DCs have been shown in multiple models to be required for the priming of CD8+ T cells, and are a critical source of the IL-12 needed to generate a protective Th1 T cell response during infection with the protozoan parasite Toxoplasma gondii. Protection from infection by T. gondii is dependent on a T cell response that can be generated by immunization the non-replicating CPS strain of T. gondii. Immunization with a low dose of CPS is capable of generating a CD4+ and CD8+ T cell response that is equivalent in magnitude to the T cell response during active infection. Surpisingly, the CD4+ and CD8+ T cell responses have both been shown to be dependent on BATF3-dependent DCs. The studies presented here aim to determine whether this DC subset is required for the processing and presentation of parasite antigen to CD4+ T cells during CPS immunization, or if these DCs serve as a required source of IL-12 for the differentiation and expansion of parasite-specific T cells.

   



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  cDC1 are essential for coordinating innate and adaptive immune responses during Toxoplasma gondii vaccination
  Presenter: David Christian
  All Authors:David Christian, Gretchen Harms Pritchard, Anita A. Koshy, Christopher A Hunter
  University of Pennsylvania
   
 

Intraperitoneal immunization with a live attenuated strain of Toxoplasma gondii generates minimal inflammation but also a robust and protective type 1 T cell response. Cytokines essential for a type I immune response (IL-12 and IFN-γ) and the Batf3-dependent type I conventional dendritic cells (cDC1s) are required to induce a parasite-specific, protective CD8+ T cell response. This CD8+ T cell response requires the spatiotemporal orchestration of antigen, antigen presenting cells, and CD4+ T cell help in draining lymphoid organs. The omentum is a visceral adipose tissue present in the peritoneal cavity containing fat associated lymphoid cluster (FALCs) termed ‘milky spots’ that serve as sites to support a rapid immune response in the peritoneum. Despite the ability of these milky spots to form organized structures capable of T and B cell priming, little is understood about how inflammatory cells from the peritoneum are recruited to these sites. Using Batf3 KO mice, cDC1s are shown here to be a required source of IL-12 immediately after CPS immunization. This IL-12 activates natural killer (NK) cells to produce IFN-γ that induces the migration of peritoneal cells containing parasite antigen to sites of T cell priming in omental milky spots. By combining a Cre-secreting, mCherry+ strain of CPS with Cre reporter mice, the role of infected cells was tracked in vivo. Immunofluorescence microscopy of these milky spots provided understanding of how the architecture of these structures enables efficient T cell priming. These data have important implications for the design of vaccine strategies capable of eliciting a protective cellular immune response, and lend insight into the role of cDC1s to initiate an adaptive immune response in atypical secondary lymphoid structures.

   



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  RNase L is a specific regulator of protein translation and limits activation of the RLR-mediated antiviral response
  Presenter: Jonathan Clingan
  All Authors:Jonathan Clingan, Sterling C. Eckard, Daphne A. Cooper, Hilario J. Ramos, Jaime Guillen-Casas, Bruno Canard, Michael Gale, Jr., Jay R. Hesselberth, David J. Barton, and Daniel B. Stetson
  University of Washington
   
 

Intracellular detection of foreign DNA or RNA in mammalian cells triggers distinct sensors that protect against virus infection: DNA activates the cyclic GMP-AMP synthase (cGAS)-STING pathway, whereas RNA activates the RIG-I-like receptor (RLR)-MAVS pathway. In addition, a family of enzymes called oligoadenylate synthetases (OAS) detect foreign RNA and catalyze the formation of 2’-5’-linked oligoadenylates (2-5A). The sole known biological function of 2-5A is to activate the endoribonuclease RNase L, which is thought to cleave cellular and viral RNAs indiscriminately into small immunostimulatory fragments that amplify the RLR response. Here, we show that contrary to this prevailing view, RNase L-deficient cells have an enhanced antiviral response to RLR ligands. We find that the in vivo specificity of RNase L is restricted to several unique sites in ribosomal RNA and that RNase L activation leads to rapid depletion of translating ribosomes, thus establishing RNase L as a specific regulator of protein translation, not an indiscriminate mediator of RNA decay. Moreover, we demonstrate that 2-5A molecules are endogenous activators of the innate antiviral response. These findings reveal that 2-5A made by OAS enzymes has differential roles for regulating the innate immune response via both RNase L-mediated translational suppression, and a direct immunostimulatory role for 2-5A itself.

   



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  Deficiency in the antimicrobial peptide cathelicidin protects against systemic Candida infection
  Presenter: Alison Coady
  All Authors:Alison Coady, Alison Coady, Simon Döhrmann, Josh Olson, Victor Nizet
  University of California, San Diego
   
 

There is an urgent need for novel treatment strategies to combat the rising incidence of antimicrobial resistance. Indeed, the increasing emergence of antifungal resistant Candida species in the clinic has led the CDC to name fluconazole-resistant Candida as a serious threat to public health. Antimicrobial peptides (AMPs) are an attractive alternative and/or supplement to antifungals due to their ability to control fungal growth. The human AMP cathelicidin (LL-37) has been shown to display direct antifungal activity in vitro against the fungal pathogen C. albicans via membrane disruption, inhibition of adherence, and modulation of biofilm formation. However, the role of LL-37 during in vivo infection remains enigmatic, where LL-37 has both antimicrobial and immunomodulatory activity. For example, while previous studies have shown that mCRAMP (the murine homolog of LL-37) is dispensable for controlling C. albicans growth in whole blood killing assays and during subcutaneous infection, more recent work demonstrated that mCRAMP can protect against C. albicans colonization and dissemination in antibiotic-treated mice co-colonized with commensal bacteria. Here, in contrast, we find that loss of mCRAMP using CRAMP-deficient mice (Cramp-/-) protects against a systemic lethal infection with C. albicans, while wild-type mice begin to succumb to infection 24 hours post inoculation. Strikingly, there is no difference in fungal burden between the Cramp-/- mice and wild-type mice at 24 hours, suggesting that wild-type mice experience a detrimental host response, such as sepsis. Production of IL-1β, an important cytokine that protects against fungal infection, is increased in Cramp-/- mice. Preincubation of human neutrophils with LL-37 reduces neutrophil response to C. albicans, suggesting that LL-37 may negatively regulate phagocyte recruitment and activation. Ongoing work includes investigating the role that cathelicidin plays in modulating phagocyte recruitment and activation in vivo. Finally, we are exploring whether cathelicidin plays a similar role in systemic infection with other Candida species, including the emerging antifungal resistant C. auris. Ultimately, this work will not only define the normal physiological role for the LL-37 AMP during systemic candidiasis but also inform the development of AMPs for therapeutic application in patients.

   



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  Long Non-Coding RNA GAPLINC Connects Macrophage Differentiation To Inflammation
  Presenter: Sergio Covarrubias
  All Authors:Sergio Covarrubias, Ran Song, Kasthuribai Viswanathan, Sol Katzman, Edward K. Wakeland, Susan Carpenter
  UC Santa Cruz
   
 

Macrophages are critical effector cells of the innate immune system essential for controlling infection and maintaining tissue homeostasis. Perturbations to these signaling pathways can have devastating consequences, leading to diseases, such as Rheumatoid Arthritis and Cancer. Macrophages arise from monocytes in a differentiation process that is tightly regulated, involving many microRNAs, proteins and stage-specific expression of transcription factors. Long non-coding RNAs (lncRNAs) represent the largest group of RNA produced from the genome and are described as transcripts greater than 200 nucleotides in length that lack protein-coding ability. LncRNAs are rapidly emerging as critical regulators of a broad range of biological processes including genomic imprinting, development, and cancer. We sought to identify novel lncRNAs involved in monocyte to macrophage differentiation. We generated comprehensive RNA-sequencing data sets from primary healthy human monocytes and differentiated macrophages and identified hundreds of lncRNAs differentially expressed during differentiation. We characterized one lncRNA, called GAPLINC, which is dramatically induced over one thousand fold transitioning from monocyte to macrophages. Interestingly, this lncRNA is rapidly downregulated upon Toll-like receptor (TLR) stimulation suggesting a connection to inflammatory pathways. Knockding down GAPLINC in primary human macrophages results in an enrichment for inflammation-related genes in our top upregulated genes, suggesting this lncRNA may negatively regulate inflammatory pathways. GAPLINC is localized to the cytoplasm of macrophages bit it does not associate with Polysomes. We are in the initial stages of understanding the mechanism by which GAPLINC is functioning in macrophages. Here we reveal an interesting connection between the regulation of macrophage differentiation and the downstream inflammation pathways.

   



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  CRISPR screening in macrophages identifies membrane-bound TNF as an autocrine-acting negative regulator of inflammation
  Presenter: Sergio Covarrubias
  All Authors:Sergio Covarrubias, Apple Vollmers, Allyson Capili, Michael Boettcher, Elektra K. Robinson, Laura O’Briain, Christopher Vollmers, James Blau, Michael McManus, Susan Carpenter
  UCSC
   
 

Excess inflammation is associated with a variety of autoimmune diseases and cancers. Macrophages are critical cells of the innate immune system involved in the recognition and destruction of invading microbes in addition to the resolution of inflammation. Understanding the entire catalog of genes involved in the inflammatory response is essential to gaining new insights into immune dysregulation that occurs in autoinflammatory diseases. NF-κB is a major transcription factor that drives the inflammatory response. Here we utilize an NF-κB reporter macrophage line to perform a fluorescence-activated cell sorting (FACS)-based whole-genome CRISPR screen. We identify 115 novel positive and negative regulators of the NF-κB pathway. Unexpectedly, we identify the pro-inflammatory cytokine, TNF, as negative regulator of inflammation and confirm that it acts in an autocrine-manner likely through binding the inhibitory p75 (Tnfrsf1b) receptor. In summary, our NF-κB-CRISPR screen uncovers novel regulators of inflammation and reveals important regulatory complexities of the TNF pathway.

   



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  Defining the transcriptional adaptations of tissue-resident CD8+ T cells to diverse non-lymphoid tissues
  Presenter: Ty Crowl
  All Authors:Ty Crowl, Clara Toma, Amir Ferry, Kyla Omilusik, Ananda Goldrath
  University of California - San Diego
   
 

CD8+ T cells are critical components of effective immune responses to intracellular pathogens and tumors. Upon recognition of infection, CD8+ T cells dramatically expand in number, drive inflammation, and kill infected cells. After infection is cleared, a small subset of long-lived, pathogen-specific CD8+ T cells remain as memory cells, providing long-term protection against previously encountered pathogens. Recently, it has been appreciated that some memory CD8+ T cells remain lodged in tissues, acting as sentinels and guarding common sites of pathogen entry. Over the last several years, many of the mechanisms by which tissue-resident memory cells (TRM) are generated and maintained have been revealed. However, TRM in diverse tissues encounter a range of microenvironments that vary widely in terms of nutrient availability, oxygen tension, pH, and cytokine milieu. The contributions of diverse tissue microenvironments to TRM differentiation are unknown, yet are likely essential for the generation of protective, localized memory responses. We have used RNA-seq, ATAC-seq, and single cell RNA-seq to understand the diversity of TRM populations in distinct tissue microenvironments and identify genes critical for tissue-specific TRM differentiation. We sequenced circulating CD8+ T cells from the spleen and blood and TRM from the small intestine, kidney, salivary gland, fat, and liver. Using these data, we identified multiple tissue-specific transcriptional programs of TRM, and we found that the transcriptional repressor Hic1 is a critical regulator of small intestine-specific TRM differentiation. Elucidating how TRM populations adapt to distinct microenvironments and the tissue-specific transcriptional regulators that mediate these changes will be critical for enhancing protective memory responses at the sites most vulnerable to infection.

   



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  Mechanisms of strand selection of microRNAs in T lymphocytes
  Presenter: Graziella Curtale
  All Authors:Graziella Curtale, Matthew E. Pipkin
  Primary Institution NameDepartment of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
   
 

MicroRNAs (miRs) are important post-transcriptional modulators of gene expression and cell differentiation. However, the mechanisms that determine which strand of the precursor miRs (pre-miRs) that ultimately get loaded into the RNA induced silencing complex (RISC) are still unclear. Here we show that whereas miR-125a-5p is predominantly loaded into the RISC in human cells, an “arm-switching” mechanism appears to exist such that the opposite miR-125a-3p strand is the predominant sequence loaded in the RISC in murine macrophages and CD8 T cells. Sequences of the human and mouse pre-miR-125a are identical, but the less mature pri-miR-125a forms differ at several bases in the 5’ and 3’ flanking regions of the pre-miR between species, suggesting mechanisms other than pre-miR thermodynamic instability accounts for specific strand loading. Notably, the flanking sequences of the murine pri-miR-125a are sufficient to switch loading of 5p and 3p arms of a heterologous miR-146b, suggesting that pre-miR flanking sequences influence strand loading. We are combining next generation sequencing and a two reporter sensor assay that detects loading of either 5p or 3p strands of a specific miR, to identify sequences that preferentially promote loading of the different miR arms from a library of pri-miRs encoding miR-146b with random flanking sequences in the context of CD8 T cells. These studies are likely to shed light on how miR strand selection is achieved and how regulation of target gene specificity is governed during immune cell development and function.

   



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  Investigating tissue-specific roles of IL-1 in behavioral and physiological metabolic adaptations during infection
  Presenter: Víctor D Cuevas
  All Authors:Víctor D Cuevas, Janelle Ayres
  Salk Institute for Biological Studies
   
 

Infections cause dramatic behavioral and physiological changes in the host that will affect host metabolism including an anorexic response, muscle and fat wasting. Infection also leads to modifications in the temperature set point of the organism to generate hyper- and hypothermic responses. Changes in thermoregulation are associated with changes in feeding behavior and changes in fat and muscle physiology. Cold exposure of mice increases feeding and metabolic rate when compared to mice housed at thermoneutrality. Moreover, cold-induced thermogenesis leads to depletion of energy stores likely to provide fuel to sustain the required temperature. The innate immune system is key not only in the defense against pathogens, but also in the regulation of metabolism, feeding and temperature. Intriguingly, the innate immune system is also central in the regulation of cold-induced thermogenesis. IL-1 signaling has been implicated in these physiological processes including the induction of anorexia, thermoregulation and mobilization of fat and muscle stores by acting via the nervous system. We have generated novel tissue specific transgenic mice that lack IL-1 signaling in the vagus nerve and hypothalamus as well as in adipose tissue to elucidate how the innate immune system orchestrates the interactions between all these different responses during infection and thermoregulation and how decisions are made to redirect the energy supply during infection.

   



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  2NBDG uptake is distinct from glucose transport and contributes to plasma cell longevity
  Presenter: Lucas J D'Souza
  All Authors:Lucas J D'Souza, Wing Y Lam, Stephen H Wright, and Deepta Bhattacharya
  Department of Immunobiology, University of Arizona College of Medicine
   
 

Plasma cells are terminally differentiated B lymphocytes whose dominant function is antibody production. These antibodies are largely protective in nature, and the durability of the humoral response against invading pathogens correlate with the lifespan of the plasma cell. However, little is known about mechanisms contributing to the long life of these cells. We have previously demonstrated that long-lived plasma cells possess a higher mitochondrial spare respiratory capacity than their short-lived counterparts, thus offering a metabolic basis for plasma cell longevity. This property depends on the on the cells’ ability to import glucose, as measured by uptake of the fluorescent glucose analog, 2NBDG. Counterintuitively, we found that expression of the glucose transporter Slc2a1 to be similar between ex vivo 2NBDG+ and 2NBDG- plasma cells. Further, genetic ablation of Slc2a1 in a myeloma line using CRISPR-Cas9 did not affect 2NBDG uptake and its kinetics despite reduced 14C-glucose uptake. Put together, our findings suggest that 2NBDG and glucose transport in plasma cells are mediated through different transporters. In addition to Slc2a1 and lesser expressed sugar transporters, abrogation of nucleoside and nucleotide-sugar transporters in a myeloma line had no effect on 2NBDG uptake; thus, ruling out a possible mimicry of naturally occurring biomolecule/s. In conclusion, as 2NBDG unequivocally marks long-lived plasma cells in vivo, identifying the transporter and pathways that mediate its uptake will be of interest in dissecting mechanisms involved in plasma cell longevity.

   



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  Control of Tumor Growth by TSLP During Colorectal Cancer
  Presenter: Steven de Jesus Carrion
  All Authors:Steven de Jesus Carrion, Steven Ziegler
  Benaroya Research Institute
   
 

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in promoting tumor growth during breast and pancreatic cancer by promoting Th2 cell-mediated inflammation. Despite being essential to maintain Treg cell homeostasis in the normal mouse and human intestine, a role for TSLP in colorectal cancer has never been shown. To determine if TSLP affects tumor growth during colorectal cancer, we utilized a murine model of colitis-associated colorectal cancer. TSLP deficient mice exhibited decreased tumor numbers when compared to the WT littermate controls. Moreover, we found that tumors in TSLP-/- mice were also significantly smaller in size than WT mouse tumors. TSLP was mainly localized in the tumor tissue, indicating that it might be produced by cancer or cancer-associated cells. Further, TSLP receptor (TSLPR) expression was significantly upregulated after development of cancer. To determine if TSLP signals directly on the tumor cells, we utilized VillincreTSLPRflox mice in which the TSLPR expression is absent only in intestinal epithelial cells (IECs). Consistent with the TSLP-/- mice, lack of TSLP signaling on IECs led to a decrease in tumor number as well as tumor size, suggesting that TSLP signals directly on the IECs to promote tumor growth. Overall, these data show a novel role for TSLP in controlling tumor progression during colorectal cancer and identify it as a potential target for immunotherapy intervention.

   



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  Regulation of Tumor Growth by TSLP During Colorectal Cancer
  Presenter: Steven De Jesus Carrion
  All Authors:Steven De Jesus Carrion, Steven F. Ziegler
  Benaroya Research Institute
   
 

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in promoting tumor growth during breast and pancreatic cancer by promoting Th2 cell-mediated inflammation. Despite being essential to maintain Treg cell homeostasis in the mouse and human intestine, a role for TSLP in colorectal cancer has never been shown. To determine if TSLP affects tumor growth during colorectal cancer, we utilized a murine model of colitis-associated colorectal cancer. TSLP deficient mice had fewer tumors when compared to WT littermate controls and TSLP receptor (TSLPR) expression was significantly upregulated after development of cancer. Further, deletion of the TSLP signal on intestinal epithelial cells (IECs) led to a significant decrease in tumor growth. In vitro, we found that TSLP signal activates the JAK2/STAT5 pathway in a colon cancer cell line, and chemical blockade of STAT5 phosphorylation led to increased apoptotic cell death, suggesting that TSLP is required for tumor survival. TSLP was also able to interact with the immune system to promote Treg cell differentiation to help the tumor grow, and in the absence of TSLP there was higher frequency of activated CD4+ T cells. Overall, these data show a novel role for TSLP in controlling tumor progression during colorectal cancer and identify it as a potential target for immunotherapy intervention.

   



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  Control of tumor growth by TSLP during colorectal cancer
  Presenter: Steven de Jesus-Carrion
  All Authors:Steven de Jesus-Carrion, Steven Ziegler
  Benaroya Research Institute
   
 

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in promoting tumor growth during breast and pancreatic cancer by promoting Th2 cell-mediated inflammation. Despite being essential to maintain Treg cell homeostasis in the mouse and human intestine, a role for TSLP in colorectal cancer has never been shown. To determine if TSLP affects tumor growth during colorectal cancer, we utilized a murine model of colitis-associated colorectal cancer. TSLP deficient mice exhibited decreased tumor numbers when compared to the WT littermate controls. Moreover, we found that tumors in TSLP-/- mice were also significantly smaller in size than WT mouse tumors. TSLP was mainly localized in the tumor tissue, indicating that it might be produced by cancer or cancer-associated cells. Further, TSLP receptor (TSLPR) expression was significantly upregulated after development of cancer. To determine if TSLP signals directly on the tumor cells, we utilized VillincreTSLPRflox mice in which the TSLPR expression is absent only in intestinal epithelial cells (IECs). Consistent with the TSLP-/- mice, lack of TSLP signaling on IECs led to a decrease in tumor number as well as tumor size, suggesting that TSLP signals directly on the IECs to promote tumor growth. Overall, these data show a novel role for TSLP in controlling tumor progression during colorectal cancer and identify it as a potential target for immunotherapy intervention.

   



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  Crucial role of FOXO1 in inflationary CD8+ T-cell responses during persistent viral infection
  Presenter: Arnaud Delpoux
  All Authors:Arnaud Delpoux, Rodrigo Hess Michelini, Shilpi Verma, Brittney Wellisch, ChenYen Lai, Chris A. Benedict, Stephen M Hedrick
  University of California, San Diego
   
 

Both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) establish persistent infections that induce the accumulation of virus-specific T-cells over time in a process called memory inflation. Although persistence of antigen (Ag) is considered essential, the factors driving memory inflation are still unclear and the molecular pathway for the inflationary memory T-cell development is poorly understood. Mainly, inflationary memory CD8+ T-cells have an effector-memory phenotype (KLRG1hi CD27-), but a low percentage of them express a central-memory phenotype (KLRG1lo CD27+), which are considered as memory precursors for inflationary CD8+ T-cells. To study the role of FOXO1 in CD8+ T-cells during a persistent viral infection, we analyzed mouse bone marrow (BM) chimeras in which FOXO1 was specifically deleted in CD8+ T-cells. These mice were infected with MCMV-?m157 strain, and the expansion and phenotype of inflationary and acute-contracting T cells was examined. The results revealed that the IE3 and M38 (two inflationary dominant epitopes) CD8+ T-cell responses do not inflate in absence of FOXO1. Furthermore, with an absence of FOXO1, there were fewer Ag-specific CD8+ T-cells that produced both IFN? and TNFa. Consistent with this lack of CD8 effector cells the mice were less able to control the virus in the spleen and the liver at day 6 after infection. A similar loss was found in salivary glands during the persistent phase. Moreover, we found that FOXO1 KO Ag-specific CD8+ T-cells fail to up-regulate the memory associated transcription factors, TCF-1 and EOMES, correlating with a lower percentage of memory precursors (KLRG1lo CD27+). Finally, we found that FOXO1 KO Ag-specific CD8+ T-cells cycle normally (Ki67+), but display an anergic state, as measured by calcium mobilization activation, and are prone to apoptosis. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing the inflationary memory program that is essential to forming long-lived effector memory cells.

   



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  Crucial role of FOXO1 in inflationary CD8+ T-cell responses during persistent viral infection
  Presenter: Arnaud Delpoux
  All Authors:Arnaud Delpoux, Arnaud Delpoux, Rodrigo Hess Michelini, Shilpi Verma, Brittney Wellisch, ChenYen Lai, Chris A. Benedict, Stephen M Hedrick
  UCSD
   
 

Cytomegalovirus (CMV) establish persistent infection that induce the accumulation of virus-specific T-cells over time in a process called memory inflation. Although persistence of antigen (Ag) is considered essential, the molecular pathway for the inflationary T-cell development is poorly understood. Mainly, inflationary CD8 T-cells have an effector-memory phenotype (KLRG1hi CD27-), but a low percentage of them express a central-memory phenotype (KLRG1lo CD27+), which are considered as their memory precursors. To study the role of FOXO1 in CD8 T cells during a persistent viral infection, we analyzed mouse bone marrow chimeras in which FOXO1 was specifically deleted in CD8 T cells. These mice were infected with MCMV-?m157 strain, and the expansion and phenotype of inflationary and acute-contracting T cells was examined. The results revealed that the IE3 and M38 CD8 T-cell responses do not inflate in absence of FOXO1. Furthermore, with an absence of FOXO1, there were fewer Ag-specific CD8 T cells that produced both IFN? and TNFa. Consistent with this lack of CD8 effector cells the mice were less able to control the virus in the spleen and the liver at day 6 after infection. Moreover, we found that FOXO1 KO Ag-specific CD8 T cells fail to up-regulate the memory associated transcription factors, TCF-1 and EOMES, correlating with a lower percentage of memory precursors (KLRG1lo CD27+). Then, we found that FOXO1 KO Ag-specific CD8 T cells cycle normally (Ki67+), but display an anergic state, as measured by calcium mobilization activation, and are prone to apoptosis. Finally, we delete FOXO1 at day 40 during the infection, we showed that the FOXO1 KO Ag-specific CD8 T cells survive less efficiently and lose their memory characteristics as well as their recall capacities. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing and maintaining the inflationary memory program that is essential to forming long-lived effector and memory cells. In addition, a memory phenotype is not a permanent differentiation state, it has to be actively maintained by the continued activity of FOXO1text here.

   



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  Role of Copa protein in thymic selection of T cells and the maintenance of tolerance
  Presenter: Zimu Deng
  All Authors:Zimu Deng, Chritopher Law,Frances Ho,Kristin Wang,Zhenlu Chong,Daniah Beleford,Anthony Shum
  University of California, San Francisco
   
 

The COPA syndrome is a recently discovered autoimmune disease caused by missense mutations of the COPA gene. COPA is a protein subunit of coat protein complex I (COPI). COPI is responsible for the retrograde movement of proteins from the Golgi apparatus to the endoplasmic reticulum. It has been unclear how a ubiquitously expressed protein that is part of a vesicular trafficking complex critical to cell homeostasis causes a loss of immune tolerance and autoimmune disease. To determine how a defect in Copa protein leads to autoimmunity we generated a germline knockin mouse bearing the same Copa missense mutation found in patients. Here we show that expression of mutant Copa within the thymus impairs the negative selection of T cells resulting in a defect in central tolerance. Mutant mice spontaneously develop a significant increase in single positive thymocytes and in peripheral tissues elevations of activated effector memory and cytokine-secreting T cells. Reciprocal bone marrow chimeras map these changes to mutant Copa expression within the thymus. Use of T cell receptor transgenic mice demonstrate that mutant Copa in the thymic epithelium impairs the processing and presentation of antigens, thereby leading to a defect in the negative selection of antigen-specific T cells. These results identify Copa as a novel molecular link between a vesicular trafficking protein and central tolerance in the thymus. Furthermore, it establishes a mechanism by which mutations in COPA may lead to autoimmune disease in COPA syndrome patients.

   



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  NRAMP1 Is Critical for Neutrophil-Mediated Control of Intracellular Pathogens
  Presenter: Vladimir Diaz-Ochoa
  All Authors:Vladimir Diaz-Ochoa, Kristen L. Lokken, Ariel Muñoz, Annica R. Stull-Lane, Jason P. Mooney, and Renee M. Tsolis
  University of California, Davis
   
 

The Natural Resistance Associated Macrophage Protein 1 (NRAMP1) is a prominent metal transporter in host defenses against pathogens. Prevailing scholarship holds that macrophages facilitate control of intracellular pathogens via NRAMP1 mediated metal starvation in the phagosome. We found that NRAMP1 in neutrophils also contributes to host control of bacterial pathogens. While investigating how vitamin A deficiency impairs immunity against systemic salmonellosis, we discovered that NRAMP1-deficient animals were equally susceptible to disseminated Salmonella as vitamin A-deficient, NRAMP1-proficient, mice. This data suggested that NRAMP1 mediated immunity to disseminated salmonellosis was dependent on vitamin A. Indeed, Vitamin A deficiency impaired infection-induced granulopoiesis, resulting in reduced expression of specific- and gelatinase granule components in neutrophils, including NRAMP1. Adoptive transfer of neutrophils from NRAMP1-proficient donors, but not NRAMP1-deficient donors, reduced the systemic Salmonella burden in vitamin A-deficient, NRAMP1-proficient, mice and in NRAMP1-deficient animals. Additionally, NRAMP1-deficient neutrophils displayed diminished killing of Salmonella ex vivo compared to NRAMP1-proficient neutrophils. During infection with another vacuolar intracellular pathogen, Brucella abortus, we found that NRAMP1 contributed to a reduction in Brucella burden of the placenta, in which neutrophils also play a prominent role. Collectively, these data suggest that NRAMP1-deficiency impairs control of intracellular pathogens by blunting neutrophil-mediated host defenses.

   



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  Dendritic cells undergo apoptosis in response to bacterial blockade of host translation to control infection
  Presenter: Jessica Doerner
  All Authors:Jessica Doerner, Jessica Doerner, Jenna Zhang, Victor Vazquez, Igor Brodsky, Sunny Shin
  University of Pennsylvania
   
 

Early responses mounted by innate immune cells are essential for host defense against bacterial pathogens. Recognition of pathogen effector molecules by the host helps to initiate this response. Macrophages and dendritic cells (DCs) are important early responders, due to their phagocytic nature and anti-microbial cytokine responses. The gram-negative bacteria Legionella pneumophila, which causes severe pneumonia, uses a type IV secretion system (T4SS) to inject effector proteins that manipulate host cell processes and enable Legionella to replicate within alveolar macrophages. Conversely, upon infection, DCs undergo rapid apoptosis in response to T4SS activity. Here, we sought to define the T4SS-translocated bacterial components that trigger apoptosis in DCs. We find a critical role for Legionella effector proteins in inducing caspase cleavage and cell death in DCs. Thus, our data suggest that DC apoptosis is an effector-triggered immune response that enables cell-intrinsic restriction of bacterial replication in DCs.

   



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  Cell death in response to bacterial effectors controls bacterial infection
  Presenter: Jessica Doerner
  All Authors:Jessica Doerner, Jenna Zhang, Igor Brodsky, Sunny Shin
  University of Pennsylvania
   
 

Early responses mounted by innate immune cells are essential for host defense against bacterial pathogens. Recognition of pathogen effector molecules by the host helps to initiate this response. Macrophages and dendritic cells (DCs) are important early responders, due to their phagocytic nature and anti-microbial cytokine responses. The gram-negative bacteria Legionella pneumophila, which causes severe pneumonia, uses a type IV secretion system (T4SS) to inject effector proteins that manipulate host cell processes and enable Legionella to replicate within alveolar macrophages. Conversely, upon infection, DCs undergo rapid apoptosis in response to T4SS activity. Here, we sought to define the T4SS-translocated bacterial components that trigger apoptosis in DCs. We find a critical role for Legionella effector proteins in inducing caspase cleavage and cell death in DCs. Thus, our data suggest that DC apoptosis is an effector-triggered immune response that enables cell-intrinsic restriction of bacterial replication in DCs.

   



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  Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma
  Presenter: Joseph S. Dolina
  All Authors:Joseph S. Dolina, Joseph S. Dolina(1), Joey Lee(1), Bjoern Peters(2), and Stephen P. Schoenberger(1)
  (1)Laboratory of Cellular Immunology, La Jolla Institute for Immunology, (2) 2 Center for Infectious Disease, La Jolla Institute for Immunology
   
 

The comparative resistance of some cancers including head and neck squamous cell carcinoma (HNSCC) to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens (NeoAg). SCCVII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity within syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation during in vitro recall assays. Prophylactic immunization with a mixture of all stimulatory peptides protected hosts from subsequent tumor challenge. However, these peptides were not therapeutically beneficial in vivo unless the Cltc NeoAg, eliciting both CD4+ and CD8+ T cell responses, was used as an immunotherapy alone. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection via boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.

   



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  Toll-like receptor 9 is required for the maintenance of CD25+FoxP3+CD4+ Treg cells during Listeria monocytogenes infection
  Presenter: Joseph S. Dolina
  All Authors:Joseph S. Dolina, Joey Lee, Stephen P. Schoenberger
  La Jolla Institute for Allergy and Immunology
   
 

It has long been appreciated, but not understood, that the CD8+ cytotoxic T lymphocyte (CTL) dependence on CD4+ T cell help (Th) is conditional; needed for some immunogens but not others. One explanation for this phenomenon envisions Th requirement as an intrinsic property of the pathogen itself rather than its introduction to the immune system. Here we show that dependence of the optimal CD8+ T cell response to Listeria monocytogenes (Lm) on CD4+ T cells is a function of the immunogen dose used for priming, with low dose Lm (LD; 50 or 10^3 CFU WT or ΔActA, respectively) inducing a primary antigen-specific CTL response profoundly dependent on CD4+ Th cells while that induced by high dose Lm (HD; 4×10^3 or 10^6 CFU WT or ΔActA, respectively) is significantly inhibited by CD4+CD25+FoxP3+ regulatory T cells (Treg). The Th-independence of HD immunization is not overcome by additional antigen but instead involves the inflammatory response to more bacteria. Evaluation of various TLR pathways as the relevant sensing mechanism showed that HD immunization in the absence of TLR9/IL-12 signaling results in a simultaneous loss of CD25+FoxP3+CD4+ Treg cells and increase in conventional CD4+ Th cells and CTLs. Our data thus reveal that the CTL response to the same pathogen is determined by distinct roles for CD4+ T cells as helpers versus regulators based on immunogen dose and demonstrate a previously undescribed role for TLR9 in the regulation of CD4+ Th and Treg cells.

   



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  Toll-like receptor 9 is required for the maintenance of CD25+FoxP3+CD4+ Treg cells during Listeria monocytogenes infection
  Presenter: Joseph S. Dolina
  All Authors:Joseph S. Dolina, Zbigniew Mikulski, Joey Lee, and Stephen P. Schoenberger
  La Jolla Institute for Allergy and Immunology
   
 

It has long been appreciated, but not understood, that the CD8+ cytotoxic T lymphocyte (CTL) dependence on CD4+ T cell help (Th) is conditional; needed for some immunogens but not others. One explanation for this phenomenon envisions Th requirement as an intrinsic property of the pathogen itself rather than its introduction to the immune system. Here we show that dependence of the optimal CD8+ T cell response to Listeria monocytogenes (Lm) on CD4+ T cells is a function of the immunogen dose used for priming, with low dose Lm (LD; 50 or 10^3 CFU WT or ?ActA, respectively) inducing a primary antigen-specific CTL response profoundly dependent on CD4+ Th cells while that induced by high dose Lm (HD; 4×10^3 or 10^6 CFU WT or ?ActA, respectively) is significantly inhibited by CD4+CD25+FoxP3+ regulatory T cells (Treg). The Th-independence of HD immunization is not overcome by additional antigen but instead involves the inflammatory response to more bacteria. Evaluation of various TLR pathways as the relevant sensing mechanism showed that TLR2 is required for CTL responses to LD immunization, and that HD immunization in the absence of TLR9 results in a simultaneous loss of CD25+FoxP3+CD4+ Treg cells and increase in conventional CD4+ Th cells and CTLs. Our data thus reveal that the CTL response to the same pathogen is determined by distinct roles for CD4+ T cells as helpers versus regulators based on immunogen dose and demonstrate a previously undescribed role for TLR9 in the regulation of CD4+ Th and Treg cells.

   



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  Cytotoxic T lymphocyte dependence on CD4+ T cell help is controlled by pathogen dose during microbial infection
  Presenter: Joseph S. Dolina
  All Authors:Joseph S. Dolina, Joey Lee, Lara Labarta-Bajo, and Stephen P. Schoenberger
  La Jolla Institute for Allergy and Immunology
   
 

The in vivo priming, expansion, and survival of CD8+ cytotoxic T lymphocytes (CTL) generally require CD4+ T cell help via ligation of CD40L expressed by activated CD4+ T helper (Th) cells with CD40 on antigen presenting cells (APCs). Whether a CD8+ T cell response is help-dependent or -independent relies on the pathogen, where help-independent CTL responses have been documented for vaccinia virus, lymphocytic choriomeningitis virus, and Listeria monocytogenes (LM). Using LM as a model, we found that the dose, not identity, of a replicating pathogen determines the dependence on CD4+ T cell help. Additional inflammation induced by more bacteria, rather than antigen, underlies this dose response, where removal of CD4+ T cells prevented the provision of help at low dose and boosted the primary CTL response at high dose. Based on immunogen dose, CD4+ T cells can thus be divided into CD4+ Th cells and T regulatory (Treg) cells. The requirement for CD4+ T cells in helping the primary response is restricted at the earliest time points, while their role in regulating it occurs at later time points. The removal of CD4+ Treg cells produces significantly larger primary CTL responses from both low and high doses, which translates into canonical help-dependent immune responses after secondary immunization with non-inflammatory cellular antigen. The paradox in the nature of help-dependence at differing bacterial doses can therefore be explained by heterogeneity seen in the CD4+ T cell response, which is comprised of both Th and Treg cells.

   



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  Exclusive expression of an exon 2-deficient Foxp3 isoform promotes IgE-driven cutaneous autoimmunity
  Presenter: Phillip Domeier
  All Authors:Phillip Domeier, Jianguang Du, Sabine Spath, Baohua Zhou and Steven Ziegler
  Benaroya Research Institute
   
 

In humans, the FOXP3 gene encodes multiple protein products from alternative splicing of the mRNA transcript, but mice only encode the full-length isoform. Regulatory T cells (Tregs) that express the exon 2-deficient isoform (FoxP3.ΔExon2) elicit impaired Treg function. Furthermore, in humans, the development of autoimmunity correlates with elevated expression of the FoxP3.ΔExon2 isoform by an undefined mechanism. To determine how the FoxP3.ΔExon2 isoform contributes to autoimmunity in vivo, we developed mice that express an exon 2-deficient isoform of the FoxP3 gene (called B6.FoxP3.ΔExon2). B6.FoxP3.ΔExon2 mice develop mild systemic autoimmune disease, characterized by splenomegaly, cutaneous lesion formation and mild kidney nephritis. Furthermore, these mice exhibit defective T follicular regulatory (Tfr) cell activity, resulting in the development of increased germinal center frequency and elevated serum IgE titers in the absence of overt infection or immunization. As compared to wild-type (C57BL/6, B6) littermates, B6.FoxP3.ΔExon2 mice have elevated Keratin 14-reactive IgE, increased deposition of IgE in the keratinocyte layer of the skin and greater numbers of IgE-producing cells in skin-draining lymph nodes. Collectively, we show that exon 2 region of the mouse Foxp3 gene is crucial for regulation of germinal center-derived IgE autoantibody formation and IgE-driven cutaneous disease.

   



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  Nrf2 Protects Against Immune Dysfunction Following Burn Injury
  Presenter: Sarah J. Dulson
  All Authors:Sarah J. Dulson, Tim Eitas, Lucas Sjeklocha, Michelle Mac, Shannon Wallet, Bruce Cairns and Robert Maile
  University of North Carolina at Chapel Hill
   
 

Patients with severe burn injury experience an acute systemic immune dysfunction. This results in increased susceptibility to potentially devastating hospital-acquired infections. Additionally, pulmonary stress can originate from systemic inflammation in burn patients and is compounded by inhalation injury. The transcription factor Nuclear Factor-Erythroid-2-Related-Factor (Nrf2) activates downstream of innate sensing receptors and engages an antioxidant and detoxifying gene program that serves to limit immunopathology. We find that Nrf2-deficient (Nrf2-/-) mice are unable to control immune responses following burn and burn + inhalation (B+I) injury, demonstrated by increased edema, vascular permeability, and mortality. Furthermore, significantly higher concentrations of circulating cytokines, including IL-6, TNFα, and MCP-1, were measured in Nrf2-/- animals following B+I injury. Interestingly, lung tissue from wildtype mice show increased expression of Nrf2 after injury, but analysis of its cellular localization revealed that most Nrf2 was confined to the cytoplasm and, thus, unable to direct gene transcription. Therefore, we conclude that Nrf2 is required following B+I injury but insufficiently activated. We propose that therapeutic enhancement of the Nrf2 pathway is a novel method to counteract immune dysregulation and could lead to improved patient outcomes following burn injury.

   



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  Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility
  Presenter: Joseph Dynes
  All Authors:Joseph Dynes, Tobias X. Dong, Shivashankar Othy, Milton L. Greenberg, Amit Jairaman, Chijioke Akunwafo, Sabrina Leverrier, Ying Yu, Ian Parker, and Michael D. Cahalan
  Department of Physiology & Biophysics, University of California, Irvine
   
 

Seemingly random patterns of movement help T cells search for antigen in the lymph node. Because prominent Ca2+ signals accompany T cell activation and lead to cell arrest and dramatic changes in cell morphology, we hypothesized that more limited Ca2+ signaling might tune patterns of interstitial T cell motility in the absence of antigen. Blocking Orai1 Ca2+ channel activity in human T cells by expression of a dominant-negative Orai1-E106A construct led to migration with higher average velocities than controls, both in reconstituted mouse lymph nodes in vivo and in confined microchannels in vitro. In particular, we found that the increase in average cell velocity was not due to an increase in maximum cell velocity, but to a reduced frequency of cell pausing accompanied by increased directional persistence, resulting in longer and straighter paths. Isolated human T cells demonstrated Ca2+ transient-associated and Orai1-dependent pauses in vitro within confined microchannels devoid of cell-extrinsic factors. Furthermore, we used a novel ratiometric genetically encoded Ca2+ indicator, Salsa6f, along with T cells from CD4-Salsa6f transgenic mice, to show that intermittent Ca2+ signals coincide with reduced cell velocity. Treatment of CD4-Salsa6f mice with MHC class-I and -II blocking antibodies substantially reduced but did not eliminate the frequent T cell Ca2+ transients seen in lymph nodes. These experiments support the existence of an Orai1-dependent cell motility program that leads to pausing and turning of T cells moving within lymph nodes. Such Orai1-dependent pauses are triggered in at least two different ways: by self-peptide MHC complexes displayed on the surface of APCs and by a novel cell intrinsic mechanism within the T cells themselves. Together, these mechanisms generate motility patterns that underlie immune surveillance in the lymph node.

   



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  AhR acts independently of Foxp3 to mediate suppression of pancreatic infiltration in NOD mice
  Presenter: Allison Ehrlich
  All Authors:Allison Ehrlich, Jamie Pennington, Xisheng Wang, Nancy I. Kerkvliet
  Oregon State University
   
 

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has been associated with potent immunosuppression. We have previously reported that suppression of type 1 diabetes by TCDD, a potent AhR ligand, is associated with an increased percentage of Foxp3+ regulatory T cells. Similar associations between AhR activation and increased Foxp3+ cells have been reported for suppression of other autoimmune diseases. Based on the ability of AhR ligands to induce Foxp3+ Tregs, AhR is a potential target for the treatment of autoimmune disease. We have recently discovered a novel, rapidly metabolized AhR ligand, 10-chloro-7H-benzimidazo[2,1-a]benzo[de]Iso-quinolin-7-one (10-Cl-BBQ), that activates AhR in CD4+ T cells in a similar manner to TCDD. Here we show oral treatment with 10-Cl-BBQ is highly effective in suppressing pancreatic islet infiltration throughout 20 weeks of age in the NOD mouse model. When we looked at changes in Foxp3+ cells, a small yet significant increase in the frequency, but not total number, of Foxp3+ cells was observed. This raises the yet unanswered question, is the increase in Foxp3+ Tregs driving AhR-mediated immune suppression? To directly assess the requirement of Foxp3+ cells in AhR induced suppression of islet infiltration, transgenic NOD.Foxp3.DTR (human diphtheria toxin receptor under the control of Foxp3) mice were employed. In the vehicle control group, Foxp3 depletion led to rapid pancreatic islet infiltration. However, treatment with 10-Cl-BBQ suppressed infiltration even in the absence of Foxp3+ cells. These results suggest that AhR functions as a Foxp3-independent transcription factor driving suppression of T cell dependent immune responses.

   



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  The intestinal lamina propria is a unique niche for regulatory T cells
  Presenter: Cruz Elisa
  All Authors:Cruz Elisa, Terri Laufer
  University of Pennsylvania
   
 

Foxp3+ regulatory T cells (Tregs) are critical to maintain intestinal homeostasis. Like all CD4+ T cells, Tregs require TCR-MHCII interactions for their development in thymus and differentiation in periphery. Past studies suggest that the TCR repertoire of intestinal lamina propria (siLP) Tregs is skewed towards food and commensal bacteria antigens. However, by using a transgenic mouse model that lacks peripheral MHCII expression and peripheral Tregs induction (K14 mice), we have demonstrated that thymically generated Tregs could enter to the siLP of weanlings and proliferate independently of MHCII to fill and maintain the compartment. Newly-generated Tregs egress from thymus as central Tregs and acquire an effector phenotype after TCR signals in periphery. However, despite the lack of MHCII in the siLP, K14 Tregs are activated and phenotypically similar to effector Tregs in WT siLP. Interestingly, transcriptional profiling reveals that many components of a TCR-dependent expression module are present in both K14 and WT siLP Tregs. Furthermore, differently to splenic Tregs, K14 siLP Tregs highly express Nur77 and IRF4, often regulated through TCR signaling. Tregs from K14 siLP localize mainly at isolated lymphoid follicles, where they are in proximity to B cells and dendritic cells; suggesting that siLP Tregs may still receive MHCII-independent signals from APCs, perhaps via costimulation molecules and/or paracrine cytokine stimulation. Indeed a short-term costimulation blockade with CTLA4-Ig reduces Nur77 expression on K14 siLP Tregs. Taken together, these results suggest that the intestinal microenvironment uniquely maintains the differentiation and homeostasis of effector Tregs independent of MHCII-TCR interactions.

   



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   CD44 controls T cell exhaustion and viral persistence during chronic viral infection.
  Presenter: Carrette Florent
  All Authors:Carrette Florent, Tinoco Roberto, Barraza Monique, Bradley Linda
  Sanford Burnham Medical Research Institute
   
 

During a chronic viral infection, inhibitory receptors play a crucial role in controlling viral persistence and T cell exhaustion. However, the role of homing molecules in this process has been poorly investigated. Using the chronic LCMV virus model, Clone 13, we found that expression of CD44, a cell surface glycoprotein broadly used to identify activated T cells, dampens antigen specific T cell responses. In CD44-deficient hosts, we observed a significant increase in antigen specific CD4 and CD8 T cells functions with decreased PD-1 expression and a striking increase in multiple cytokine production. T cell accumulation was not due to increased proliferation based on BrdU incorporation, and the increased CD8 T cell response required CD4 T cell help because CD8 T cell exhaustion was maintained in CD4 depleted CD44-deficient mice. Using a bone marrow chimera approach, we found that restricting the CD44 deficiency in the non-hematopoietic compartment was sufficient to reproduce the observations made in the complete CD44 deficient hosts. Finally CD44-deficiency resulted in viral clearance by d15pi. Importantly, treatment of WT mice with a CD44-blocking antibody increased antigen specific CD4 and CD8 T cell recovery and some aspects of T cell function as early as d9pi. Taken together, these results indicate that CD44 is a novel inhibitory receptor that can be targeted to improve T cell response during chronic viral infections.

   



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  Type I Interferon-mediated Activation of IRF1 Underlies the Distinct Inflammatory Responses Elicited by Type I and Type III Interferons
  Presenter: Adriana Forero
  All Authors:Adriana Forero, Snehal Ozarkar, Hongchuan Li, Chia H Lee, Emily A Hemann, Marija S Nadjsombati, Matthew R Hendricks, Lomon So, Richard Green, Chandra N Roy, Saumendra N Sarkar, Jakob von Moltke, Stephen K Anderson, Michael Gale Jr, Ram Savan
  University of Washington, Department of Immunology
   
 

The epithelium is a highly proliferative tissue under constant exposure to pathogenic and commensal microbes. There type I (α/β) and III interferons (λ) (IFNs) activate similar downstream signaling cascades that result in kinetic differences in the expression of antiviral IFN-stimulated genes (ISG). We address the fundamental question of why these two IFN families are required to prevent viral dissemination at barrier sites by providing some of the molecular mechanisms underlying the disparity in their ability to induce inflammation. IFNα/β singularly induce expression of the transcription factor IRF1, with little to no IRF1 induction observed after IFN? treatment. Through genome-wide expression analysis, we demonstrate that while IRF1 expression is dispensable for the antiviral activity of IFNα/β, it is necessary for the induction of inflammatory chemokines and immune cell infiltration. The muted induction of IRF1 by IFNλ is due to insufficient STAT1 activation and homodimerization given the limited IFNλ receptor 1 subunit (IFNLR1) expression which is refractory to inflammatory stimuli or viral challenge. However, exogenous overexpression of IFNLR1 can enhance both antiviral and chemokine gene expression through the induction of IRF1 demonstrating how IFNLR1 expression regulation is crucial for preventing deleterious inflammation. On the other hand, our gene expression profiling identified genes that were expressed uniquely in IFNλ stimulated cells. Gene set enrichment and regulatory analyses suggested that many of these genes are involved in pathways consistent with the resolution of inflammation and the maintenance of barrier function. Overall, we show that IFNα/β and IFNλ work in concert to sustain antiviral immunity and limit tissue damage at sites of pathogen exposure. The transient inflammatory responses to IFNα/β help recruit immune effectors to promote protective immunity while IFN? act as potent antiviral effectors and restricting tissue damage.

   



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  Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer
  Presenter: Anusha Preethi Ganesan1, 2
  All Authors:Anusha Preethi Ganesan1, 2, Oliver Wood3, Eva M Garrido-Martin4, Serena Chee3, Toby Mellows4, James Clarke3, Daniela Samaniego-Castruita1, Divya Singh1, Grégory Seumois1, Aiman Alzetani5, Edwin Woo5, Peter S Friedmann4, Gareth J Thomas3, Emma V King3, Tilman Sanchez-Elsner4, Pandurangan Vijayanand1,4,6, Christian H Ottensmeier 3,5,6.
  1La Jolla Institute for Allergy & Immunology, La Jolla, CA, USA. 2Division of Pediatric Hematology Oncology, Rady Children’s Hospital, University of California San Diego, San Diego, CA, USA. 3Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom. 4 Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine University of Southampton, Southampton, United Kingdom. 5Southampton University Hospitals NHS foundation Trust, Southampton, United Kingdom. 6These authors jointly directed this work.
   
 

Therapies that boost the anti-tumor responses of cytotoxic lymphocytes (CTLs) have shown promise in the clinic. However, clinical responses to currently available immunotherapeutic agents vary considerably, for which the molecular basis is unclear. To date, unbiased transcriptomic studies of CTLs in human cancers have been performed in whole tumors or cells obtained from peripheral blood or metastatic sites of heavily pre-treated patients. We performed global transcriptional profiling of CTLs in tumors and adjacent non-tumor tissue from treatment-naïve patients with early stage lung cancer to define the molecular features associated with robustness of anti-tumor immune responses. We observed major differences in the transcriptional program of tumor-infiltrating CTLs that are shared across tumor subtypes. Pathway analysis revealed enrichment of genes in cell cycle, T cell receptor (TCR) activation and co-stimulation pathways, indicating tumor-driven expansion of presumed tumor antigen-specific CTLs. We also observed marked heterogeneity in the expression of molecules associated with TCR activation and immune checkpoints such as 4-1BB, PD1, TIM3, and their expression was positively correlated with the density of tumor-infiltrating CTLs. Interestingly, transcripts linked to tissue-resident memory cells (TRM), such as CD103, were enriched in tumors containing a high density of CTLs, and CTLs from CD103high tumors displayed features of enhanced cytotoxicity, implying better anti-tumor activity. In an independent cohort of 689 lung cancer patients, we confirmed that patients with CD103high (TRM rich) tumors survived significantly longer. In summary, we define the molecular fingerprint of tumor-infiltrating CTLs at the site of primary tumor and identify a number of novel targets that may be important in modulating the magnitude and specificity of anti-tumor immune responses in lung cancer.

   



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  TLR4 activation triggers energy conservation programs in mice
  Presenter: Kirthana Ganeshan
  All Authors:Kirthana Ganeshan, Kevin Man, Yoshitaka Sogawa, Xiaojin Cui, Ajay Chawla
  UCSF
   
 

Organisms acquire and allocate energy to support programs of growth, reproduction and tissue maintenance. Activation of immunity is a high-cost stress response that can force the organism to reallocate available energy to support programs that improve host fitness. Indeed, the activation of immunity can often pose a significant metabolic challenge for the host, and as such; it is likely that immunity may compete with host maintenance programs for energy. However, the nature of these physiologic trade-offs and their significance remain poorly understood. To investigate this question, we activated immunity in mice that had a competing need for energy to maintain homeothermy (mice housed under sub-thermoneutral conditions (22°C)). Using LPS-activated immunity as a model system, we determined that mice housed at 22°C choose to lower metabolic rate and abandon homeothermy in an effort to conserve energy as compared to thermoneutral mice (housed at 30°C). The activation of this energy conservation program is dependent on hematopoietic sensing of LPS. Metabolomic analysis of plasma from the entry and exit phases of this programed hypothermia revealed that entry into hypothermia is supported by the catabolism of amino acids, while exit is fueled by fatty acids and the activation of UCP1-dependent thermogenesis. The decision to engage in such an energy-conserving program ultimately enhances organismal fitness, providing a potential mechanism by which mammals overcome unanticipated environmental adversities.

   



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  Egr2 is required for the activation of the TH17 pathogenetic program
  Presenter: Yuanyuan Gao
  All Authors:Yuanyuan Gao, Yan Wang[1], Nicolas Bouladoux[2], Yasmine Belkaid[2], and Vanja Lazarevic[1]
  [1]Experimental Immunology Branch, Center of Cancer Research, NCI/NIH[2]Mucosal Immunology Section, Laboratory of Parasitic Diseases, NIAID/NIH
   
 

TH17 lineage cells are a heterogenous population of CD4+ effector cells. Some subsets of TH17 cells play an important role in the host defense, while others participate in the pathogenesis of many inflammatory and autoimmune diseases. TH17 cells can alter their differentiation program ultimately giving rise to either protective non-pathogenic or pro-inflammatory pathogenic cells. Molecular switches that control the development of “pathogenic” versus “non-pathogenic” TH17 cells remain largely unknown. In this study, we identified the transcription factor Egr2 as a critical regulator of the TH17 pathogenic program. We found Egr2 was transiently expressed in the early-intermediate stage (24-48 hours) of TH17 differentiation. When ectopically expressed under TH17-polarizing conditions, Egr2 significantly enhanced the expression of TH17 signature genes in a RORγt-dependent manner. Although Egr2 was dispensable for TH17 lineage commitment, its expression was required for the generation of pathogenetic TH17 cells. Mice with T cell-specific deletion of Egr2 were less susceptible to experimental autoimmune encephalomyelitis (EAE) than WT mice. Myelin-specific Egr2-deficeint TH17 cells failed to induce a chronic inflammatory response in the CNS. Transcriptional analysis revealed that Egr2 promoted the pathogenicity of TH17 cells by regulating the expression of pathogenicity-associated genes. Interestingly, Egr2 was not required for the effector function of protective gut-resident TH17 cells in response to Citrobacter rodentium challenge. These findings indicate that Egr2 represents an attractive candidate for the therapeutic targeting of pathogenic TH17 cells while preserving tissue-protective functions of TH17 cells at barrier sites.

   



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  mTORC1 signaling activates a proactive UPR in plasma cell differentiation prior to XBP1 splicing.
  Presenter: Brian Gaudette
  All Authors:Brian Gaudette, David Allman
  University of Pennsylvania
   
 

The generation of antibody-secreting plasma cells (PCs) from mature, naïve B cells requires a shift in transcriptional programs leading to striking changes in cellular morphology and physiology. It is thought that the lynchpin of this process is initiation of the unfolded protein response (UPR) through activation of the XBP1 transcription factor downstream of Blimp1-dependent increases in immunoglobulin protein translation. The paradox of a purely reactive, endoplasmic reticulum (ER)-stress-dependent UPR in PC differentiation is the observed lack of PERK-dependent translation inhibition, which is intrinsic to the canonical UPR. By contrasting the UPR activation in two subsets of naïve B cells with different kinetics of PC differentiation, follicular and marginal zone (MZ) B cells, we are able to differentiate between mTORC1-dependent and ER stress-dependent activation of canonical UPR targets. Our results indicate that MZ B cells, which more rapidly differentiate to PCs, have increased mTORC1 activity at baseline, which is accompanied by increased expression of ER protein folding machinery including negative regulators of PERK activation. This priming of the UPR in MZ B cells is independent of extrinsic activation as it occurs in germ free mice and in mice deficient in TLR and BCR signaling. Furthermore, we demonstrate an mTORC1-dependent increase in expression of canonical UPR targets prior to both Blimp1 and XBP1 activity in naïve B cells under PC-inductive conditions. Thus, activation of the mTORC1 pathway in early PC differentiation primes the ER to allow for rapid increase in protein production in the absence of canonical ER stress.

   



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  The Notch-Myc axis sets the division-differentiation clock in marginal zone B cells
  Presenter: Brian Gaudette
  All Authors:Brian Gaudette, Carly Roman, Daniela Gómez Atria, Ivan Maillard, David Allman
  University of Pennsylvania
   
 

Lymphocyte differentiation is often tightly linked to mitosis. Clonal bursts due to antigen- or TLR-driven responses increase numbers of responding cells and may also facilitate changes in gene expression and chromatin needed for effector cell differentiation. Alternatively, to guard against rapid infection, lymphocyte pools may also contain cells poised for effector differentiation with minimal proliferation. Using cell cycle inhibitors or induced mutation of CDK1, we directly compared the impact of arresting mitosis on early plasma cell (PC) differentiation for naive follicular and marginal zone (MZ) B cells. MZ B cells reside in the marginal sinus of the spleen where they are positioned to respond rapidly to blood borne microbes. Whereas PC differentiation from follicular B cells occurred after only 4 or more divisions and was highly dependent on mitosis, MZ B cells yielded PCs much faster and despite full cell cycle blockade. Furthermore, short-term withdrawal of Notch signaling in MZ B cells in vivo caused rapid and robust down-regulation of large numbers of established Myc-regulated genes, and resulted in division/differentiation kinetics that mirrored follicular B cells. Altogether these results suggest that ongoing Notch signaling establishes a differentiation-poised state in MZ B cells needed for rapid division-independent antibody responses.

   



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  Integration of IFN[g] and integrin signaling pathways regulates CD8 T cell differentiation
  Presenter: Audrey Gerard
  All Authors:Audrey Gerard, Matthew F Krummel
  UCSF
   
 

CD8 T cell responses to IFNγ result in diverse and sometimes antagonizing functions. In particular, IFNγ has a dual function in T cell homeostasis, promoting priming, expansion and differentiation of CD8 T cells as well as apoptosis of effector T cells. The cellular and molecular mechanisms underlying the functional dichotomy of IFNγ have not yet been identified. To answer this question, we characterized IFNγ secretion and function over time following Listeria infection. We observed a first wave of IFNγ production early after infection, which was specifically implicated in CD8 T cell differentiation. Whereas both NK cells and early activated CD8 T cells produced IFNγ, only IFNγ produced by CD8 T cells affected CD8 T cell differentiation. IFNγ is shared between recently activated CD8 T cells in the context of secondary T-T synapses, which are required for IFNγ-induced CD8 T cell differentiation. We demonstrated that integrin signaling shapes IFNγ signaling in this context. Recently activated CD8 T cells have impaired Stat1 tyrosine, but not serine, phosphorylation following IFNγ exposure. Integrins can rescue Stat1 tyrosine phosphorylation through Src kinases activation. This suggests that IFNγ leads to differential gene expression patterns according to the microenvironment (i.e whether IFNγ is received in the context of contacts), thereby dictating the functional outcome of recently activated CD8 T cells. Deciphering how IFNγ specific outcomes are established and regulated will be crucial to design better-targeted therapies, allowing us to trigger a specific functional outcome.

   



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  Interleukin-8 receptor-dependent B cell migration governs postnatal Peyer’s patch development and confers protection to Salmonella infection
  Presenter: Romana R. Gerner
  All Authors:Romana R. Gerner, Suzi Klaus, Kareem Siada, Purnima Sharma, Flavian Thelen, Araceli Perez-Lopez, Marcus P. Wong, Victor Lei, Robert A. Edwards, Richard Ransohoff, Tom E. Lane, David Lo, Sean-Paul Nuccio, Elina Zuniga, and Manuela Raffatellu
  Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, La Jolla, CA
   
 

Chemokine-dependent signaling in immune cells is a major mechanism to recruit leukocytes to sites of infection or tissue damage but also guides homeostatic leukocyte trafficking and immune cell positioning. The chemokines Cxcl1 and Cxcl2 are highly up regulated in mouse cecal tissue during Salmonella-induced colitis. Its cognate receptor CXC-chemokine receptor-2 (CXCR2), also known as the Interleukin-8 receptor beta (IL-8RB), is required for neutrophil recruitment and plays critical roles in immunity. Herein, we investigated CXCR2’s contribution to the mucosal immune response during intestinal Salmonella infection and found a novel neutrophil-independent role for CXCR2 on B cells. In line with earlier reports, Cxcr2-/- mice exhibited a strongly impaired neutrophil recruitment to the gut resulting in increased susceptibility to Salmonella dissemination. Moreover, we observed a 100-fold higher colonization in Peyer’s patches (PP) compared to wildtype littermates, which could be phenocopied by treatment with CXCR2-neutralizing antibodies, but not in mice selectively lacking Cxcr2 in granulocytes, or upon antibody-mediated neutrophil depletion. Cxcr2-/- mice did not display PP hypertrophy, a hallmark of intestinal Salmonella infection, and instead exhibited a strong decrease in PP B cell numbers. At baseline, Cxcr2-/- mice also exhibited hypoplastic PP with lower B cell numbers, but similar numbers of PP compared to wildtype mice, indicating that CXCR2 is not involved in the prenatal phase of PP formation. Mixed bone marrow (BM) chimera experiments confirmed that wildtype BM was sufficient to repopulate PP in B cell-deficient recipients (i.e., muMT-/-), whereas Cxcr2-/- BM did not result in PP formation, suggesting that CXCR2 is critical for B cell homing to PP. Furthermore, CXCR2-mediated B cell migration to PP was dependent on microbial signals, as germ-free mice developed normal PP after colonization with a low complexity microbiota, which was prevented in mice concomitantly treated with CXCR2-neutralizing antibody.Collectively, our study identifies CXCR2 as a previously unrecognized B cell receptor, and uncovers its important role in B cell homing to Peyer’s patches and in host defense against a gut pathogen.

   



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  Pathogenic Th1 and Th17 cells during EAE are influencing regulatory T cells functions
  Presenter: Simon GLATIGNY
  All Authors:Simon GLATIGNY, Simon Glatigny, Estelle Bettelli
  Benaroya Research Institute
   
 

Multiple sclerosis (MS) is an autoimmune, inflammatory neurodegenerative disease of the central nervous system (CNS) characterized by demyelination and progressive loss of motor functions. Clinical relapses are thought to be due to multifocal infiltration of immune cells, including Thelper (Th) cells, and B cells, resulting in severe demyelination and ultimately leading to degeneration of neuronal structures. Auto-reactive CD4+ Th cells play an important role in the initiation and progression of MS and its mouse model experimental autoimmune encephalomyelitis (EAE). Pathogenic CD4+ T cells are characterized by specific effector functions and secretion of specific cytokines. Inflammatory T cell functions can be limited effectively by regulatory T cells (Treg). However, Treg from MS subjects show poor suppressive capacities. New research indicates that the function of Tregs on their target is in part due to characteristics of the Treg and the effector T cell (Th1, Th17 or Th GM-CSF) populations to suppress. In recent years, it has been suggested that Tregs function can be compromised under inflammatory conditions and that this effect is due in part to their sensing of proinflammatory cytokines. Among them, IL-6 has been shown to promote the differentiation of Th17 cells and inhibit regulatory T cells. IL-6 signals through a receptor composed of a specific chain (IL-6Ra) and a common Glycoprotein 130 (gp130) chain. Gp130 is a signal-transducing receptor that forms part of the receptor complex for several pro and anti-inflammatory cytokines including IL-6, IL-27, IL-11, LIF and OSM. IL-6R and gp130 have been targeted for the development of improved disease modifying therapies in MS and other autoimmune diseases. However, it is not known whether neutralization of their activity has similar effect on regulatory T cell functions and the development of EAE. The aim of our study was to investigate the role of Treg specific elimination of IL-6R or gp130 and their consequences for the development of EAE. We induced EAE by the adoptive transfer of myelin oligodendrocyte glycoprotein (MOG35-55) specific Th cells into recipient mice with genetic ablation of gp130 or IL6R specifically in Treg (Foxp3Cre Gp130floxed or Foxp3Cre IL6Rfloxed). We show here that the transfer of Th1 cells induce comparable disease in control Foxp3Cre mice and Foxp3Cre IL6Rfloxed mice. In contrast, Foxp3Cre Gp130floxed mice developed exacerbated disease compared to Foxp3Cre IL6Rfloxed mice and control animals. Altogether, our data show that modulating IL-6R and gp130 signaling in Treg has differential effect on their function and on EAE development. Our data suggests that the modulation of IL-6 signaling in Treg does not impact their capacity to control Th1 responses. However, signaling through gp130 promote Treg functions and limit the development of EAE. Future experiments will focus on determining the mechanisms by which gp130 mediated signaling might enhance Treg functions and the progression of EAE and identify which gp130 signaling cytokine(s) can promote Treg activity in the context of EAE and MS.

   



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  Integrin alpha-4 is required for regulatory B cell control of experimental autoimmune encephalomyelitis
  Presenter: Simon GLATIGNY
  All Authors:Simon GLATIGNY, Catriona A Wagner and Estelle Bettelli
  Benaroya Research Institute
   
 

The neutralization of alpha-4 integrin (Itga4) is currently used as treatment in multiple sclerosis. While most studies focused on its function on lymphocyte migration to the central nervous system, we have uncovered the importance of Itga4 expression on B cells for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell targeting therapies.

   



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  Regulation of B cell functions by Integrin alpha 4 during EAE
  Presenter: Simon GLATIGNY
  All Authors:Simon GLATIGNY, Simon Glatigny, Katie Wagner, Rebekka Duhen, Carlos A Arbelaez, Mohamed Oukka and Estelle Bettelli
  Benaroya Research Institute
   
 

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), mediated by autoreactive myelin-specific T cells. B cells have been implicated in the pathogenesis of MS and EAE. While antigen-activated B cells can differentiate into antibody-secreting plasma cells and serve as potent antigen presenting cells (APCs), other B cell subsets are substantially weaker APCs and may exert anti-inflammatory properties, modulating the effector functions of other immune cells. Furthermore, B cells have been shown to play a regulatory role during EAE as B-cell deficient animals failed to recover and the transfer of a splenic CD1dHigh CD5+ B cell subset promoted the recovery of mice from EAE. Itga4 is important for the entry of lymphocytes in the CNS under inflammatory conditions. However, data from our laboratory showed that elimination or neutralization of Itga4 differentially affect the entry of Th1, Th17 and Treg cells into the CNS. The goal of this study was to determine how Itga4 modulates B cell homing and functions during the course of EAE. Using mice with a specific deletion of Itga4 on B cells (CD19Cre Itga4flox mice), we show that Itga4 control EAE severity since CD19Cre Itga4flox mice develop more severe EAE than control mice. CD19Cre Itga4flox mice have limited number of splenic marginal zone B cells and IL-10 producing B cells infiltrating the CNS compared to control mice. In contrast, Itga4 deficient transitional and follicular splenic B cells induce a stronger T cell response than their Itga4 sufficient counterparts. This study reveals that Itga4 act as a critical regulator of the homing and function of B cell subsets during EAE.

   



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  Activation of human dendritic cells in malaria
  Presenter: A. Goetz
  All Authors:A. Goetz, Maureen Ty, Ana Rodriguez
  NYU School of Medicine
   
 

Malaria is characterized by cyclical fevers and high levels of inflammation, and while an early inflammatory response contributes to parasite clearance, excessive and persistent inflammation can lead to severe forms of the disease. At the same time, malaria infections fail to induce durable immunological memory and knowledge of anti-malarial immunity is incomplete. Dendritic cells (DCs) are not only important for the early cytokine responses but also essential for bridging and regulating the innate and adaptive immune responses to pathogenic infections. Very little is known about the role DCs play in the immune response to Plasmodium. Results of studies in both human and animals have been contradictory. To address the role of DCs in malaria immune response we purified human DCs from peripheral blood of healthy donors and co-incubated them with P. falciparum schizonts in vitro. While DCs up-regulated surface expression of the activation markers HLA-DR, CD80, CD86, and CD40, they failed to secrete significant amounts of cytokines, namely IL-8, IL-6, IL-10, TNF, IL-1beta, and IL-12p70. Analysis of the two main human DC sub-populations, plasmacytoid (pDCs) and myeloid DCs (mDCs), revealed that mDCs up-regulated all co-stimulatory markers mentioned above while pDCs only up-regulated HLA-DR. To examine whether P. falciparum-activated human DCs are able to activate and polarize T cells without producing significant amounts of cytokines we will co-incubate DCs with autologous T cells and analyze proliferation and cytokine production. Considering the inadequate immune response to the parasite, Plasmodium could evade the immune system by activating DCs in a suboptimal way, especially in early stages of the disease or low parasitemia.

   



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  Dendritic-T cell interactions in blood stage malaria: the role of reactive oxygen species
  Presenter: Anton Goetz
  All Authors:Anton Goetz, Maureen Ty, Ana Rodriguez
  NYU School of Medicine
   
 

Malaria is characterized by high levels of inflammation, and while an early inflammatory response contributes to parasite clearance, excessive and persistent inflammation can lead to severe forms of the disease. At the same time, malaria infections fail to induce durable immunological memory and knowledge of antimalarial immunity is incomplete. Very little is known about the role dendritic cells (DCs) play in the immune response to Plasmodium and how they contribute to activation of CD4+ T cells during blood stage malaria. To address the role of this critical part of the malaria immune response we purified primary human DCs from peripheral blood of healthy naïve donors and co-incubated them with blood stage P. falciparum in vitro. The Although DCs up-regulated surface expression of HLA-DR and co-stimulatory markers, they did not secrete significant amounts of cytokines. Since the lack of cytokine production seems contradictory to the pathogenesis of the disease, especially accompanying up-regulation of activation markers on DCs, we hypothesized that there might be another inflammatory factor involved in vivo. Xanthine oxidase (XO), an enzyme that produces reactive oxygen species, is increased during malaria infection, positively correlates with the severity of the disease, and can activate immune cells. Supplementation of the cultures with XO rescued cytokine secretion by DCs to LPS control levels. We then co-incubated P. falciparum-activated DCs with naïve autologous CD4+ T cells. Although parasite-activated DCs secreted only low levels of cytokines, they were able to activate and polarize naïve CD4+ T cells into Th1 effector cells. A re-stimulation with autologous P. falciparum-activated DCs specifically increased proliferation and cytokine secretion of those primed CD4+ T cells compared to control DCs. Addition of XO to co-cultures increased an initial proliferation response but ultimately lead to low total numbers of responsive T cells, indicating a modulatory effect of XO-produced reactive oxygen species on the immune response to malaria. Our findings might contribute to a better understanding of the mechanisms leading to the absence of sterile immunity in malaria which is relevant for future vaccine designs and therapeutic interventions.

   



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  Adipose tissue-resident ILC2 dysregulation underlies age-related metabolic impairments
  Presenter: Emily Goldberg
  All Authors:Emily Goldberg, Irina Shchukina, Tamara Dlugos, Yoon-Hee Youm, Christina D. Camell, Maxim Artyomov, Vishwa Deep Dixit
  Yale University
   
 

Aging is characterized by persistent low-grade inflammation believed to drive many chronic diseases, including insulin resistance, cardiovascular diseases, Alzheimer’s disease, and immunesenescence. A primary candidate source of this increased inflammation is the increased visceral adipose tissue that accumulates during aging. The adipose-resident immune compartment is responsible for maintaining tissue homeostasis and limiting inflammation. We therefore hypothesized adipose-resident immune changes during aging were responsible for elevated inflammation leading to collapse of metabolic health. To test this, we used scRNAseq to broadly assess age-related changes exclusively in adipose-resident immune cells in young and old mice. These experiments revealed a pronounced loss of type 2 innate lymphoid cells (ILC2s), which are critical regulators of adipose tissue metabolic homeostasis. We identified dysregulation of IL-33, an important regulator of adipose ILC2s, as a driving factor leading to loss of adipose ILC2s during aging. Notably, treating old mice with IL-33 restored ILC2 numbers in adipose tissue. However, metabolic health in old mice was not improved after IL-33 treatment and actually increased susceptibility of old mice to cold challenge. Instead, using RNAseq and adoptive transfer experiments, we found that aged ILC2 are intrinsically altered during aging to become pro-inflammatory and incapable of maintaining adipose tissue homeostasis. Altogether, our work reveals a novel immune-metabolic axis that controls adipose tissue inflammation and metabolic health during aging.

   



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  Ketogenic diet activates protective [g][d] T cell responses against influenza virus infection
  Presenter: Emily Goldberg
  All Authors:Emily Goldberg, Ryan D. Molony, Sviatoslav Sidorov, Eriko Kudo, Vishwa Deep Dixit, and Akiko Iwasaki
  Yale
   
 

Influenza A virus (IAV) infection-associated morbidity and mortality are a key global healthcare concern, necessitating the identification of novel therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing anti-viral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet, as neither feeding mice a high-fat high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD mediated immune-metabolic integration represents a viable avenue towards preventing or alleviating influenza disease.

   



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  Regulation of human Th17 cell differentiation by the DNA-binding transcription factor TEAD4
  Presenter: Jacob Gorman
  All Authors:Jacob Gorman, Christina Stracener, Bonnie Swerdlow, Geoffrey L. Stephens, Lorraine Clarke, Michael Fung, and Tomas Mustelin
  MedImmune
   
 

CD4 T cells differentiate into various subsets of cytokine-secreting cells based on the external cytokine cues they receive from antigen presenting cells. The cytokines that CD4 T cells express will ultimately determine how they influence the immune response. For example, IL-4-producing Th2 cells have been shown to be important for immune responses large multicellular organisms such as Helminths. However, Th2 cells also contribute to allergic asthma. Alternatively, IL-17-producing Th17 cells are important for the elimination of extracellular pathogens such as fungi. However, Th17 cells along with IFN-γ-producing Th1 cells contribute to autoimmune diseases such as multiple sclerosis (MS). Alternatively, in the mouse model of model of MS, experimental autoimmune encephalomyelitis, IL-4 produced by Th2 cells has been shown to be protective. It has been demonstrated that there is plasticity in CD4 T helper subsets and that CD4 T cells are able to convert into different cytokine-producing subsets. Therefore understanding the molecular mechanisms that regulate CD4 T cell plasticity will be instrumental in diverting CD4 T cells into a protective phenotype. We have found that the DNA-binding transcription factor, TEAD4 to be highly expressed in Th17 cells relative to Th1 or Th2 cells. TEAD4 lacks a catalytic domain and is dependent on the recruitment of cofactors to regulate gene expression. Induced overexpression of TEAD family transcription factors has been shown inhibit gene expression by titrating out coactivator gene induction. We have shown that TEAD4 overexpression during in vitro differentiation of human Th17 cells impairs their expression of IFN-? and IL-17A. Similarly, IL-17a expression is reduced when TEAD4 is overexpressed in murine Th17 cells. Consistent with regulation of Th17 differentiation, TEAD4 expression is induced by TGF-β stimulation of activated CD4 T cells. Understanding the mechanisms by which TEAD4 regulates Th17 cell differentiation will be critical to harness CD4 T cell plasticity and to divert these cells to protective phenotype.

   



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  A role for hypoxia inducible factors in tissue specific anti-viral CD8+ T cell responses
  Presenter: John Goulding
  All Authors:John Goulding, Anthony Phan, Siavash Namiranian, Ananda Goldrath
  Dept. Biological Sciences, UCSD
   
 

It has recently been established that components of the transcriptional response to hypoxia can influence the extent to which CD8+ T cells control persistent viral infection and neoplastic growth. These findings suggest that micro-environmental cues can regulate effector CD8+ T cell responses by controlling both differentiation and function. To further investigate this possibility, we used a localized Vaccinia virus (VACV) infection model to further dissect the implication of these signals on polyclonal CD8+ T cell responses. We now demonstrate that a lack of hypoxia-inducible factor (HIF) 1a and 2a expression in CD8+ T cells protects against illness severity, as measured by weight loss, and limits lung pathology. In addition, HIF 1a and 2a expression influences effector and memory subset differentiation and effector molecule expression in VACV specific CD8+ T cells in the lung. Collectively these data demonstrate the importance, and growing need, to better understand how local micro-environmental cues can influence the many transcriptional programs and molecular pathways that regulate CD8+ T cell differentiation. This understanding will inform efforts to better develop efficacious vaccines and therapeutic cell therapies.

   



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  A role for hypoxia inducible factors in CD4+ T cell differentiation
  Presenter: John Goulding
  All Authors:John Goulding, Laura Shaw, Kyla Omilusik & Ananda Goldrath
  University of California, San Diego
   
 

Immune cells are unique in their ability to migrate and respond dynamically to tissue specific contextual cues. It is increasingly apparent that established and transient micro-environmental niches that exist during homeostasis and tissue inflammation may influence cell fate decisions and subsequent function. Recent evidence suggests that hypoxia-inducible factors (HIFs), components of the transcriptional response to low oxygen levels, can influence the extent to which CD4+ T cells differentiate into specific T helper subsets. To further investigate this possibility, we utilized CD4+ T cells that lacked components of the HIF / Von Hippel-Lindau (VHL) pathway to dissect the implication of these signals on CD4+ T cell responses. We find that CD4+ T cells lacking HIF-1α and HIF-2α have altered CD4+ T cell subset differentiation as measured by archetypical cytokine production and master regulator transcription factor expression. Collectively these data demonstrate the importance, and growing need, to better understand how local micro-environmental cues can influence the many transcriptional programs and molecular pathways that regulate CD4+ T cell differentiation. This understanding will inform efforts to better develop efficacious vaccines and therapeutic cell therapies.

   



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  Functional Analysis of DNA Sensors in the Interferon Stimulatory DNA (ISD) Pathway
  Presenter: Elizabeth Gray
  All Authors:Elizabeth Gray, Dan Stetson
  University of Washington
   
 

Detection of viral DNA and production of type I interferons (IFNs) are essential for antiviral immunity; on the other hand, inappropriate immune responses to self DNA results in autoimmunity. Intracellular DNA sensing activates the STING-dependent IFN stimulatory DNA (ISD) pathway. The DNA receptors involved in activating this pathway are not fully characterized, although multiple sensors have been proposed. Recent work has defined cyclic GMP-AMP synthase (cGAS) as a key DNA sensor in the ISD pathway, and cGAS-deficient cells have a dramatically impaired IFN response to transfected DNA ligands, DNA viruses, and retroviruses. In addition, the AIM2-like receptors (ALRs) are an evolutionary diverse family of DNA-binding receptors that are also thought to participate in the ISD pathway. Several mouse and human ALRs activate the ISD pathway when overexpressed, and multiple studies have suggested that ALRs are required for an optimal IFN response to various DNA pathogens. On the other hand, others have found that the ISD pathway is intact following ALR knockdown. Thus, while cGAS has clearly emerged as a key DNA sensor in the ISD pathway, the function of the ALRs is still unclear. We have developed novel tools to define the relative contributions of the ALRs and cGAS to the ISD pathway, namely mice lacking all thirteen ALR genes and cGAS-deficient mice. In addition, we have used CRISPR gene targeting to disrupt cGAS and all four human ALRs in human cells. Analysis of IFN induction following transfection of DNA ligands or infection with DNA viruses has revealed that cGAS is absolutely required for activation of the ISD pathway in both mouse and human cells, consistent with recent studies. We are currently examining the ISD pathway in our ALR-deficient mouse and human cells to define how the ALRs function in the innate response to DNA virus infection.

   



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  The AIM2-like receptors are dispensable for activation of the interferon stimulatory DNA (ISD) pathway.
  Presenter: Elizabeth Gray
  All Authors:Elizabeth Gray, Dan Stetson
  University of Washington
   
 

Detection of intracellular DNA triggers activation of the STING-dependent interferon-stimulatory DNA (ISD) pathway, which is essential for antiviral immune responses; on the other hand, inappropriate immune responses to self DNA result in autoimmunity. Multiple DNA sensors have been proposed to activate the ISD pathway, including cyclic GMP-AMP synthase (cGAS) and a family of DNA binding receptors called the AIM2-like receptors (ALRs). Analysis of cGAS-deficient mice has revealed that cGAS is a key DNA sensor that is required for activation of the ISD pathway; however, whether the ALRs contribute to this pathway remains unclear. Here, we generated mice lacking all 13 mouse ALR genes as a novel tool to explore the function of the ALRs. We show that all ALRs are dispensable for the type I interferon (IFN) response to transfected DNA ligands, DNA virus infection, and lentivirus infection. We also show that the DNA sensor cGAS, but not the ALRs, is required to drive autoimmune disease in the Trex1-deficient mouse model of Aicardi-Goutieres Syndrome. Finally, we used CRISPR to disrupt the human AIM2-like receptor IFI16 in primary human fibroblasts and show that IFI16 is dispensable for the IFN response to transfected DNA ligands as well as human cytomegalovirus (HCMV) infection. Thus, our data reveal that ALRs are dispensable for activation of the ISD pathway and demonstrate that cGAS is the primary DNA sensor that drives the IFN response to DNA.

   



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  Coordinate Transcriptomic and Epigenetic Analysis Reveals Novel Adaptations to Chronic and Acute Viral infection in Innate immune populations.
  Presenter: Trever T. Greene
  All Authors:Trever T. Greene, Yeara Jo, Elina Zuniga
  University of California San Diego
   
 

Chronic viral infections such as HIV, HBV, and HCV are significant burdens on human health. Recently it has become apparent that the early responses to chronic viral infections in the innate immune system shape the immune responses against these viruses. However, there has been no systemic analysis of how cells of the innate immune system respond to a non-resolving systemic infection, and specifically how this differs to their response to an acute infection. Here we use RNA sequencing in combination with ATAC-seq to systematically analyze the transcriptomes and chromatin accessibility of four innate populations important for the control of viral infection (plasmacytoid DC, CD8+ DC, CD11b+ DC & Macrophages) from mice infected with acute and chronic isolates of lymphocytic choriomeningitis virus (LCMV). Our results reveal unique transcriptional programs driven by acute and chronic infection in these cell types, not only confirming previously established mechanisms of immune cell adjustment to persistent pathogen replication, but also identifying several never before described adaptations. In addition, in contrast to what has been described for other immune cells, and despite large scale changes in gene expression, we demonstrate that acute and chronic LCMV infection drive largely similar changes in chromatin accessibility, suggesting a model in which innate cell adaptations to either chronic and acute infection involve similar chromatin environment and rely on other systems to support their unique transcriptional adaptations.

   



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  CD40L-carrying HIV-virions stimulate B cell activation in human immune system mice
  Presenter: Gordana Halec
  All Authors:Gordana Halec, Jonathan Said, Otoniel Martínez-Maza, Marta Epeldegui
  Obstetrics & Gynecology, David Geffen School of Medicine at UCLA, Los Angeles, California
   
 

Background: HIV infection is known to be associated with chronic B cell hyperactivation. This has significant pathogenic relevance, as B cell activation is associated with enhanced risk for non-Hodgkin lymphoma (NHL), the most common malignancy in AIDS individuals in the post-HAART era. One of the underlying mechanisms of AIDS-NHL is a constant stimulation of B cells by immune stimulatory molecules present in HIV plasma membrane envelope after budding of virions from infected cells. In vitro work from our (Epeldegui et al., 2010) and other laboratories, has demonstrated that one of these crucial molecules is CD40L/CD154. Aim: To confirm the in vitro findings, we aimed to mimic in vivo HIV-induced B cell hyperactivation and early lymphomagenic effects using human immune system (HIS) mouse model which uses NOD/SCIDcG-/- (hNSG) mice implanted with human thymus/liver tissue at 6-8 weeks of age, and subsequently (3 weeks later) injected intravenously with human fetal liver CD34+ hematopoietic stem cells (huBLT). We have injected HIS mice with HIV virions carrying functional human CD40L (CD40L+ HIV), or non-functional CD40L mutant (T147N+ HIV), and compared them with the non-stimulated HIS mice (MOCK group). Results: All mice (total 15 mice with 5 mice per group) were successfully reconstituted and expressed =40% of human CD45+ cells in their blood across the span of 12 weeks as measured by flow cytometry. In addition, in spleens of all mice, we could confirm formation of while pulp into structures corresponding to germinal centers in the human spleen. Immunohistochemical staining of murine spleens revealed a pattern of clustered cells stained positive for cell membrane associated CD10 protein, while positivity for BCL-6 protein was found scattered across the tissue in <1% of cells. When compared to T147N and MOCK mice, mice exposed to CD40L+ HIV showed continuous elevated blood levels of CD19+ cells (representing B cells) and decreased levels of CD3+ cells (representing T cells), together with higher levels of cells expressing lymphocyte activation markers (CD10 and CD38). The same effects were observed in spleens and lymph nodes of HIS mice exposed to CD40L+ HIV versus those exposed to T147N+ HIV and MOCK mice. Analysis of murine plasma for the expression of cytokines known to be associated with B cell activation and early lymphomagenic events in HIV+ individuals, is pending. Conclusion: This initial study indicates that HIS mice can be a promising system to investigate early lymphomagenic effects stimulated by CD40L-carrying virions.

   



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  CD11c+ and CD4+ cells are important for TSLP-dependent response to hapten in chronic CHS
  Presenter: Angie L. Hertz
  All Authors:Angie L. Hertz, Steven F. Ziegler
  Benaroya Research Institute
   
 

The epithelial-derived cytokine thymic stromal lymphopoieitin (TSLP) has been implicated in the initiation and progression of allergic inflammation through its ability to activate dendritic cells, among other cells. A mouse model of allergic contact dermatitis, also known as contact hypersensitivity (CHS), is a useful tool for dissecting the molecular mechanisms of the allergic response. Our mouse model elicits CHS through sensitization and repeated application of low-dose dinitrofluorobenzene (DNFB). Using various Cre strains coupled to TSLPR-flox to enable deletion of the TSLP receptor on specific subsets of cells we are able to demonstrate a role of TSLP in CHS targeting CD11c+ cells and CD4+ cells. Mice with a deletion of TSLPR on CD4+ cells showed a modest decrease in ear swelling, an indicator of the severity of inflammation. However, mice with a deletion of TSLPR on CD11c+ cells displayed a dramatic decrease in ear swelling, nearly down to the levels of the global TSLPR knockout. These preliminary results indicate that CD11c+ cells may be main driver of TSLP-dependent inflammation in this model of allergic dermatitis.

   



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  Polyfunctionality of CD8 T Cell Responses to Cytomegalovirus in Transplant Patients
  Presenter: Lauren E. Higdon
  All Authors:Lauren E. Higdon, Kenneth B. Margulies, Jonathan S. Maltzman
  Stanford University, University of Pennsylvania
   
 

Polyfunctionality, or production of multiple cytokines and effector molecules, is important in the control of chronic viral infections, such as cytomegalovirus (CMV). CMV infection causes significant morbidity and allograft damage in transplant recipients. Primary CMV infection is rapidly resolved in healthy individuals, and memory T cells control reactivation of latent virus. IFNγ production by CD8 T cells is important for control of CMV, but other CD8 effector functions have not been evaluated in detail in the context of immunosuppression and transplantation. We have analyzed memory T cell responses to two CMV polypeptides, pp65 and IE-1, focusing on the effect of transplant-associated lymphodepletion on T cell specificity and polyfunctionality. In this study, polyfunctionality was defined as co-expression of IFNγ, TNFα, and/or the degranulation marker CD107a. Our analyses were conducted using pre- and up to one year post-transplant PBMC from renal and cardiac transplant patients. All patients received anti-viral prophylaxis and immunosuppression consisting of steroid, calcineurin inhibitor, and cell cycle inhibitor. A subset of transplant patients also received T cell depleting induction therapy. Our results show that pre-transplant and in normal donors, the 11 patients and 4 controls studied thus far can be divided into three groups based on dominant antigen specificity: IE-1, pp65, or equal response to both. The antigen-specific CD8 T cell population contained a similar proportion of cells expressing all three functions (~35%) in patients with IE-1 and pp65 dominance, but a lack of dominance was correlated with a lower proportion of polyfunctional cells (~20%). Cells expressing IFNγ were more likely to be polyfunctional than monofunctional. In addition, the cells expressing two of the three markers were almost uniformly TNFα positive. Some patients had early lymphodepletion-associated decreases in polyfunctional cells, but all returned to a pre-transplant proportion of CD8 T cells by day 180 post-transplant. In addition, the skewing of fine specificity to IE-1 or pp65 was maintained over the first year post-transplant in 8 of 11 patients. In conclusion, pre-transplant TNFα expression is predictive of polyfunctionality both pre- and post-transplant. While long-term maintenance of polyfunctional cells appears to be unaffected by lymphodepletion, further study will be needed to determine whether lymphodepletion has an effect on fine antigen specificity.

   



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  Fast Identification and Assembly of HIV-1-Specific T Cell Receptor Gene Therapy Lentiviral Vectors
  Presenter: Christian Hofmann
  All Authors:Christian Hofmann, Christian Hofmann, Christian-Raul Aguilera-Sandoval, Balamurugan Arumugam, Priya K. Patel, Brian Diep, Sangeun Park, Hwee Ng, Otto O. Yang
  Division of Infectious Diseases, Department of Medicine, UCLA, Los Angeles
   
 

Background: Immune surveillance through HIV-1-specific CD8+ cytotoxic T lymphocytes (CTL) is crucial for the long-term control of HIV-1 replication in vivo. However, most HIV-1-infected persons lack the ability to control the virus with their T cell receptor (TCR) repertoire, due to incomplete coverage of epitope variation. A potential immunotherapeutic strategy that would provide patients with the ability to control the virus without taking medication could be the adoptive transfer of gene-modified T cells, equipped with a combination of TCRs covering all common variants of their targeted epitope. We developed a rapid methodology to identify epitope-specific TCR sequences and insert them into lentiviral vectors without the need for HLA tetramers, cell sorting, or cell cloning. Methods: We focused on five HIV-1 Gag epitopes based on common or protective HLA type and epitope conservation: KRWIILGLNK263-272 (KK10, HLA-B*2705), KAFSPEVIPMF162-172 (KF11, B*5701), GLNKIVRMY269-277 (GY9, B*1501), RQANFLGKI429-437 (RI9, B*1302), and WASRELERF36-44¬ (WF9, B*3501). We identified Gag-specific TCR sequences by expansion after epitope stimulation using a quantitative spectratyping method. Via a one-step reaction we cloned both TCR α and β chains into a lentiviral vector and the functionality of these TCRs was subsequently determined by a Jurkat cell NFAT-dependent GFP reporter assay. Additionally, TCR-transduced primary CD8+ T cells were tested in a chromium release assay against HIV-1-infected T1 cells transduced with the relevant HLA genes. Results: We cloned and functionally tested seventeen TCRs against different HIV-1-specific epioptes (six for KK10, four for KF11, five for GY9, one for RI9, and one for WF9). Several TCRs have been screened for their recognition of epitope variants and functional avidity using synthetic peptides, Conclusions: Our data demonstrate proof-of-concept for rapid TCR cloning into vectors suitable for gene therapy and capacity to screen these TCRs for the ability to recognize epitope variants. This work sets the foundation for combination TCR gene therapy to cover epitope variation.

   



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  Longitudinal analysis of Plasmodium falciparum-specific atypical and classical memory B cell responses to natural malaria infection in children and adults
  Presenter: Christine S. Hopp
  All Authors:Christine S. Hopp, Akshay T. Krishnamurty, Silvia Portugal, Chris Thouvenel, Ogobara K. Doumbo, Boubacar Traore, Susan K. Pierce, David J. Rawlings, Marion Pepper, Peter D. Crompton
  NIH, NIAID
   
 

Antibodies play a key role in naturally acquired immunity to malaria; however, antibodies that reliably protect from malaria are only acquired after years of repeated Plasmodium falciparum (Pf) infections. We have shown in Mali that this inefficiency in humoral immunity to malaria is associated with short-lived plasma cell responses; a gradual, incremental increase in Pf-specific memory B cells (MBCs) over many years; and an expansion of CD21loCD27- ‘atypical’ MBCs that are isotype-switched and somatically hypermutated, but that exhibit markedly reduced B cell receptor (BCR) signaling and effector function. A similar subset of ‘exhausted’ MBCs has been observed in patients with chronic viral infections and autoimmune diseases. However, in the context of malaria, it remains unclear whether atypical MBC expansion is Pf-specific or represents a global expansion as a result of chronic immune activation from repeated Pf infections and/or co-infections that are common in malaria-endemic areas. Using PfMSP1 and PfAMA1 B cell tetramer staining we recently identified an expanded population of Pf-specific MBCs in malaria-exposed individuals in Mali that were 60 fold more frequent as compared to healthy U.S. adults. Preliminary findings show that monoclonal antibodies expressed from individually sorted PfMSP1-specific MBCs are indeed PfMSP1-specific by ELISA. Using this B cell tetramer approach, we are tracking Pf-specific classical and atypical MBCs in a longitudinal cohort study of children and adults in Mali. Using B cell tetramers specific for tetanus and Influenza we will determine the relative role of Pf in driving atypical MBC expansion and also compare the magnitude and kinetics of Pf-, tetanus- and influenza-specific atypical and classical MBC responses from age 3 months to 25 years of age at well-defined time points before, during and after acute Pf infection. This analysis should provide important new insights into the mechanisms underlying the delayed acquisition of naturally acquired immunity to malaria in children, and may also aid in the development of an urgently needed malaria vaccine.

   



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  Investigating lipid-metabolic requirements in anti-inflammatory macrophage activation
  Presenter: Wei-Yuan Hsieh
  All Authors:Wei-Yuan Hsieh, Tin Doung, Kevin Williams, Quan Zhou, Viet Bui, Joseph Argus, and Steve Bensinger
  Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles
   
 

Macrophages are immune cells responsible for a broad range of processes such as killing invading pathogens, clearing apoptotic cells and subcellular debris, initiating wound-healing programs, and presenting antigens to T cells. To achieve these different functions, macrophages possess the capacity to acquire pro- and anti-inflammatory programs by altering fluxes through various metabolic pathways. It is well understood that interferon (β/γ) and specific TLR agonists suppress macrophage lipid synthesis to promote classical-proinflammatory activation. However, little is understood how anti-inflammatory polarizing signals, such as IL-4, coordinate macrophage lipid-metabolic reprogramming. Here, we show that IL-4 signaling co-opts the activation of SREBP-SCAP pathway in bone marrow derived macrophages (BMDMs) to induce lipid synthesis. M(IL-4) polarization is significantly impaired in the absence of SCAP. 13C tracer analysis of fatty acid methyl esters (FAMEs) of M(IL-4) revealed the accumulation of specific long chain fatty acids that are required for macrophage polarization. The location of these fatty acids in complex lipids is further characterized through the use of lipidomics. These results provide a new role for lipid metabolism in supporting macrophage activation and function.

   



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  Light-based screening for determining genetic and transcriptional underpinnings of cellular heterogeneity
  Presenter: Kenneth Hu
  All Authors:Kenneth Hu, Matthew Krummel
  UCSF Department of Pathology
   
 

Microscopy can provide valuable information on parameters such as cell morphology, time-dependent behavior as well as spatial relations between cells. The ability to correlate a cell viewed on the microscope to a transcriptional profile or a knock out locus would be a powerful capability. Light serves as an excellent tool for selection of cells based on its high spatial resolution. We present several approaches to using light to tag cells based on visualized criteria on the microscope to distinguish them in downstream applications. One application we propose is the ability to perform a vision-based screen for genes in T cells that control formation and behavior of microvilli at the immune synapse which govern the ability of the T cell to scan for and recognize cognate antigen. A range of synapse phenotypes are expected in a population of T cells given a knockout library; cells exhibiting a synapse with a morphology of interest can be identified with photoactivatable GFP (PA-GFP). We are developing an automated system for identification of aberrant phenotypes and subsequent photoactivation of GFP in these cells, allowing these cells to be identified and sorted out downstream. The genetic loci underlying those aberrant phenotypes can thus be identified from these now GFP+ cells. Furthermore, this approach can be applied to discerning the transcriptional states underlying heterogeneities within a seemingly uniform cell population. By analyzing the expression profiles of photoactivated cells compared to unactivated, heterogeneities in cell morphology or behavior such as migratory capacity can be characterized as distinct positions within transcriptome space. For example, T cells from a seemingly uniform population adopt varying morphologies as they migrate and display differential migratory speeds and processivity. This could represent a number of metastable states in transcription space which T cells can occupy and would be impossible to correlate to phenotype without the capability to distinguish them.

   



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  Reversal of Fatal Autoimmunity by Regulatory T cells
  Presenter: Wei Hu
  All Authors:Wei Hu, Zhong-Min X. Wang, Yongqiang Feng, Michael Schizas, Beatrice E. Hoyos, Jacob Verter, Alexander Y. Rudensky
  Memorial Sloan Kettering Cancer Center
   
 

Regulatory T (Treg) cells play a non-redundant role in suppressing aberrant immune activation towards self. Treg therapy is therefore an attractive candidate for treating autoimmune as well as allergic inflammations. However Treg therapy based on infusion of ex vivo expanded Treg cells has only shown limited success. It has been observed that Treg cells become less stable and lose their suppressive capacity under inflammatory conditions, raising the question whether Treg cells maintain their identity and functionality in autoimmune lesions and suppress ongoing inflammation. Moreover, it is unclear how long infused Treg cells can persist and remain functional. Here using mice expressing a convertible Foxp3 reporter/null allele, we show that a single cohort of Treg cells are fully capable of suppressing fatal multi-organ autoimmune disease. These Treg cells, upon sensing the inflammatory environment, can rapidly expand, become more activated, and exhibit greater suppressive capacity. Following contraction of the otherwise lethal autoimmune disease, these Treg cells persist for an extended period of time, at least 7 months, and continue to protect the host against activation of self-reactive cells. Single cell transcriptomic profiling shows that these long-lived Treg cells remain phenotypically and functionally more active, with no survival defect or signs of exhaustion. Interestingly, a subpopulation of long-lived Treg cells with similar phenotypes can be found in a homeostatic Treg cell pool. These results demonstrate the stability and functionality of Treg cells under severe inflammatory conditions, and may shed light on the design of better Treg therapies.

   



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  Natural microbial exposure shapes the memory CD8 T cell pool and immune responsiveness to new challenges
  Presenter: Matthew A. Huggins
  All Authors:Matthew A. Huggins, Mark Pierson, Sara E. Hamilton
  University of Minnesota
   
 

Over time CD8 T cells encounter multiple infections independently from recognizing cognate antigen through their T cell receptor. Inflammatory events caused by microbial exposures can be quite numerous and may impact future immune responses to vaccines or resistance to infection. Although many studies have investigated how repeated antigen stimulation causes T cell exhaustion, repeated bystander inflammation has not been well characterized. In this study, we compare identical CD8 T cell populations in specific pathogen free (SPF) mice to those in ‘dirty’ mice—laboratory mice co-housed with microbially diverse pet store mice. Our published data indicate that dirty mice acquire a diverse set of microbes upon cohousing with pet store mice and exhibit increased pro-inflammatory gene signatures in the blood. In ongoing work, we demonstrate that cohoused mice have increases in baseline levels of numerous inflammatory cytokines and chemokines. These differences correspond with widespread changes in the cellular composition of immune cells. Specifically, cohousing skews the memory T cell population towards long-lived effector cells (LLEC). This subset is characterized by expression of terminal effector differentiation markers (KLRG1), cytotoxic molecules (granzyme B), and unique chemokine receptor expression (CX3CR1). Importantly, memory CD8 T cells isolated from dirty mice provide significantly improved pathogen clearance after intentional infection with Listeria monocytogenes (LM). Cohoused T cells provided enhanced protection to both antigen specific and antigen-independent challenges. Ongoing studies are focused on defining the extrinsic inflammatory signals that contribute to altered CD8 T cell functionality as well as the T cell intrinsic mechanisms of enhanced pathogen protection. A more complete understanding of how previous antigen exposure shapes the immune response could help optimize the vaccination and treatment of humans with varying degrees of microbial burden.

   



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  Age-related changes in organization and function of lymph node microenvironment
  Presenter: Ilija Jeftic
  All Authors:Ilija Jeftic, Nico A Contreras, Richa Jain, Heather L Thompson and Janko Nikolich-Žugich
  Department of Immunobiology and the University of Arizona Center on Aging, University of Arizona College of Medicine-Tucson, Tucson, Arizona, United States of America
   
 

Elderly patients are more vulnerable to infectious diseases and show diminished responses to vaccination compared to adult counterparts. While age-related changes in primary lymphoid organs are well described, the morphological and functional changes affecting secondary lymphoid organs have been less well characterized. Lymph node (LN) structure, including its stromal cells, is crucial to its function, that consists of lymphocyte maintenance, as well as of directing antigen, antigen-presenting cells, and antigen-specific lymphocytes into intimate communication necessary to mount immune responses. Experimental evidence indicates that LNs undergo alterations during aging, including drastic reduction in both lymphoid and stromal LN cell numbers and organization. Additionally, we show an age-dependent replacement of areas populated with immune cells by connective tissue and an increased degree of fibrosis in LNs compared to adult counterparts. Aged LNs exhibited higher expression of fibrosis-related cytokines such as TGF-b. Treatment of aged animals with trophic factors reversed age-related lymph node defects and fibrosis, with an expansion of both stromal cells and naïve T cells. Collectively, these results demonstrate an age-related increase in LN fibrosis, implicate profibrogenic pathways, and begin to establish targeted strategies to improve immune homeostasis, immune responses and vaccine efficacy in the elderly.

   



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  The Influence of Antigen-Specific TH1 and TH17 Cells in Shaping Neuro-inflammatory Patterns in Patients with Multiple Sclerosis
  Presenter: Mark Johnson
  All Authors:Mark Johnson, Emily R. Pierson, Mariko Kita, Jane Buckner and Joan M. Goverman
  Buckner: Benaroya Research Institute Kita: Virginia Mason All else: Department of Immunology, University of Washington
   
 

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although pathology and clinical course vary amongst MS patients, the majority of lesions are found within the brain. However, a small subset of MS patients (2-10%) exhibit inflammation localized to the spinal cord, without extensive involvement of the brain. The mechanisms defining these unique neuro-inflammatory patterns are poorly understood. Using the EAE model, we previously showed that the response to inflammatory, effector T cells is regulated differently in the brain and spinal cord. Specifically, we showed that IL-17 promotes, while IFN? inhibits, brain localized inflammation through a mechanism involving the recruitment of neutrophils by the ELR chemokine CXCL2. In contrast, IFN? promotes CXCL2 expression and subsequent inflammation in the spinal cord. Collectively, these data suggest that the relative abundance and localization of TH17 and TH1 cells are critical determinants in the manifestation of brain and/or spinal cord inflammation in EAE via their disparate effects on CXCL2 induction in these microenvironments. To translate our findings to humans, we investigated the frequency of myelin protein-specific TH1 and TH17 cells in the blood of MS patients with different neuroinflammatory patterns. We hypothesized that patients with lesions only in the brain would have a higher TH17:TH1 ratio compared to patients with predominantly spinal cord lesions. Surprisingly, our results revealed a significant decrease in myelin basic protein (MBP)-specific TH1 cells in patients with predominantly spinal cord lesions compared to patients with lesions only in the brain. A similar trend for reduced numbers of TH1 cells specific for myelin oligodendrocyte glycoprotein (MOG) was observed in spinal cord-predominant patients, as well. In contrast, the majority of patients with brain-only lesions exhibited reduced numbers of MOG-specific TH17 cells compared to patients with predominantly spinal cord lesions. These distinct TH1 and TH17 patterns in response to two myelin proteins in the peripheral blood suggest that the pathogenesis of MS may differ in patients exhibiting brain vs. spinal cord localized lesions.

   



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  Histone Deacetylase 7 Controls the Thymic Development of iNKT Cells and Other Innate-like Lymphocytes
  Presenter: Herbert Kasler
  All Authors:Herbert Kasler, Intelly S. Lee, Hyung W. Lim, Eric Verdin
  Gladstone Institute of Virology and Immunology
   
 

The growing family of innate-like tissue-resident lymphocytes, comprising several types of agonist-selected T cells as well as innate lymphoid cells, constitutes a vital first line of defense at mucosal barriers. These cells are particularly abundant in the digestive tract and associated organs, which constitute a crucial interface with the potentially dangerous microbiota of the gut lumen. There, they mount rapid effector responses in response to a variety of stimuli, in a manner more characteristic of innate immune cells than T cells. We have discovered that Histone Deacetylase 7 (HDAC7), a class IIa HDAC that we previously found to regulate TCR-dependent apoptosis during negative thymic selection of conventional T cells, also controls the development of invariant natural killer T cells (iNKT cells), a CD1d-restricted population of lymphocytes expressing an invariant Va14Ja18 TCR. These and other related cell types require expression of the BTB-POZ family protein PLZF, which has been shown previously to interact with HDAC7, for their development and acquisition of innate-like effector function. Mice expressing a dominant gain-of-function mutant of HDAC7 (HDAC7-?P) in the thymus completely lack iNKT cells, while positive selection of conventional T cells in unimpaired. Conversely, thymocytes lacking HDAC7 acquire rapid effector function and the expression of memory markers constitutively, a finding that strongly mirrors the phenotype observed due to enforced thymic expression of PLZF. Moreover, we have found that HDAC7 and PLZF regulate highly overlapping sets of genes in thymocytes that are highly relevant to the development of innate-like function in lymphocytes. Lastly, mice expressing a HDAC7-?P in the thymus, despite exhibiting a global defect in negative thymic selection and autoantibodies to a wide array of tissue-specific antigens, only develop autoimmunity in the anatomic compartments normally most populated by innate-like lymphocytes. Our findings establish HDAC7 as a key gatekeeper of the innate-like lymphocyte effector program via modulation of PLZF activity, and also provide potentially important insights into the relationship between innate-like effector function and the maintenance of immune self-tolerance.

   



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  Uptake of tumor-antigen induces myeloid cell reprogramming in lung metastasis
  Presenter: Kelly Kersten
  All Authors:Kelly Kersten, Ran You, Mark Headley, Sophia Liang, Matthew F. Krummel
  Department of Pathology, University of California San Francisco
   
 

Metastatic spread is one of the deadliest aspect of cancer. Yet little is known about the fate of disseminated tumor cells when they arrive in and colonize distant organs, or how they interact with the immune system during the early steps of metastasis. Previously, our laboratory has developed a model for stable intravital two-photon lung imaging in mice to capture the early interactions of disseminated tumor cells with host immune cells during pulmonary metastasis1. We showed that soon after they enter the lung tissue, tumor cells continuously shed microparticles or cytoplasts in the lung capillaries that move independently along the lung vasculature1. Distinct populations of myeloid cells ingested these tumor microparticles and sequentially accumulate in the lung interstitium, possibly providing a supportive niche for the outgrowth of metastases. Although the numbers of myeloid cell subsets rise globally in the early metastatic lung, gene expression analysis of macrophages – the most predominant tumor particle-ingesting myeloid population – revealed that ingestion of tumor particles results in a phenotypic switch. However, the functional significance of this phenotype change remains unclear. Here we show that macrophages that have ingested tumor particles down-regulate markers including Flt3, Btla and Zbtb46 that are associated with “dendritic cell maturation”, suggesting a reduced capacity to activate anti-tumor T cell responses. In addition, genes associated with “cell adhesion and extracellular matrix pathway” were upregulated in tumor-ingested macrophages compared to non-tumor-ingested macrophages. In vitro co-cultures of bone marrow-derived macrophages and B16.ZsGreen tumor cells recapitulated our in vivo findings and revealed that the upregulation of VCAM, CD63 and CD38 in macrophages occurs rapidly after ingestion of ZsGreen+ tumor particles. Moreover, Ingenuity pathway analysis revealed enrichment of mTOR signaling – a major regulator of cell metabolism – in tumor-ingesting macrophages. In vivo blockade of mTOR signaling with rapamycin abrogated the upregulation of VCAM, CD63 and CD38 in ZsGreen+ macrophages, suggesting that the phenotypic change observed in tumor-ingesting macrophages might be mTOR-dependent. Future experiments are focused on deciphering the functional significance of the phenotypic switch of macrophages after ingestion of tumor material. These results will increase our understanding of the interactions between disseminated tumor cells and immune cells, and will eventually help the development of novel immunotherapeutics to prevent metastatic disease. 1. Headley, M. B. et al. Visualization of immediate immune responses to pioneer metastatic cells in the lung. Nature 531, 513–517 (2016).

   



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  ECD8 T cell response is delayed and dampened upon vaginal versus systemic LCMV infection
  Presenter: Shahzada Khan
  All Authors:Shahzada Khan, Ben Cohn, Miles Oliva, Timothy C Borbet, Ashley Hughey, Emily Deal, Martin Trepcar, Elze Rackaytete, Erik Woodruff, Julie Luong, and Shomyseh Sanjabi
  Gladstone Institutes, UCSF
   
 

We currently lack vaccines against many sexually transmitted pathogens, and we know little about the mechanisms of how effective antigen-specific CD8+ memory T cells are induced and maintained in the female reproductive tract (FRT). FRT has an immunologically tolerogenic environment that is equipped to tolerate allogeneic sperm and support the growth of semi-allogenic fetus, but is also the portal of entry for many sexually transmitted pathogens. Our goal is to understand the immune cell populations and mechanisms that orchestrate the fine balance between this tolerogenic environment and the inflammation that is induced in response to vaginal viral infections. We have established a vaginal lymphocytic choriomeningitis virus (LCMV) infection model, allowing us to compare the mucosal versus systemic immune response that is elicited against this pathogen. We have found that intravaginal LCMV infection leads to a delayed and dampened CD8 cytotoxic T lymphocyte (CTL) response compared with systemic infection. The delayed priming of CD8 T cells after intravaginal infection is due to the amount of time that LCMV takes to reach the iliac lymph node (iLN) from the lower FRT. Inoculation of a higher viral dose resulted in a higher CTL response, but could not overcome the delay in CD8 T cell priming. The poor magnitude in the CTL response after vaginal infection was preceded by a dampened type I interferon response, and a reduced recruitment and activation of CD11b+ dendritic cells (DCs) in the FRT and the draining lymph nodes. Priming and activation of CD8 T cells positively correlated with viral clearance in the LNs but not in the lower FRT during the first week of infection. These observations describe a fundamental “defect” in the FRT to mount timely and effective CTL responses against vaginal viral infections, which may provide a “window of opportunity” to sexually transmitted pathogens. Identifying the cell populations and/or tolerogenic mechanisms involved in dampening the CTL response in the FRT would inform future studies for better therapeutic/preventive interventions.

   



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  Development of novel cell treatment method for type 1 diabetes based on CAR T-cell therapy
  Presenter: Shio Kobayashi
  All Authors:Shio Kobayashi, Martin Thelin, Alborz Kaimzadeh, Neha Deshpande, Heather Parrish, Mark Lee, Michael Kuhns, and Thomas Serwold
  Joslin Diabetes Center
   
 

Chimeric antigen receptor (CAR) T-cell therapy is a new form of immunotherapy against cancer. A CAR is a single-module fusion protein containing a tumor-specific antigen-binding domain of an antibody, which is fused directly to signaling components of the T cell receptor (TCR) signaling pathway. CARs, when expressed by CD8+ cytotoxic T cells, confer the ability to recognize and kill target tumor cells specifically. We adapted this engineering strategy to target autoreactive T cells in a mouse model of type1 diabetes (T1D). We engineered a chimeric receptor consisting of peptide-loaded major histocompatibility complex (pMHC) fused to the proximal, and transmembrane domains of the TCRα and TCRβ chains. The pMHC-TCR fusions paired with the endogenous CD3 subunits. We also engineered a chimeric co-receptor designed to bring LCK into the signaling complex, creating a 5-module chimeric receptor (5M-CAR). 5M-CAR T-cells loaded with an MHC II-restricted autoantigen commonly targeted in the NOD mouse model of T1D, killed their target CD4 T cells in an antigen-specific manner both in vitro and in vivo. Infusion of 5M-CAR T-cells also prevented diabetes in a NOD-SCID mouse transferred with autoimmune T cells in an antigen-specific manner. These findings indicate that 5M-CAR T-cells have broad potential for elimination of harmful populations of monoclonal and oligoclonal T cells in autoimmune diseases.

   



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  TLR-induced anti-commensal IgG antibodies regulate gut immune homeostasis
  Presenter: Meghan Koch
  All Authors:Meghan Koch, Gabrielle Reiner, Kyler Lugo, Thaddeus Sehar, William Ludington, Gregory Barton
  University of California, Berkeley
   
 

Maternal antibodies have been suggested to be important for establishing intestinal immune homeostasis in developing offspring, yet the mechanisms by which maternal antibodies achieve this remain unclear. We analyzed the gut immune response in mice born to antibody-deficient dams and found that maternal antibodies regulate the activation of mucosal CD4+ T cells. Interestingly, maternal IgA, the dominant mucosal antibody isotype, was not necessary for this regulation. As such, we developed an unbiased, flow cytometric based assay to assess the microbiota-specific antibody response, thereby allowing us to investigate if other antibody isotypes are important for enforcing intestinal immune homeostasis. Unexpectedly, we found that mice generate a robust anti-commensal IgG2b- and IgG3- response. This commensal-specific IgG response occurs through a pathway independent of T cells, yet dependent on signaling through TLR2 and TLR4. These TLR-dependent anti-commensal IgG antibodies are transmitted from mothers to their offspring. There, they function to regulate T follicular helper responses and subsequently dampen germinal center B cell responses. This work reveals a feedback loop whereby T cell-independent antibody responses function to regulate mucosal CD4+ T cell responses and thereby establish equilibrium between the host and its resident microbiota.

   



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  Aire and Brg1 Define a Fulcrum for Ectopic Gene Expression in the Thymus to Provide Precise Control of Immune Tolerance
  Presenter: Andrew Koh
  All Authors:Andrew Koh, Erik Miller, Jason Buenrostro, David Moskowitz, William Greenleaf, Howard Chang, Gerald Crabtree
  Stanford University
   
 

The thymus mirrors the peripheral self by expressing thousands of tissue-specific antigens to purge dangerous self-reactive clones from the T cell repertoire. How thymic epithelia gain initial access to these tissue-specific genes and activate them at levels that prevent physiologic harm is unknown. Here, we identify Brg1 as an essential determinant promoting accessibility at tissue-specific loci to poise transcription and impose central tolerance. Surprisingly, we show that Aire harbors an intrinsic repressive function that restricts chromatin accessibility and transcriptional amplitude. Aire imposes this repressive influence within minutes upon recruitment to chromatin and operates over the genome in opposition to the mSWI/SNF chromatin remodeling complex. Human autoimmune mutations that impair Aire’s multimerizing and histone-binding activities also inhibit this repressive influence indicating dual roles of Aire’s functional domains. Together, Brg1 and Aire define a fulcrum in thymic epithelial development that allows the access and activation of tissue-specific genes at levels that prevent toxicity, yet facilitate immunological tolerance.

   



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  Immune-complex independent Targeting of Native Antigen to Follicular Dendritic Cells accelerates humoral immune responses
  Presenter: Jan Kranich
  All Authors:Jan Kranich, Ashreta Latha, Agnieska Foltyn-Arfa Kia, Lisa Rausch and Thomas Brocker
  LMU Munich
   
 

Detection of dying cells in vivo is challenging. Annexin V, a phosphatidylserine (PS)-binding protein requires high Ca2+ concentrations. Attempts to use Annexin V in vivo therefore were not satisfactory. To circumvent this, we developed a green fluorescent protein (GFP)-based fusion protein of Mfge8, which, upon injection stains dying cells in tissues and organs independently of Ca2+ with high sensitivity and specificity under physiological conditions. In addition, recombinant Mfge8-GFP also visualizes PS+ apoptotic bodies and extracellular vesicles (EVs). Upon injection of this reagent into mice, Mfge8-GFP initially identifies selectively marginal zone B cells in the spleen, which are extensively coated with PS+ EVs, but are not apoptotic themselves. Thereafter, Mfge8-GFP was cleared from the marginal zone and accumulated on FDC in B-cell follicles, indicating a transport of EVs from the marginal zone into the follicle. Here, affinity maturation of B cells is triggered by continuous recognition of native antigen complexed with Ag-specific antibodies that are displayed on FDC. Upon injection chimeric Mfge8 fused to antigenic protein rapidly accumulated on FDCs resulting in drastically accelerated B-cell responses to the fused antigen. In contrast to immunizations with conventional antigenic protein, were accumulation on FDCs only occured in immune mice, where antigen specific antibodies were already present, Mfge8-fusion proteins accumulated on FDCs in naive mice and induced extremely early occurrence of isotype switched antigen-specific IgG-antibodies. The fusion of antigen to Mfge8 can be exploited to deliver native antigen to FDCs via EVs. This immune-complex independent way of antigen delivery to FDCs could be used to develop vaccines for accelerated generation of neutralizing antibodies.

   



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  Thymic stromal lymphopoietin promotes interplay between tumor cells and myeloid cells to regulate breast tumor progression
  Presenter: Emma Kuan
  All Authors:Emma Kuan, Steven Ziegler
  Benaroya Research Institute
   
 

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in controlling various human cancer development through regulating Th2 responses by directly acting on CD4+ T cells. However, it is still largely unknown the role of TSLP in other cell types within tumors since TSLP receptor (TSLPR) is widely expressed on many cell types, including many hematopoietic cells and epithelial cells under inflammatory conditions. We found that TSLP signaling in both human and mouse breast tumor cells is important to maintain their survival and their capacity to produce TSLP in vitro. By using murine metastatic breast tumor models we found that the lack of TSLP in hematopoietic cells or TSLPR, but interestingly not TSLP, in breast tumor cells, leads to significantly smaller primary tumor size. We identified the critical TSLP source is myeloid cells within tumors. We also showed that asthmatic mice with TSLP locally expressing in lungs leads to more tumor metastasis to lungs and TSLP blockage in lungs significantly reduced tumor metastasis. Besides tumor cells, we discovered that TSLP signaling in Ly6Chi monocytes is also crucial for promoting tumor progression by regulating monocyte suppressor functions and their ability to differentiate into tumor associated macrophages. Our work is the first to show myeloid cell derived-TSLP plays an important role in directly promoting breast tumor progression via maintaining tumor cell survival. We also provide another novel mechanism of the requirement of TSLP signaling in regulating the pro-tumor functions in Ly6Chi monocytes. These studies define a novel TSLP-mediated crosstalk between tumor-infiltrating myeloid cells and tumor cells and provide an effective therapeutic intervention in metastatic breast cancer.

   



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  Non-tumor derived thymic stromal lymphopoietin regulates breast tumor progression through Ly6Chi monocytes
  Presenter: Emma Kuan
  All Authors:Emma Kuan, Steven Ziegler
  Benaroya Research Institute
   
 

Various cancers in humans display a tumor promoting T helper type 2 (Th2)-like inflammation. One possible mechanism suggested in human breast cancers is thymic stromal lymphopoietin (TSLP ) secreted by breast tumor cells which can drive Th2 responses via tumor-associated dendritic cells. However, how Th2 responses develop and if other immune cells involved in breast tumor growth and metastasis remains unclear. We found in breast tumor-bearing mice TSLP can be produced by both two pro-tumor myeloid cell populations, neutrophils and Ly6Chi monocytes, which are also known as granulocytic and monocytic myeloid derived suppressor cells, respectively. This non-tumor derived TSLP is crucial for both primary breast tumor development and tumor metastasis to lungs. One important mechanism for this is through directly acting on tumor cells as TSLP stimulation to breast tumor cells increases their ability to produce more TSLP and maintains their survival. Mice transplanted with TSLPR deficient breast tumor cells developed smaller primary breast tumors and fewer lung metastases. Conversely, mice that constitutively express TSLP in lungs transplanted with breast tumor cells have markedly increased lung metastases, and blocking TSLP in the lung resulted in a dramatic reduction in the number of lung metastases. We observed similar results in an autochthonous breast tumor model using MMTV-PyMT (MTAG) mice, where TSLP deficient MTAG mice develop delayed and smaller breast tumor and fewer lung metastases. We further discovered that in Ly6Chi monocytes TSLPR signaling is crucial for their suppressor functions and their ability to differentiate into macrophages in tumors. Transferred TSLPR deficient Ly6Chi monocytes significantly reduce lung metastases in tumor bearing mice by changing T cell populations in lungs and in tumor. Our work is the first to indicate non tumor-derived TSLP plays an important role in promoting both breast tumor growth and metastasis via maintaining tumor cell survival. We also provide a novel mechanism of the requirement of TSLPR signaling in differentiation and suppressor functions in Ly6Chi monocytes.

   



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  Thymic stromal lymphopoietin promotes interplay between tumor cells, myeloid cells, and CD8+ T cells in regulation of breast cancer
  Presenter: Emma Kuan
  All Authors:Emma Kuan, Steven Ziegler
  Benaroya Research Institute
   
 

A type-2 inflammation cytokine thymic stromal lymphopoietin (TSLP) recently has been shown involved in various human cancer development. In breast cancer, TSLP is reported to regulate breast tumor cells, dendritic cells, and CD4+ T cells to promote tumor progression. However, it is still largely unknown the role of TSLP in other cell types within breast cancer since TSLP receptor (TSLPR) is widely expressed on various cell types. We recently showed that breast tumor cells express functional TSLPR and myeloid cell-derived TSLP, but not tumor-derived TSLP, is the critical source to maintain tumor cell survival in vitro and in vivo. Although the lack of tumor-derived TSLP did not alter their survival in vivo, inflammatory monocytes (also called monocytic myeloid derived suppressor cells) from these mice displayed reduced suppressive function in an in vitro suppression assay. Indeed, we found inflammatory monocytes expressed TSLPR and TSLPR deficient inflammatory monocytes derived from tumor-bearing mice displayed reduced suppressive function in suppression assay. In vivo or in vitro stimulation of inflammatory monocytes with TSLP increased their suppressive function. Most importantly, conditional depletion of TSLPR only in monocytes in tumor-bearing mice resulted in reduced tumor-associated macrophages and increased expression of exhaustion marker PD-1 in tumor-infiltrating CD8+ T cells that further led to smaller primary tumors and less tumor metastasis to lungs, suggesting a pro-tumor role of TSLP in regulating monocytes in breast cancer. Interestingly, missing TSLP signaling directly in CD8+ T cells in tumor-bearing mice led to a greater primary tumor size that was due to more PD-1 expression on tumor-infiltrating CD8+ T cells, indicative of an opposite role of TSLP in CD8+ T cells in breast cancer. Our work reveals the complex regulation of TSLP in various cell populations within breast tumors and is the first to define the in vivo role of TSLP signaling in monocytes, CD8+ T cells, and breast tumor cells that can serve as a cornerstone for therapeutic intervention in breast cancer.

   



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  Differential regulation of TGF-[b] activation and Th17 cell generation by DC and macrophage subpopulations
  Presenter: David Kugler
  All Authors:David Kugler, Helena Paidassi, Fiona Raso, Mridu Acharya, Adam Lacy-Hulbert
  Benaroya Research Institute
   
 

Th17 cells play important protective roles during bacterial and fungal infection at mucosal surfaces, but also contribute to inflammatory and autoimmune disorders. How DCs and other APCs regulate the differentiation and maintenance of Th17 cells and related T cell subsets to achieve this delicate balance between defense and damage remains poorly understood. Differentiation of both Th17 cells and closely related peripheral Tregs relies on TGF-β signaling. We have shown that this is critically dependent on expression of integrin αv β8 by DCs, which activates latent TGF-β for signaling to interacting T cells. Consequently, mice lacking αv in DCs have impaired peripheral Tregs and develop spontaneous colitis, but also very few Th17 cells and are protected from Th17-mediated autoimmunity. While DC αv was necessary for development of both cell types, it is unclear if the same population of DCs supports both fates. To clarify this we are studying the role of DC αv during infection with Citrobacter rodentium, an intestinal pathogen that stimulates strong Th17 immunity. We find that deletion of αv using CD11c-CRE and LysM-CRE lines, which delete predominantly in DCs and macrophages respectively, resulted in distinct effects on Treg and Th17 differentiation. Most notably, DC-specific deletion led to a profound loss of RORγT+ IL-17A+ T cells and intestinal Tregs, which was less pronounced in macrophage-specific αv knockouts. In contrast, both mouse strains showed an increase in IL17A+ IFN-γ+ T cells, which have been implicated in Th17-mediated pathology. Our data suggest that distinct populations of DCs are responsible for generation of IL17A+ and IL17A+ IFN-γ+ Th17 as well as peripheral Treg cells. In ongoing experiments, we are determining the identity of these subpopulations and the signals that induce their expression of αvβ8. This project is supported by NIH grant DK093695 to ALH

   



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  Investigating the host-pathogen interactions during nosocomial methicillin resistant Staphylococcus aureus pneumonia
  Presenter: Keenan Lacey
  All Authors:Keenan Lacey, Frank Yeung, Bo Shopsin, Ken Cadwell, Victor J. Torres
  NYU School of Medicine
   
 

Pneumonia accounts for more deaths than any other infectious disease worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of hospital-acquired pneumonia. The prevalence of MRSA is increasing worldwide. To confront the growing problem of MRSA, we require a greater understanding of the host-pathogen interactions during infection. This remains poorly understood partly due to the lack of in vivo models relevant to infections occurring in healthcare settings. Most research to date has focused on highly virulent and cytotoxic MRSA strains, despite the fact that many nosocomial infections are caused by MRSA isolates that exhibit low cytotoxicity in ex vivo models and low virulence in mice. This study aims to functionally dissect host- and bacterial-directed mechanisms that lead to mortality in nosocomial settings. We have developed a nosocomial murine model of antibiotic conditioning, which we found lowers the barrier to infection, mimicking the nosocomial setting. We demonstrate that exposure to antibiotics mitigates the impact of reduced bacterial virulence in mice, allowing for a permissive environment for hospital adapted HA-MRSA isolates. These findings establish a robust model that is enabling us, for the first time, to probe the bacterial and host factors important during nosocomial infections, which are relevant to hospitalized patients that are also subjected to antibiotic regimes.

   



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  Precocious expression and engagement of mature [a][b] TCR in CD4-CD8- T cell progenitors can drive NOTCH1 mutation and T-ALL transformation
  Presenter: Kimberly Laffey
  All Authors:Kimberly Laffey, Robert J. Stiles; Melissa Ludescher; Tessa R. Davis; Shariq S. Khwaja; Richard J. Bram; Peter J. Wettstein; Venkataraman Ramachandran; Cory E. Johnson; Richard D. Hammer; Diana Gil; Adam G. Schrum
  University of Missouri
   
 

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformation of thymocytes. Most T-ALL involves activating NOTCH1 mutations but the drivers of mutation and transformation are incompletely understood. Here we report a rare but natural oncogenically susceptible stage during T cell development in mice, and apparently in humans. Most conventional thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, a few thymocytes in the conventional pathway rearrange both at CD4-CD8- double-negative (DN) stage. We show that such ‘precocious’ αβ TCR expression, when engaged by self-antigens, can risk T-ALL transformation. First, ~0.01-0.05% of mouse and human thymocytes are precocious αβ TCR DN. Second, we show that both mouse and human can present T-ALL tumors with their earliest developmental phenotype matching this stage. Third, the OT1 TCR transgene is expressed in mice with parallel timing and level with the natural, TCR-precocious subset, providing a model to study antigen-dependent signaling and transformation. We show that DN OT1 thymocytes can upregulate CD69 in response to antigen presentation independent of CD8 receptor, in vitro. Furthermore, OT1 and OT1.RAG2-/- mice, but not OT1.RAG2-/-.β2M-/- mice, spontaneously develop T-ALL in vivo with activating NOTCH1 mutations, showing that MHC-I is required and pre-TCR and other signals are insufficient for oncogenesis. Transplant experiments corroborate a requirement for recipient MHC for young tumors, but a multi-passaged tumor with increased aggression lost the requirement for MHC. Finally, the earliest developmental stage identified for OT1 tumors was the precocious DN stage, in common with the human clinical case. These data together suggest that precocious TCR expression and antigen signaling can cause T-ALL tumorigenesis.

   



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  Crucial role for CD11c+ interstitial macrophages in the development of TSLP-mediated allergic airway inflammation
  Presenter: Jen-Feng Lai
  All Authors:Jen-Feng Lai, Lucas J. Thompson, Steven F. Ziegler
  Benaroya Research Institute
   
 

Allergic asthma is a common chronic Th2-driven airway inflammatory disease. TSLP is an epithelial-derived cytokine recognized as a potent factor in the development of asthma. TSLP is known to influence the ability of dendritic cells to enhance Th2 T cell responses, however many of the mechanisms underlying TSLP-induced airway inflammation remain undefined. In addition to dendritic cells, the lung tissues include other MHC class II-expressing phagocytic populations that may influence T cell responses. Here we demonstrate that pulmonary CD11c+ interstitial macrophages (IM) (CD11cintF4/80+Nur77+CD11b+CX3CR1hiCCR2loMHCII+/-) express high levels of TSLPR and recruited to the lung tissue in response airway priming in the context of TSLP and antigen. TSLP-mediated eosinophilic allergic airway inflammation is attenuated by ablation of CD11c+ IM or by selective deficiency of TSLPR signaling in these cells. More importantly, CD11c+ IM are sufficient for the development of this response in the absence of DC. These findings indicate a crucial role for CD11c+ IM in the development of TSLP-mediated allergic airway inflammation. By elucidating the cellular mechanisms of airway inflammation, these data may contribute to more effective therapeutic approaches for treatment of asthmatic disease.

   



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  Tumor-infiltrating CD39+ MAIT cells possess a Treg-like subset and bridge the gap between gut microbiome and Colorectal Cancer
  Presenter: Shamin Li
  All Authors:Shamin Li, Yannick Simoni, Etienne Becht, Chiew Yee Loh, Bernett Lee, Teck Por Lim, Niranjan Nagarajan, Daniel Tan SW, Iain Tan BH, Evan W Newell
  Fred Hutchinson Cancer Research Center
   
 

Recently, our group has shown that CD8+ tumor-infiltrating lymphocytes (TILs) are composed of bystander and tumor-specific T cells, and CD39 can be used to identify each population. Interestingly, tumor tissue is also infiltrated by a group of unconventional T cells called Mucosal-associated invariant T (MAIT) cells. Since MAIT cells recognize riboflavin metabolites produced by many species of bacteria and there is mounting evidence that gut microbiota composition can strongly influence the antitumor efficacy of drugs, we explored whether the response of these cells in tumors could be antigen-driven. The transcriptomic profiles of tumor-infiltrating MAIT cells showed an enrichment for genes related to apoptotic and TCR signaling pathways, supporting the importance of MAIT cells in tumor response. We then distinguished two different populations among tumor-infiltrating MAIT cells that are either CD39+ or CD39-, and showed that in vitro, CD39 expression is induced in a TCR-dependent manner and not by non-TCR cytokine-dependent signaling. To investigate the relationship between MAIT cells and the gut microbiome in Colorectal Cancer, we have estimated the microbial composition in each bulk tumor using whole genome sequencing data. We observed a positive correlation between CD39+ MAIT cell frequency and tumor bacterial load, suggesting that the phenotype and function of tumor-infiltrating MAIT cells could be shaped by the gut microbial composition. Lastly, using mass-cytometry to deeply profile CD39+ MAIT cells, we observed a higher expression of the inhibitory receptor CTLA-4 and identified a subset of Treg-like MAIT cells (CD4+FoxP3+) specific to the tumors. The presence of these Treg-like MAIT cells is associated with a high infiltration in the tumors of bacteria from Bacteroidetes and Fusobacteria phyla. This study has allowed to identify two distinct populations in tumor-infiltrating MAIT cells that are CD39+ and CD39-, and revealed an unreported Treg-like subset. It will not only provide new insights in the T cell tumor immune responses, but also pave the way for using MAIT cells in gut microbiome manipulations.

   



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  SIRT1 deacetylates ROR[g]t and enhances Th17 cell generation
  Presenter: Hyungwook Lim
  All Authors:Hyungwook Lim, Seung Goo Kang, Jae Kyu Ryu, Birgit Schilling, Mingjian Fei, Intelly S. Lee, Amanuel Kehasse, Kotaro Shirakawa, Masaru Yokoyama, Martina Schnölzer, Herbert G. Kasler, Hye-Sook Kwon, Bradford W. Gibson, Hironori Sato, Katerina Akassoglou, Changchun Xiao, Dan R. Littman, Melanie Ott, and Eric Verdin
  Gladstone Institutes
   
 

The balance of effector and regulatory T cell function, dependent on multiple signals and epigenetic regulators, is critical to immune self-tolerance. Dysregulation of T helper 17 (Th17) effector cells is associated with multiple autoimmune diseases, including multiple sclerosis. Here we report that Sirtuin 1 (SIRT1), a protein deacetylase previously reported to have an anti-inflammatory function, in fact promotes autoimmunity by deacetylating RORγt, the signature transcription factor of Th17 cells. SIRT1 increases RORγt transcriptional activity, enhancing Th17 cell generation and function. Both T cell-specific Sirt1 deletion and treatment with pharmacologic SIRT1 inhibitors suppress Th17 differentiation and are protective in a mouse model of multiple sclerosis. Moreover, analysis of infiltrating cell populations during disease induction in mixed hematopoietic chimeras shows a marked bias against Sirt1-deficient Th17 cells. These findings reveal an unexpected pro-inflammatory role of SIRT1 and importantly support the possible therapeutic use of SIRT1 inhibitors against autoimmunity.

   



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  Factors controlling the induction of commensal specific CD8+ T cells in skin
  Presenter: Jonathan L. Linehan
  All Authors:Jonathan L. Linehan, Nicolas Bouladoux, Shruti Naik, Seong-Ji Han, Allyson Byrd, Yasmine Belkaid
  NIH-NIAID
   
 

Our laboratory recently demonstrated that skin-resident commensals control steady state immunity and host protective responses to cutaneous infection. However, little is known about the mechanisms by which this occurs, including which arms of the immune system are involved. We found that the human skin commensal Staphylococcus epidermidis (S.epi) promoted a robust Interleukin-17A (IL-17A) producing CD8+ T cell response when applied to the skin of mice. This CD8+ T cell response provided bystander protection from Candida albicans infection. These cells migrated to the epidermis and expressed surface molecules akin to pathogen-specific resident memory T (TRM) cells. Strikingly, transcriptional analysis identified a profile for commensal elicited CD8+ T cells distinct from skin-homing TRM cells, including some upregulated transcripts typically associated with regulatory T cells. Additionally, transcripts associated with canonical CD8+ T cells, including Runx3, were downregulated. We sought to determine which factors were required for the development of commensal specific IL-17A+ CD8+ T cells in the skin. Similar to TRM cells, there was a requirement for TGF-β signaling for the IL-17A+ CD8+ T cell phenotype. Next, we investigated whether these cells were restricted to classical or nonclassical MHCI molecules. CD8+ T cells isolated from skin produced IL-17A in the presence of S.epi loaded wild-type and KbDb-/- dendritic cells (DCs), but not H2-M3-/- DCs, indicating that these cells are specific for S.epi antigens and restricted to the nonclassical MHCI molecule H2-M3, which binds microbe derived formylated peptides. These results reveal that a common human skin commensal can drive a unique cutaneous CD8+ T cell response and highlight a previously unappreciated role for alternative modes of CD8+ T cell differentiation. These findings have important implications for our understanding of tissue immunity and the tissue specific factors controlling responses to the microbiota.

   



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  Unc93b1 recruits syntenin-1 to control TLR7 signaling and prevent autoimmunity
  Presenter: Bo Liu
  All Authors:Bo Liu, Olivia Majer, Lieselotte SM. Kreuk, Nevan Krogan, Gregory M. Barton
  Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
   
 

Nucleic acid (NA)-sensing Toll-like receptors, such as TLR7 and TLR9, are tightly regulated to enable discrimination between self and foreign NAs and thus prevent autoimmunity. Despite the structural and functional similarities between these receptors, evidence from mouse models that TLR7 and TLR9 can have opposing effects in autoimmune diseases suggests that individual TLRs may have distinct regulatory mechanisms. The chaperone protein Unc93b1 mediates the trafficking of multiple TLRs from the endoplasmic reticulum to endosomes. We performed a mutagenesis screen of Unc93b1 for TLR signaling in macrophages and discovered a new function of Unc93b1 that specifically limits TLR7 but not TLR9 signaling. Mutations in Unc93b1 that lead to enhanced TLR7 signaling also disrupt binding of Unc93b1 to syntenin-1, which has been implicated in trafficking of transmembrane proteins into multivesicular bodies (MVBs). Both Unc93b1 and TLR7 can be detected in exosomes, suggesting that recruitment of syntenin-1 facilitates the sorting of TLR7 into intralumenal vesicles of MVBs, which terminates signaling. Binding of syntenin-1 requires phosphorylation of Unc93b1 and provides a mechanism for dynamic regulation of TLR7 activation and signaling. Disruption of the Unc93b1/syntenin-1 interaction in mice results in TLR7-dependent autoimmunity. Thus, Unc93b1 not only enables proper trafficking of NA-sensing TLRs but also sets the activation threshold of these potentially self-reactive receptors. Our findings suggest that Unc93b1 and its associated proteins could be potential therapeutic targets that when dysregulated can lead to chronic inflammation or autoimmunity.

   



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  Crosstalk between lung epithelial cells and myeloid cells in innate immune defense against bacterial pneumonia
  Presenter: Xin Liu
  All Authors:Xin Liu, Mark A Boyer, and Sunny Shin
  University of Pennsylvania
   
 

Pathogens have evolved a wide range of strategies to allow survival and subsequently cause disease in humans. However, it is still poorly understood how the immune system successfully overcomes pathogen-induced disturbance and enable robust immune responses against infection. The intracellular bacterium Legionella pneumophila has the ability to block global protein synthesis in target macrophages, but the host can still strongly evoke an IL-1-dependent inflammatory response that clears infection. Here, we show that IL-1 signals directly to type II alveolar epithelial cells, which potently drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by these cells. Importantly, GM-CSF metabolically reprogrammed myeloid cells to undergo aerobic glycolysis, which was required for maximal pro-inflammatory cytokine production. Our findings reveal that lung epithelial cells act as a key intermediary to facilitate communication between infected and bystander myeloid cells and orchestrate antimicrobial defense.

   



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  Evolutionary constraint on LAT phosphorylation to optimize T cell ligand discrimination
  Presenter: Wan-Lin Lo
  All Authors:Wan-Lin Lo, Neel H. Shah; Sara Rubin; Weiguo Zhang; Veronika Horkova; Ina Fallahee; Ondrej Stepanek; Leonard Zon; John Kuriyan; Arthur Weiss
  University of California, San Francisco
   
 

Self/non-self discrimination is the core of T cell-mediated immune responses. The kinetic proofreading model, as a plausible mechanism, requires that the TCR binds to a cognate pMHC for a sufficient duration to allow a series of biochemical modification (e.g., phosphorylation) to reach competent states sufficient for successful T cell activation. However, the molecular identities of these proofreading events are yet to be defined. Here, our data suggest a previously unappreciated role of the adaptor protein, LAT, as a TCR signaling “proofreader” through its slow phosphorylation kinetics at Y132. Upon TCR stimulation, the T cell kinase Zap70 is activated. Among all of Zap70’s substrates, Y132 in LAT is the only one that exhibits slow phosphorylation kinetics. But this “slowly-phosphorylating” Y132 site is unique among the five LAT phosphorylation sites that is responsible for a critically important function, the recruitment of PLC?1. LAT-recruited PLCγ1 is phosphorylated and activated which results in subsequent calcium mobilization and, through diacylglycerol, contributes to Ras and PKC activation leading to successful T cell activation. Our data showed that replacement of this glycine with an aspartate accelerated Y132 phosphorylation. Consequently, G131D enhanced PLCγ1 activation, increased calcium flux, and resulted in greater ligand sensitivity. But the G131D mutation also compromised T cells’ abilities to discriminate self/non-self. Mouse peripheral OT-I CD8 T cells ectopically expressing G135D (homologous to human G131) LAT exhibited stronger phenotypical and functional activation characteristics than wild-type LAT expressing cells. G135D-expressing OT-I CD8 T cells were able to upregulate the activation marker CD69 and induce Erk phosphorylation, express key transcriptional factor IRF4, and produce the cytokine IFNγ in respond to the weak agonist G4 or a self-peptide to which wild-type LAT expressing cells could not respond. Interestingly, sequences conferring more rapid and efficient phosphorylation of LAT Y132 could be found in the LAT homolog of some cold-blooded animals, such as zebrafish. We showed that zebrafish thymocytes were less sensitive to cold temperatures than mammalian LAT. Thus, cold-blooded animals may require more efficient phosphorylation of the Y132 homologous sequence to ensure a strong PLCγ1 dependent responses in cold environments. Expression of G131D in OT-I TCR+ Jurkat variants was able to endow cells the ability to respond to OVA stimulation at room temperature, whereas the wild-type glycine at position 131 in LAT prevented cells from responding. Thus, our data suggested that the slow phosphorylation kinetics of human Y132 (mouse Y136) in LAT evolved to be a poor site of phosphorylation to in order to serve as a critical signaling step to control kinetic proofreading in mammalian T cells, while the cold-blooded animals require a more optimal site of phosphorylation at Y132 homolog due to the constraint imposed by cold temperature. Our data offers insight into a novel mechanism to allow mammals to fine tune their T cell response threshold and contributes to T cell antigen discrimination.

   



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  Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens
  Presenter: Erin W. Meermeier
  All Authors:Erin W. Meermeier, Aneta Worley, Bruno F. Laugel, Andrew K. Sewell, Alexandra J. Corbett, Jamie Rossjohn, James McCluskey, Melanie J. Harriff, Tamera Franks, Marielle C. Gold & David M. Lewinsohn
  Oregon Health and Science University
   
 

Mucosal-associated invariant T (MAIT) cells detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing T cell receptor (TCR)α. However, whether other TCRs can recognize MR1-antigen is unknown. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2+ MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2+ clone recognizes unique antigens. We provide preliminary evidence that MR1-restricted TCR usage differs between blood and a mucosal tissue site. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defense against infection by broadening the recognition of microbial metabolites.

   



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  Immune Subversion by the Protozoan Parasite Trichomonas vaginalis
  Presenter: Frances Mercer
  All Authors:Frances Mercer, Fitz-Gerald Diala, Shek Hang Ng, and Patricia J. Johnson
  UCLA
   
 

Trichomonas vaginalis is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection. While acute symptoms in women may include vaginitis, asymptomatic or untreated infections can persist and are associated with increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation is attributed to complications of T. vaginalis infection, however effective cellular immune responses to the parasite have not been characterized. Additionally, re-infection is common; suggesting poor effectiveness of an adaptive immune response. In vivo studies in humans and mice have indicated neutrophil (PMN) recruitment as the major cellular immune response to T. vaginalis infection, but PMN effectiveness at combating the parasite is not known. We established an in vitro co-culture system to assess the interaction between T. vaginalis and primary human leukocytes, using Flow Cytometry and Imaging Flow Cytometry. Cytokine analysis of supernatants from co-culture of T. vaginalis with human monocytes shows a cytokine program dominated almost exclusively by IL-8 secretion, a PMN recruitment chemokine. We also noted some striking differences between common laboratory strains and some clinical isolates of T. vaginalis. Firstly, some clinical isolates are able to lyse T-cells and B-cells, pointing to lymphocyte lysis as a potential adaptive immune subversion strategy. Secondly, PMN are able to efficiently kill laboratory strains of T. vaginalis; however several clinical isolates are resistant. Preliminary data indicate that PMN killing of T. vaginalis proceeds through an engulfment mechanism. Future experiments will determine the molecular players in PMN engulfment and killing of T. vaginalis, and the molecular determinants of PMN subversion by resistant strains.

   



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  Early immune responses to necroptotic death.
  Presenter: Michelle Messmer
  All Authors:Michelle Messmer, Annelise G. Snyder, Michael Y. Gerner, Andrew Oberst
  University of Washington
   
 

Necroptosis, a programmed form of cell death that results in significant cytokine and chemokine production as well as abundant release of cellular contents to extracellular space, has been demonstrated to more efficiently prime antigen-specific CD8+ T cell responses compared to other forms of cellular death. However, the immunogenic pathway connecting necroptotic cell-derived antigen to T cell priming remains poorly understood. This project seeks to define early immune responders targeted by specific forms of cell death. Using novel activatable pro-death constructs coupled with high-content imaging approaches, we show that necroptotic cell debris rapidly drains to lymph nodes and is taken up by myeloid subsets primarily in the subcapsular sinus, although antigen-bearing myeloid cells are also detectable within the deep T cell zone. Necroptotic antigen-bearing myeloid cells have higher expression of co-stimulatory markers following dying cell injection, concurrent with observed T cell clustering in the lymph node. Better understanding of the mechanisms underlying enhanced T cell priming by necroptotic cells will clarify settings such as in tumor therapy, where specific death induction could be optimally paired with immune interventions.

   



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  CCL21 defect impairs dendritic cell trafficking in SAMP1/YitFc mice
  Presenter: Zbigniew Mikulski
  All Authors:Zbigniew Mikulski, Rebecca Johnson, Iftach Shaked, Gisen Kim, Heba Nowyhed, Wendy Goodman, Grzegorz Chodaczek, Theresa Pizarro, Fabio Cominelli, Klaus Ley
  La Jolla Institute
   
 

SAMP1/YitFc (SAMP) mice develop chronic ileitis similar to Crohn’s disease. The disease mechanism is unknown but thought to involve adaptive and innate immune responses. Here, we discovered that the chemokine CCL21, a ligand for CCR7, is almost completely absent in mesenteric lymphatics and other tissues of SAMP mice. Lymphatic CCL21 is known to be required for dendritic cell (DC) trafficking. Absence of CCL21 results in a severe defect of CD11b+CD103+ DC migration from the ileal lamina propria to the mesenteric lymph nodes (MLN), similar to the defect seen in CCR7-deficient mice. The ability of DCs to produce retinoic acid supporting regulatory T cells (Tregs) is also drastically reduced in SAMP mice. In young mice, the defects in CCL21 expression and DC trafficking preceded the clinical manifestation of ileitis. As a therapeutic intervention, we mobilized DCs by oral treatment with the TLR7 ligand R848, which increased Tregs in MLN and dramatically improved disease scores. Thus, absence of CCL21 expression represents a major pathogenic defect contributing to ileitis in SAMP mice. Our data suggest that therapies aimed at improved DC trafficking might be useful in patients with Crohn’s disease.

   



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  Thymic tuft cells promote a medullary microenvironment rich in type 2 cytokines and shape T cell development
  Presenter: Corey Miller
  All Authors:Corey Miller, Irina Proekt, Jakob von Moltke, Haiguang Wang, Kristin Rattay, Kristen Lyn Wells, Imran Khan, Bruno Kyewski, Kristin Hogquist, Lars Steinmetz, Richard Locksley, Mark Anderson
  University of California, San Francisco
   
 

The thymus is responsible for generating a diverse yet self-tolerant T cell pool. While the thymic medulla is largely composed of developing and mature AIRE+ epithelial cells, recent evidence suggests far greater heterogeneity amongst medullary thymic epithelial cells than previously appreciated. Here we describe in detail an epithelial subset which shares striking similarity to peripheral tuft cells found at mucosal barriers. As in the periphery, thymic tuft cells express the canonical taste transduction pathway and IL25. However, they are unique in their ability to present antigen and express a broad diversity of taste receptors. A subset of thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2 for their development. Remarkably, cellular depolarization is required for their thymic function where they support the development and function of thymic iNKT cells and act to establish a medullary microenvironment rich in type 2 cytokines. These findings reveal a dynamic medullary environment where differentiation of a minor and highly-specialized epithelial subset plays a non-redundant role in shaping thymic function.

   



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  Mesenchymal stem cell treatment modulates the CD8 T cell response in feline chronic gingivostomatitis
  Presenter: Emily Mills Ko
  All Authors:Emily Mills Ko, Emily Mills Ko, Boaz Arzi, Amir Kol, Naomi Walker, Kaitlin Clark, Dori Borjesson
  UC Davis
   
 

Mesenchymal stem cells (MSCs) have potent immunomodulatory and trophic properties. Feline chronic gingivostomatitis (FCGS), a naturally occurring, chronic oral inflammatory disease marked by severe lymphoplasmacytic inflammation of the oral mucosa and gingiva. Intravenous (IV) administration of autologous MSCs modulates the immune response, reduces disease severity, and regenerates the oral mucosa in cats with non-responsive FCGS. Of the 8 cats treated with 2 doses each, 3 were completely cured, 3 had significant clinical improvement and 2 did not respond to treatment. Cats that were completely cured had high blood CD8+ T cell numbers at presentation with marked clinical improvement associated with a correction of elevated CD8+ T cell numbers at 6 months post MSC therapy (p<0.05). We hypothesize that MSCs regulate the proliferation and cytotoxicity of CD8+ T cells. We also hypothesize that MSCs reduce the expression by CD4 T cells of key signals required to maintain memory populations which sustain the CD8+ T cell response over time. MSC administration to cats with spontaneous FCGS may serve as a novel animal model to better understand MSC interactions with the immune system in vivo, and to translate regenerative medicine therapies to treat chronic human oral inflammatory disorders.

   



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  PATHOGEN SENSING BY THE NLRP1 INFLAMMASOME
  Presenter: Patrick Mitchell
  All Authors:Patrick Mitchell, Andrew Sandstrom, Lisa Goers , Edward W. Mu, Cammie F. Lesser, Russell E. Vance
  University of California, Berkeley
   
 

Inflammasomes are innate immune complexes that orchestrate downstream inflammatory signaling. Upon pathogen detection, inflammasome assembly leads to Caspase-1 dependent processing of downstream effectors (e.g., IL-1β). We have proposed that inflammasomes play a critical role in innate defense via detection of “patterns of pathogenesis” – unique, pathogen-encoded activities that distinguish pathogens from harmless microbes. Many inflammasomes sense and are subsequently activated by binding to microbial ligands, analogous to pattern recognition receptor binding of pathogen-associated molecular patterns. In contrast, the NLRP1 inflammasome is activated by proteolytic cleavage of its N-terminus. However, the mechanism by which N-terminal cleavage converts NLRP1 from its inactive to active state was unknown. Here we describe a unique mechanism of NLRP1 inflammasome activation by “functional degradation.” We find that cleavage results in proteasome-mediated degradation of the N-terminal domains of NLRP1, liberating a C-terminal fragment that is a potent Caspase-1 activator. By inducing the specific degradation of NLRP1, we show that proteasome-mediated degradation of NLRP1 is not only necessary but also sufficient for NLRP1 inflammasome activation. Moreover, our new ‘functional degradation’ model led us to identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1 degradation and activation. Our results provide a unified mechanism for NLRP1 activation by diverse pathogen-encoded enzymatic activities.

   



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  Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse
  Presenter: Stephen Mok
  All Authors:Stephen Mok, Colm R. Duffy, Nana-Ama A.S. Anang, James P. Allison
  MD Anderson Cancer Center
   
 

The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1). Blocking either pathway relieves the negative regulation of T-cells resulting in significant antitumor responses in patients with melanoma, lung cancer, and a growing list of malignancies. Both anti-CTLA-4 and anti-PD-1 therapy induce durable responses and improve survival rate. As the number of patients being administered checkpoint blockade antibodies increases, the total number of patients who respond for a period of time before tumor relapse will also increase. Approximately 25% of patients who initially respond to treatment relapse within 2 years. Although several tumor relapse mechanisms have been described, the long-term immunity mediated by immunotherapies remains unclear. We hypothesized that anti-CTLA-4 and anti-PD-1 have differential effects on memory T-cell differentiation. We used anti-CTLA-4 or anti-PD-1 therapy in combination with irradiated cancer cell vaccine in mice and re-challenged those mice with the same cancer cell line. We observed that in murine tumor models, anti-CTLA-4 mediates a more durable memory antitumor response than anti-PD-1. By tracing the antigen-specific CD8 T-cells throughout the memory phase to re-challenge, we demonstrated that during re-challenge, the memory T-cells generated by anti-CTLA-4 1) expand in greater frequency, 2) have more cytokine production, and 3) differentiate into the population of KLRG1+ effector CD8 T-cells among effectors compared with anti-PD-1. Our studies enable us to identify the long-term effect of specific immunotherapies on the immune system.

   



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  C5aR Blockade Increases Intratumoral Clonal T Cell Response in Squamous Carcinomas
  Presenter: Dhaarini Murugan
  All Authors:Dhaarini Murugan, Wesley Horton, Terry R. Medler, Sushil Kumar, Tiziana Cotechini, Patrick Leyshock, Alexandra M. Forsyth, Justin J. Leitenberger, Molly Kulesz-Martin, Adam Margolin, Zena Werb, and Lisa M. Coussens
  Oregon Health and Science University
   
 

Activation of complement cascade factors in neoplastic tissues has been paradoxically linked to both cancer progression, and cancer restriction. As a protumoral mediator, activation of terminal complement proteins (C5a) leads to recruitment and activation of leukocytes into “damaged” tissues. To investigate potential T cell-suppressive properties of complement mediators, we have utilized the K14-HPV16 transgenic mouse model of squamous carcinogenesis to evaluate functional mediators of complement activation, and downstream effector programs regulating T cell functionality. Our data have revealed that urokinase (uPA)-positive macrophages regulate C3-independent activation of complement C5a in neoplastic tissue, that in turn mediate suppression of CD8+ T cell cytotoxicity. Therapeutic inhibition of C5a receptor (C5aR1) with a peptide antagonist improved efficacy to paclitaxel chemotherapy associated with CXCR3-dependent CD8+ T cell activation. To investigate the effects of combination therapy on the T cell repertoires infiltrating responding tumors and in peripheral blood, we performed deep sequencing of the complementarity-determining region (CDR) 3 of the T cell receptor (TCR)β chain in matched tumor lysates and peripheral blood mononuclear cells (PBMCs). Combination therapy induced a significant reduction in both circulating and intratumoral TCRβ diversity indicating selective enrichment of specific T cell clonotypes. In addition, clonal homeostasis analyses revealed increased hyperexpanded clones in tumor, and a corresponding increase in the medium frequency clones in PBMCs. Further, combination therapy increased TCRβ clonotype sequence commonality between tumor and PBMCs as identified by pairwise analyses. Together, these data indicate that C5aR1-blockade in combination with paclitaxel chemotherapy facilitates infiltration of antigen-specific T cell clones into tumors and associated with decreased tumor burden and slowed tumor kinetics. In addition, these data highlight the importance of targeting C5aR1-dependent signaling pathways as important immune modulatory pathways in squamous carcinomas for anti-cancer immunotherapy. Acknowledgement: T.R.M. acknowledges support from the American Cancer Society – Friends of Robert Kinas Postdoctoral Fellowship (PF-14-221-01-MPC), NIH/NCI Postdoctoral Training Grant (CA106195), The Cathy and Jim Rudd Career Development Award for Cancer Research, and the Medical Research Foundation. M.K.M. acknowledges support from NIH/NCI (CA192405). Z.W. acknowledges support from the NIH/NCI (CA057621). L.M.C. acknowledges support from the NIH/NCI (CA130980, CA155331, CA163123), a DOD BCRP Era of Hope Scholar Expansion Award (W81XWH-08-PRMRP-IIRA), the Susan G Komen Foundation (KG110560), and the Breast Cancer Research Foundation, the Breast Cancer Research Foundation, the Brenden-Colson Center for Pancreatic Health, and a Stand Up To Cancer – Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant.

   



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  Characterization of metabolic profiles in follicular B cells post activation
  Presenter: Arpita Myles
  All Authors:Arpita Myles, Michael Cancro
  University of Pennsylvania
   
 

Immune cell activation is accompanied with bioenergetic shifts that support proliferation, growth and differentiation. The metabolic profile of B lymphocytes in response to activation is not well characterized. We report that follicular B cells (CD23+) upregulate their mitochondrial mass upon stimulation through the B cell receptor (BCR) or toll-like receptor 9 (TLR9). This is accompanied by increase in levels of reactive oxygen species (ROS), basal respiration, ATP production and spare respiratory capacity. In vitro, addition of anti-CD40 (to mimic co-stimulatory signals) causes a reduction in glycolysis and mitochondrial respiration. Since B cell differentiation in vivo is tailored by cytokines like IL-4 and IL-21, we examined their effects on metabolic profiles of ex vivo activated follicular B cells. IL-4 reduced mitochondrial metabolism in context of both BCR + anti-CD40 and TLR9 stimulation. IL-21, on the other hand, upregulated metabolism of CpG (TLR9 ligand) treated cells but did not substantially alter properties of BCR + anti-CD40 group. These findings suggest that B cell activation in vitro is accompanied by metabolic shifts, which are tuned by co-stimulatory and cytokine signals. The mechanism underlying these shifts, as well as their functional relevance in the context of B cell differentiation in vivo models of infection and immunization requires further investigation.

   



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  Identifying and engineering TCR specificity against mutated self
  Presenter: Martin Naradikian
  All Authors:Martin Naradikian, Leslie Montero, Samantha Hall, Claire Chen, Milad Bahmanof, Luise Sternberg, Jerome Lane, Zeynep Kosaloglu-Yalcin, Manasa Lanka, Aaron Miller, Bejorn Peters, Ezra Cohen, Stephen Schoenberger
  La Jolla Institute for Allergy and Immunology
   
 

With the recent successes of checkpoint inhibitors and chimeric-antigen receptor (CAR) T cell therapy against various malignancies, immunotherapy has emerged as a new standard of care in the field of oncology. However, these approaches fail to impart specificity against cancer neo-antigens. Indeed, while CD19-specific CAR T cells do eliminate B cell leukemia, they also eradicate normal B cells. Adopting a similar approach for solid tumors will prove difficult because most malignancies do not have lineage-determining surface markers as well restricted as CD19 is to B cells and are typically shared with vital tissues and organs. Thus, there exists a need to engineer specificity against “mutated self”. Here, we report a novel methodology for determining immunogenic, mutated-self peptides in multiple cancers using whole exome and transcriptome sequencing of patient-derived tumor biopsies. Importantly, using these peptides, we empirically show that patients harbor neo-antigen specific CD4 and CD8 T cells in both TIL and blood. By sorting cytokine-secreting T cells and performing single-cell sequencing, we have identified TCRs that recognize common driver mutations. Moreover, we validate these TCR constructs by engineering T cells and stimulating them with proper MHC-restricted antigen presenting cells. Lastly, we have established inducible cancer stem cell lines from patients and transplanted them into PDX mouse models. Future studies will address whether engineering sufficient numbers of neo-antigen specific T cells will lead not only to the recognition of established tumors but also control and eradication.

   



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  Identifying and engineering TCR specificity against solid tumor neoantigens
  Presenter: Martin S Naradikian
  All Authors:Martin S Naradikian, Leslie Montero, Samantha Hall, Rukman Thota, Milad Bahmanof, Luise Sternberg, Jerome Lane, Angela Frentzen, Zeynep Kosaloglu-Yalcin, Aaron Miller, Bjoern Peters, Ezra Cohen, Stephen Schoenberger
  La Jolla Institute for Immunology
   
 

Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient’s immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient’s set of HLA molecules. These methods have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient’s own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV16+ Head and Neck Squamous Cell Carcinoma lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07:02. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy.

   



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  Reversing T cell anergy by removing E3 ubiquitin ligases Cbl-b.
  Presenter: Trang Nguyen
  All Authors:Trang Nguyen, Zhi-En Wang, Lin Shen, Arthur Weiss.
  University of California, San Francisco
   
 

T cell anergy is an intrinsically inactive state resulting from absent co-stimulation and/or high co-inhibitory tone. T cell anergy is an important peripheral tolerance mechanism to prevent self-reactive T cells from attacking host tissues. Unresponsiveness in anergic T cells has been attributed to the induction of multiple negative regulators, including E3 ubiquitin ligases, that target T cell receptor (TCR) signaling pathways. However, it is not generally understood how T cell anergy states are established and maintained. We generated a model of T cell anergy by introducing the Zap70 hypermorphic mutant, W131A, into the OT2 transgenic background (W131AOT2) which resulted in high numbers of anergic and Treg CD4 T cells. Peripheral W131AOT2 anergic CD4 T cells had impaired TCR signaling and failed to produce IL-2 or up-regulate CD69, CD25, and Nur77 after antigen or TCR stimulation. Interestingly, single positive CD4 thymocytes from these mice exhibited normal TCR signaling and normal upregulation of CD25, CD69, and Nur77, as well as IL2 production in response to antigen or anti-CD3 stimulation. Thus, the anergic phenotype is acquired in the periphery. The E3 ubiquitin ligase Cbl-b (Cbl Proto-Oncogene B) is highly expressed in anergic T cells and contributes to establishing the unresponsive state. Cbl-b-/- T cells are largely resistant to T cell anergy induction and Cbl-b-deficient (Cbl-b-/-) mice have enhanced susceptibility to develop spontaneous autoimmunity. Thus, Cbl-b plays a central role in the development of autoimmunity and setting a threshold for T cell activation. Peripheral W131AOT2 T cells had large increases in Cbl-b mRNA and protein expression compared to control OT2 T cells. To assess functions of Cbl-b in maintaining T cell anergy, W131AOT2 were crossed to Cbl-b deficient (Cbl-b-/-) mice to generate W131AOT2 Cbl-b-/- mice. Interestingly, W131AOT2 Cbl-b-/- exhibited similar frequencies of phenotypically anergic, Treg CD4 T cells compared to W131AOT2 mice. However, loss of Cbl-b in W131AOT2 mice reversed peripheral T cell unresponsiveness to antigen or anti-CD3 stimulation, including up-regulation of phosphorylated Erk (pErk), phosphorylated Akt (pAkt), T cell activation markers CD69, CD25, and the proportion of IL-2 secreting CD4 T cells and their proliferation. Together, these results reveal that T cell anergy is induced in the periphery and Cbl-b plays an essential role in the regulation of peripheral tolerance and anergy of T cells.

   



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  T cell abnormalities in Good Syndrome: not just a B cell deficiency
  Presenter: Hiromi Ogawa
  All Authors:Hiromi Ogawa, Forum Patel, Michiko Shimoda, Emanual Maverakis
  UC DAVIS school of Medicine
   
 

Good syndrome is a rare immunologic disease characterized by hypogammaglobulinemia in the setting of thymoma. It was first described by Dr. Robert Good in 1954. Clinical studies have demonstrated that Good syndrome is associated with hematological disorders, which include anemia in over 50% patients, a low white blood cell count, thrombocytopenia and neutropenia. The immunological studies reveal low to absent B cells in the peripheral blood and an inverted CD4/CD8 T cell ratio. Patients with Good syndrome often experience pathogenic infections as well as other presentations common with other B cell deficiencies such as X linked agammaglobulinaemia (XLA) and common variable immune deficiency (CVID). For example, patients with Good syndrome are susceptible to opportunistic infections and other disorders that are linked to cell-mediated immunity, which indicates that these patients have dysregulation in both their humoral and cellular immunity. In this study, we examined the immune profile of PBMCs in Good syndrome patients by flow cytometry and their cytokine secretion profiles following stimulation with anti-CD3/28 and/or rIL-2. Our results demonstrate that Good syndrome CD4 T cells express higher levels of HLA-DR compared to healthy controls. In addition, Good syndrome patients had significantly elevated Th2 type cytokine production following anti-CD3/28 stimulation. On the other hand, Th1 type cytokine production was significantly reduced in PBMCs following stimulation with rIL-2 and/or with anti-CD3/28. These findings indicate that, in addition to their lack of a humoral response, Good syndrome patients also have imbalanced cytokine production by T cells, which may be one reason why they are predisposed to infections linked to cell-mediated immunity. Developing a better understanding of the mechanism of immune dysregulation might be helpful for treating patients with Good syndrome.

   



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  The Role of Ubiquitin Specific Protease 1 in Promoting Id Protein Stability During T Cell Responses
  Presenter: Kyla Omilusik
  All Authors:Kyla Omilusik, Kyla D. Omilusik, Marija Nadjsombati and Ananda W. Goldrath
  University of California, San Diego
   
 

CD8+ T cells are necessary components of immune responses against intracellular pathogens and tumours. The activation of effector T cells and subsequent differentiation into memory populations is a complex process with dramatic changes in gene expression. Of interest, E protein transcription factors and their inhibitors, Id proteins, play key roles in lymphocyte development, differentiation, and maintenance. Specifically, one Id protein, Id2, is upregulated in effector CD8+ T cells and maintained in memory cells presumably to induce effector function. Interestingly, Id2 overexpression is also associated with lymphomas. However, the mechanisms controlling normal Id2 expression and how misregulation leads to transformation is largely unknown. Recently, USP1, a deubiquitinase, was shown to stabilize Id2 in osteosarcomas which resulted in decreased E protein activity. Analogously, we propose a role for USP1 in posttranslational control of Id2 in T cells. Here, we show that USP1 is upregulated in T cells following infection and this expression change is mediated through the T cell receptor. Furthermore, we establish a direct interaction of Id2 and USP1 following T cell activation. By defining a regulatory role for USP1 mediated deubiquitination of Id2 and subsequently E protein activity, we will have a more complete understanding of mechanisms required for controlled survival and expansion of healthy T cells and how failure of this process can lead to tumorigenesis. Ultimately, identifying USP1 as an oncogene in lymphomas will provide a novel target for therapeutic interventions.

   



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  c-Ski mediates Th17 and Treg responses to TGFß
  Presenter: SuFey Ong
  All Authors:SuFey Ong, Mathais Hauri-Hohl, Steven F Ziegler
  Benaroya Research Institute
   
 

TGFß is involved in both induced Treg and Th17 differentiation via its induction of Foxp3 and ROR?t. The oncogene c-ski regulates TGFß function by inhibiting SMAD2/3 from both binding to SMAD4 and to DNA, repressing the transcription of TGFß-inducible genes. Previously, we have shown that the absence of ski in medullary thymic epithelial cells (mTECs) limits cellular growth due to unchecked TGFß signaling. Given that central role of Treg and Th17 cells in the development of autoimmune disease, we explored the role of Ski and TGFß signaling in T cell function and differentiation. We demonstrate that the absence of Ski in CD4+ T cells leads to amplified TGFß sensitivity as shown by increased Th17 differentiation at low levels of TGFß in vitro. Additionally, Foxp3CreSkifl/fl T cells polarized in the presence of TGFß and IL-2 in vitro lead to increased Treg conversion in culture, suggesting that Ski may also play a role in Treg stability. Both of these strains show decreased EAE pathogenicity compared to control mice. In conclusion, we have shown that Ski functions as a modifier of TGFß signaling in CD4+ T cells and can directly influence the outcome of autoimmune disease pathogenesis.

   



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  T Lymphocyte Calcium Dynamics Using a Ratiometric tdTomato-GCaMP6f Transgenic Reporter Mouse
  Presenter: Shivashankar Othy
  All Authors:Shivashankar Othy, Tobias X. Dong, Amit Jairaman, Jonathan Skupsky, Angel Zavala, Ian Parker, Joseph L. Dynes, and Michael D. Cahalan
  Department of Physiology and Biophysics, University of California Irvine, CA 92697-4561, USA
   
 

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Currently available synthetic calcium indicators such as fura-2 and fluo-4 are unsuitable for long-term studies due to leakage out of cells, and fluorescent protein-based GECI such as GCaMP6 (non-ratiometric) are problematic for calcium imaging in motile cells where fluorescence changes resulting from movement may be indistinguishable from actual changes in calcium levels. Here, we describe development and characterization of “Salsa6f”, a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic calcium. We created a transgenic mouse strain in which Salsa6f is expressed under cell-specific genetic control in Cre-driver strains using the Rosa26-Cre recombinase system, and we have used this system to monitor cytosolic calcium in CD4-Salsa6f T cells. Salsa6f expression is non-perturbing; we saw no effects of Salsa6f expression in T cell surface phenotype, cell proliferation, differentiation, homing, and motility in the lymph node. Salsa6f is distributed uniformly throughout the cytosol; its exclusion from the nucleus provides reliable and selective reporting of cytosolic calcium signaling. We describe single cell calcium signals reported by Salsa6f during T cell receptor activation in naïve T cells, Th17 cells and regulatory T cells, and calcium signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of calcium signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular calcium transients (“sparkles”) as cells migrate. Altogether, our results demonstrate the sensitivity, brightness, uniformity of labelling, and ease of detecting calcium signals in moving T cells using Salsa6f.

   



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  Allosteric sensing of peptide-MHC recognition by the T cell Receptor
  Presenter: Sarah Overall
  All Authors:Sarah Overall, Andrew McShan, Kannan Natarajan, Jinasheng Jiang, Vlad Kumirov, Rui Wang, Huaying Zhao, Peter Shuck, Mulualem Tilahun, Jinfa Ying, Louise Boyd, Ad Bax, David Marguilles, Nikolaos Sgourakis
  University of California Santa Cruz
   
 

The recognition of peptide-MHC (pMHC) by the TCR is a critical first step in the activation and differentiation of T cells. Mechanistically, it is still unclear how antigenic sensing by the TCR is achieved, in particular, how the TCR communicates ligand binding to CD3, which forms the entire signaling component of the TCR:CD3 complex. We have characterized perturbations of the TCR constant domains by NMR and show distinct changes upon pMHC recognition in both the alpha and beta chains, indicative of structural or dynamic changes in these regions. Perturbations in the alpha chain cluster within the hinge region between the variable and constant domains suggestive of interdomain motion following pMHC engagement. Further characterization of the bond angles within the two domains of the unliganded alpha chain further suggests that in solution, the two domains may be oriented differently relative to the crystal structure. In contrast, the beta chain shows distinct perturbations of residues E130 and T138 of the constant domain upon pMHC binding. Mutations of these residues in T cell lines inhibits receptor expression, however mutations of adjacent residues in the H3 helix of the beta chain abrogates IL-2 production, demonstrating the critical role these residues play in propagating ligand binding events from the TCR to CD3 and providing evidence that the TCR undergoes allosteric changes upon pMHC recognition.

   



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  Macrophage-pDC dynamics and interferon production during viral infection
  Presenter: Oriana Perez
  All Authors:Oriana Perez, Stephen T. Yeung, Kamal Khanna, Mohamed Oukka, Daniel Stetson, Boris Reizis
  NYU Medical School
   
 

Plasmacytoid dendritic cells (pDCs) are a specialized subset of dendritic cells that produce robust quantities of type I interferons (IFNs) early after viral infection. The early production of IFNs is critical for initiating a series of antiviral host responses driven in part through the activation of innate immune cells and the formation of adaptive immunity. Previous in vitro studies have shown that IFN production by pDCs can be triggered by interaction with live virus-infected cells, however there is currently no evidence demonstrating pDC activation in vivo. Thus, the exact cellular dynamics regulating pDC activation in vivo are not fully understood. To address this current gap in our understanding we sought to visualize pDC activation in vivo following viral infection. For this purpose we utilized novel reporters to visualize interferon beta (IFNB) responses and pDC dynamics following local vesicular stomatitis virus (VSV) skin infection. Our recent studies showed that CD169+ macrophages in the skin draining lymph nodes (LNs) regulated pDC activation and IFNB responses. For these studies we locally depleted CD169+ macrophages (CD169-DTR mice) and showed that in the absence of CD169+ macrophages pDC activation was significantly impaired following VSV infection. Histological analysis suggests pDCs in close proximity to VSV-infected CD169+ macrophages were the primary producers of IFNB and in the absence of CD169+ macrophages the spatial organization of IFN?-producing pDCs was altered. These finding reveal a dynamic functional dichotomy between the role of macrophages and pDCs in host protection against viral infection.

   



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  Interleukin-1 signaling drives hematopoietic stem cells towards precocious myeloid differentiation at the expense of self-renewal
  Presenter: Eric Pietras
  All Authors:Eric Pietras, Cristina Mirantes-Barbeito, Sarah Fong, Dirk Löffler*, Ranjani Lakshminarasimhan, Chih Peng Chin, José-Marc Techner, Britta Will°, Claus Nerlov#, Ulrich Steidl°, Timm Schroeder*, and Emmanuelle Passegué
  The Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, USA. *Institute for Stem Cell Research, Swiss Institute of Technology, Basel, Switzerland. °Department of Cell Biology, Albert Einstein Medical College, Queens, New York, USA, #Weatherall Institute of Molecular Medicine, Oxford University, Oxford, UK
   
 

Chronic inflammatory diseases such as gout, rheumatoid arthritis and type II diabetes are debilitating conditions driven by aberrant immune system activation. Importantly, these diseases are characterized by deregulated production of blood cells leading to severe hematological complications, including anemia and overproduction of myeloid cells, which contribute to disease pathology. Although increased levels of the pro-inflammatory cytokine interleukin-1 (IL-1) is a key driver of these diseases, its role in regulating their hematological features is largely unknown. Here, we demonstrate that IL-1 directly impacts the fate of rare hematopoietic stem cells (HSCs) that normally provide lifelong maintenance of all blood cell lineages. Using continuous single-cell tracking technology and molecular analyses, we show that IL-1 accelerates HSC cell division and directly instructs HSC differentiation along the myeloid lineage via rapid, precocious activation of the myeloid transcription factor, PU.1. We find that this mechanism ‘primes’ HSCs to rapidly produce myeloid cells in response to acute need following myeloablative chemotherapy. Importantly however, chronic exposure to IL-1 severely restricts HSC lineage output, leading to continued myeloid overproduction at the expense of the lymphoid and erythroid lineages. Moreover, IL-1 severely erodes the self-renewal and regenerative capacity of HSCs, resulting in their failure under replicative challenges such as serial transplantation. Thus, our results identify a critical regulatory circuit by which IL-1 directly regulates myeloid differentiation in HSCs, and demonstrate that IL-1 functions as a double-edged sword, accelerating myeloid production in response to acute needs while disrupting HSC self-renewal and lineage output in the context of chronic exposure. We are now identifying the contribution of this circuit to deregulated blood production in several models of IL-1-driven chronic inflammatory disease, investigating the mechanisms by which IL-1 signaling activates PU.1, and further characterizing the changes in HSC fitness caused by chronic IL-1 exposure. Collectively, these findings provide critical insight into the effects of IL-1 on HSC function and blood homeostasis. They also provide a basis for understanding and targeting deregulated blood production that contributes to the pathogenesis of a wide array of IL-1-driven chronic inflammatory diseases.

   



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  Therapeutic targeting of airways epithelium for the prevention of rhinovirus induced asthma exacerbation
  Presenter: Kathryn Pothoven
  All Authors:Kathryn Pothoven, Rane S. Creasy BSc, Kaitlyn A. Barrow BSc, Matthew C. Altman MD, Jason S. Debley MD, Steven F. Ziegler PhD
  Bnaroya Research institute
   
 

Human rhinovirus (HRV) infection is a common viral trigger of asthma exacerbation. Airway epithelial cells express ICAM1, the entry receptor for the A serotype of HRV and they are the first cell-type to encounter HRV upon infection. Epithelial cells play an important role in general tissue homeostasis, repair of tissue injury, and responses to inflammatory stimuli. Additionally, epithelial cells from asthmatic patients have been shown to be structurally dysfunctional compared to healthy epithelia. We hypothesize that the airway epithelial response to HRV primes a subsequent asthma exacerbation and that the clinical timepoint of onset of rhinovirus symptoms may be a potential therapeutic intervention point aimed at preventing or reducing the symptoms of rhinovirus induced exacerbations. Airway epithelial cells were obtained from control (H) and asthmatic pediatric patients. The asthmatic patients can be further categorized based on whether the patient has a previous history of exacerbation. Patients with a previous history of exacerbation (E) have more severe disease that patients that do not have a history of exacerbation (A). The nasal epithelial brushings from all patients were grown at air-liquid interface (ALI) and they were either left uninfected or infected with HRV16 and at 24 hours post infection RNA was harvested for RNAseq analysis. A Venn diagram comparing the H, A and E groups showed 36 increased and 43 decreased genes in the H group, 34 increased and 39 decreased genes in the A group, and 475 increased and 389 decreased genes in the E group. The E group is distinct from the other two groups, supporting our hypothesis that the epithelial cells from patients with a history of previous exacerbation may prime a subsequent exacerbation. Clustering analysis of the 475 upregulated genes in the E group identified a unique cluster of cytokines and cytokine receptors, OSMR, IL1A, IL4R, INFGR2, IL23A, IFNL1, IFNL2, and IFNL3 that are attractive therapeutic candidates. Mechanistic studies will be conducted to determine the efficacy of inhibition of these therapeutic candidates in the context of airways disease.

   



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  Neutrophil-derived Oncostatin M is elevated in eosinophilic mucosal disease and may promote epithelial barrier dysfunction through the induction of epithelial mesenchymal transition.
  Presenter: Kathryn Pothoven
  All Authors:Kathryn Pothoven, James Norton, Lydia Suh, Roderick Carter, Bruce K Tan, Robert P Schleimer
  Northwestern University Feinberg School of Medicine
   
 

Epithelial barrier dysfunction is thought to play a role in many mucosal diseases including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis (EoE). Oncostatin M (OSM), was elevated in both mRNA (28.3 fold, p<.01, n=12) and protein (4.4 fold, p<.05, n=12-19) in the nasal polyps of CRS patients compared to control tissue. OSM protein was also elevated in induced sputum from asthmatic patients compared to control (7 fold, p<.05, n=11-12). Additionally, OSM mRNA was elevated in esophageal biopsies of EoE patients vs. controls, (3 fold, p<.01). OSM stimulation of differentiated airway epithelium induced barrier dysfunction, measured by decreased transepithelial electrical resistance (63% reduction, p<.0001, n=11) and increased permeability to 10kD dextran (2.45 fold, p<.05, n=5). Levels of OSM protein in tissue lysates from CRS and controls correlated with levels of a2-macroglobulin, a marker of epithelial leak, in matched nasal secretions (r=.5055, p<.01), suggesting that OSM may play a role in epithelial barrier dysfunction in mucosal disease. To determine which cell type was producing OSM, nasal polyp sections were stained for OSM and hematopoetic cell specific markers. OSM showed co-localization with neutrophil elastase (n=10), however OSM did not co-localize with markers for eosinophils, macrophages, T cells or B cells (n=3-5). GM-CSF has previously been shown to induce OSM. GM-CSF protein was elevated in nasal polyps compared to control. GM-CSF was sufficient to induce OSM protein in cell culture supernatants of ex vivo cultured neutrophils. Interestingly, immunofluorescent staining for GM-CSF showed that the source of GM-CSF were the OSM+ neutrophils, suggesting that these neutrophils were driving their own production of OSM (n=5). Additionally, bronchial biopsy sections were stained for OSM and neutrophil elastase. None of the control patients (n-4), 60% of the moderate asthmatics (n=5), and 100% of the severe asthmatics (n=6) had OSM+ neutrophils within the biopsy section, suggesting that neutrophil derived OSM may mediate barrier dysfunction in both CRS and asthma. To determine the mechanism of OSM mediated epithelial barrier dysfunction, we measured mRNA expression of OSM and markers of epithelial mesenchymal transition (EMT). OSM expression positively correlated with mesenchymal phenotype markers, (VIM, SERPINH1, ITGA5, and DDR2), EMT transcription factors, (TWIST1, TWIST2, ZEB1, and ZEB2) and the EMT inducer, TGFB1, suggesting that OSM expression and EMT may be related. OSM stimulation of differentiated airway epithelial cultures induced protein expression of mesenchymal markers, S100A4 and αSMA, and cellular proliferation marker ki67 shown through immunofluorescence, suggesting that OSM may mediate barrier dysfunction through the induction of EMT. Therapeutic targeting of OSM, its signaling, mediators of EMT, neutrophils, or GM-CSF may be beneficial in the treatment of eosinophilic mucosal disease.

   



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  Airway epithelium from asthmatic patients have an elevated immune response to human rhinovirus infection compared to healthy patients
  Presenter: Kathryn Pothoven
  All Authors:Kathryn Pothoven, Kaitlyn A. Barrow BSc, Matthew C. Altman MD, Jason Debley MD, Steven F. Ziegler PhD
  Benaroya Research Institute
   
 

Human rhinovirus (HRV) infection is a common viral trigger of asthma exacerbation. Airway epithelial cells express ICAM1, the entry receptor for most strains of HRV and are the first cell-type to encounter HRV upon infection. Epithelial cells play an important role in general tissue homeostasis, repair of tissue injury, and responses to inflammatory stimuli. Additionally, epithelial cells from asthmatic patients have been shown to be structurally dysfunctional compared to healthy epithelia. We hypothesize that the airway epithelial response to HRV infection in asthma patients is altered compared to control patients. Airway epithelial cells were obtained from control and asthmatic pediatric patients, grown at air-liquid interface (ALI) and left uninfected or infected with HRV16. RNAseq was performed on the samples and the data was further analyzed using an algorithm assigning differentially regulated genes to modules of genes that all have similar expression patterns. The collective gene expression of these modules were then compared between media control and HRV infection in both healthy and asthmatic patients. Two modules were identified to be elevated in ALI cultures derived from asthmatic patients upon HRV infection compared to ALI cultures from healthy patients. Gene ontology (GO) analysis identified that the first module was comprised of genes associated with type 1 interferon (IFN) signaling, response to IFNβ, negative regulation of viral genome replication and IFNγ signaling, indicating that the epithelium from asthmatic patients had an elevated antiviral immune response compared to controls upon infection. GO analysis also identified that the second module was comprised of genes associated with antigen presentation and processing via MHC class I, leukocyte and neutrophil chemotaxis, and I-κB kinase and NF-κB signaling that was elevated in ALI cultures from asthmatic patients compared to ALI cultures from healthy patients. These data suggest that epithelia from asthmatic patients have an elevated immune response to HRV compared to controls which may contribute to the development of subsequent asthma exacerbations.

   



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  Airway epithelium from asthmatic patients have altered immune responses to rhinovirus infection
  Presenter: Kathryn L Pothoven
  All Authors:Kathryn L Pothoven, Kaitlyn A Barrow, Jason S Debley, Steven F Ziegler
  Benaroya Research Institute, Seattle Children's Research Institute
   
 

Airway epithelia are the interface between the lungs and the environment. They play important roles in homeostasis of the airways, the immune response to pathogens and in inflammation. Epithelia from asthmatic patients have been shown to be structurally dysfunctional through the loss of differentiated cells, impaired barrier function, and altered cytokine expression including elevated IL-33 and TSLP. Human rhinovirus (HRV) respiratory infection is a common trigger of asthma exacerbation in both children and adults. We hypothesize that the airway epithelial immune response to HRV contributes to subsequent exacerbations in asthma patients. Airway epithelium from asthmatic patients and healthy controls were grown at air liquid interface (ALI) to allow the epithelial cells to fully differentiate as they would in vivo. Once the ALI cultures were fully differentiated, they were either left uninfected or infected with a 0.1 MOI of HRV16 for 48 or 96 hours. RNA was collected and analyzed. Both healthy and asthmatic epithelial cultures expressed elevated CXCL10 transcript compared to uninfected controls at 48 hours, suggesting that ALI cultures had been infected. However, ALIs from asthma patients expressed more CXCL10 compared to ALIs from healthy controls. Baseline expression of the type 2 epithelial cytokines TSLP and IL33 was also elevated in ALIs from asthma patients compared to controls and did not change upon infection at 48 hours, suggesting that asthmatic epithelia may have an elevation in both type 1 and type 2 immune responses to HRV infection. Interestingly, at both 48 and 96 hours the epithelial mesenchymal transition (EMT) marker, vimentin (VIM) was elevated in both HRV infected and uninfected epithelia from asthmatic patients compared to controls, suggesting that EMT may play a role in the pathogenesis of asthma. Additionally, the pro-repair growth factor, amphiregulin (AREG) was decreased in asthmatic epithelia following HRV infection. These data suggest that asthmatic epithelia may have an elevated type 1 and type 2 immune response, and may also have dysfunctional repair mechanisms in response to HRV infection.

   



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  Characterizing the metabolic interaction of Legionella pneumophila with its protozoan and metazoan hosts.
  Presenter: Jordan Price
  All Authors:Jordan Price, Russell Vance
  UC Berkeley
   
 

The intersection of infection and host cell metabolism is an underexplored realm of host-pathogen interaction. For many intracellular bacterial pathogens, is unclear how the metabolic environment within host cells affects bacterial survival, replication, and pathogenicity. The mammalian macrophage displays a high degree of metabolic plasticity, and is the preferred host cell for many species of pathogenic intracellular bacteria. The capacity of macrophages to remodel their metabolism in response to diverse stimuli may be one reason why these cells are routinely exploited by enterprising intracellular pathogens as a replicative niche. The facultative Gram-negative intracellular bacteria Legionella pneumophila is particularly well-suited for studying the interaction between host cell metabolism and bacterial pathogenesis, as it can infect a wide range of host cells by targeting conserved cellular processes, including host metabolism. While it naturally infects free-living amoebae, Legionella can also infect human alveolar macrophages and cause the respiratory illness known as Legionnaires’ disease. Many previous studies of Legionella infection have focused on how the bacteria manipulate host trafficking machinery to establish a replicative niche within its host cell, the so-called Legionella-containing vacuole. Less attention has been given to the metabolic state of the Legionella-infected macrophage, the nutrients Legionella consumes during infection, and how the mammalian immune response has evolved to respond to pathogenic metabolic perturbation. Here we present our preliminary findings describing the metabolic state of Legionella-infected macrophages in vitro, as well as data suggesting that perturbation of macrophage metabolism during infection has a dramatic impact on the ability of Legionella to replicate intracellularly. These studies expand our understanding of the dynamic metabolic interchange between host and intracellular pathogen during infection.

   



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  Antigen presentation by thymic stromal cells enforces central tolerance
  Presenter: Irina Proekt
  All Authors:Irina Proekt, Corey Miller, Mark Anderson
  UCSF
   
 

Abnormal selection of self-reactive T cells in the thymus is a key step to initiation of autoimmune disease. During development, single positive CD4+ T cells are exposed to a wide array of tissue-specific antigens (TSAs), which are produced by thymic medullary epithelial cells (mTECs) under the control of Autoimmune Regulator (Aire). TSAs can be presented in the context of major histocompatibility complex class II molecules (MHCIIs) by either mTECs or thymic dendritic cells. T cells that recognize these TSA-MHCII complexes are either deleted or positively selected to become regulatory T cells (Tregs). Although Aire+ mTECs express high levels of MHCII, the role of direct presentation to CD4+ thymocytes by these cells is not well understood. To address that, our lab has created a mouse model that allows for inducible genetic ablation of MHCII specifically in the Aire-expressing mTEC subset by using a tamoxifen-inducible Cre recombinase under the control of the endogenous Aire locus (iAire-cre) in combination with mice homozygous for an MHC Class II locus flanked by loxP sites (MHCIIfl/fl). We have shown that this model allows for efficient and specific ablation of MHCII from the surface of mTECs without affecting other thymic antigen-presenting cell subsets or perturbing thymocyte development. In addition, ablation of MHCII expression on mTECs completely disrupts negative selection and Treg conversion in the Rip-mOVA x OTII model. Furthermore, lack of MHCII on mTECs leads to loss of peripheral tolerance to an endogenous Aire-regulated retinal antigen, IRBP. These data suggest that direct presentation of self-antigens by mTECs is a major tolerance mechanism during thymic T-cell development.

   



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  Coupling intestinal iron uptake and innate immunity in C. elegans
  Presenter: Malini Rajan
  All Authors:Malini Rajan, Malini Rajan, Cole P. Anderson, Paul M. Rindler, S. Joshua Romney, Maria C. Ferreira dos Santos, Jason Gertz, Elizabeth A. Leibold
  University of Utah
   
 

Iron is essential for survival of most organisms, but is toxic in excess. All organisms have thus developed mechanisms to sense, acquire and sequester iron. In C. elegans, iron uptake and sequestration are regulated by HIF-1. We previously showed that hif-1 loss of function mutants are developmentally delayed when grown under iron limitation. Here we identify nhr-14, encoding a nuclear receptor homologous to vertebrate HNF4, in a genetic screen conducted for mutations that rescue the developmental delay of hif-1 mutants under iron limitation. NHR-14 is highly expressed in intestinal cell nuclei and in cells in the head, and its subcellular localization and expression are not regulated by iron. Loss of nhr-14 leads to the upregulation of the intestinal metal transporter SMF-3 that increases iron uptake in hif-1 mutants, rescuing the low iron-dependent developmental delay. Loss of nhr-14 also promotes the nuclear localization of the zinc-finger transcription factor PQM-1, which activates smf-3 through the interaction with GATA-like DAF-16-associated elements (DAEs) in the smf-3 promoter. In addition to smf-3, RNA-seq analysis revealed upregulation of innate immune response genes as well as DAF-16/FoxO-suppressed Class 2 genes, which are known to be regulated by PQM-1. Consistent with the upregulation of innate immune response genes, nhr-14 mutants showed enhanced resistance to the human pathogen Pseudomonas aeruginosa that depends in part on the upregulation of smf-3 and iron uptake as well as the upregulation of innate immune response genes. P. aeruginosa reduces expression of nhr-14 in wild-type N2 worms, which is associated with PQM-1 nuclear localization. We propose that increased iron uptake by SMF-3 is a strategy to limit the acquisition of intestinal iron by pathogens, and may serve as a critical component of the host innate immune response. Our data provide insight into how C. elegans utilizes nuclear receptors to regulate innate immunity and iron availability, and show iron sequestration as an important component of the innate immune response.

   



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  NAIP proteins are essential for inflammasome formation in response to cytosolic flagellin and type III secretion system components
  Presenter: Isabella Rauch
  All Authors:Isabella Rauch, Kang Chulho, Vance Russel E.
  UC Berkeley
   
 

Inflammasomes are a family of multiprotein cytosolic complexes that activate inflammatory caspases and host defense upon pathogen detection. The NLRC4 inflammasome can be activated by cytosolic presence of flagellin, or by the type III secretion system (T3SS) rod or needle proteins of bacteria. Recognition of these pathogen ligands is mediated by the NAIP family of NLRs, with NLRC4 serving as an adaptor to recruit Caspase 1 downstream of the NAIPs. Due to the highly homologous nature of the mouse NAIP genes arranged in one cluster at chromosome 13, targeting individual NAIPs for deletion with conventional methods has only been successful for NAIP5. Due to the redundancy of NAIP5 with NAIP6 in binding flagellin, Naip5–/– animals are only partially defective for responses to flagellin. The in vivo role of individual NAIPs is still unclear. For further clarification of the role of NAIPs in vivo, specific knockout strains are essential. The newly developed CRISPR/Cas9 method of genome editing allows to specifically target small regions in the genome, making it the ideal method to create such knockouts. We designed a guide RNA targeting all mouse NAIPs, thereby creating several different knockout lines with one targeting. We successfully created Naip1-/-, Naip2-/- and Naip 1-6 (cluster) -/- mouse lines. Infection of Naip1-/-, Naip2-/-, Naip5-/- and Naip1-6 -/- macrophages with Salmonella typhimurium shows that no single NAIP is required for pyroptosis upon Salmonella infection. However, loss of NAIP1-6 phenocopies NLRC4 deficiency. In vivo treatment of NAIP1-6 deficient animals with FlaTox (intracellular delivered flagellin) shows a complete loss of reaction to this toxin comparable to NLRC4 deficient animals, proving that NAIPs are essential for intracellular flagellin recognition. Further analysis of these mouse lines will give valuable insight into the role of individual NAIPs in pathogen recognition.

   



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  IL-17A inhibits expression of IL-17 lineage cytokines through a negative feedback loop involving IL-24, and controls autoimmune disease
  Presenter: Kumarkrishna Raychaudhuri
  All Authors:Kumarkrishna Raychaudhuri, Wai Po Chong, Reiko Horai, Phyllis B. Silver, Yingyos Jittayasothorn, Chi-Chao Chan, Jun Chen, Rachel R. Caspi
  NEI. NIH
   
 

The Th17 response has been associated with autoimmune diseases in patients and in animal models. IL-17A is recognized as the Th17 signature cytokine and IL-17-producing T cells are pathogenic effectors in models of autoimmunity, including experimental autoimmune uveitis (EAU). Paradoxically, injection of IL-17 was shown by others to ameliorate the disease. We used a model of spontaneous uveitis in IRBP T cell receptor transgenic R161H mice to investigate the susceptibility to disease of these mice on an IL-17A-/- background. Surprisingly, IL-17A-/- R161H mice developed essentially undiminished uveitis and IL-17-/- R161H T cells, polarized to Th17 and infused into wild type recipients, induced similar disease to IL-17A sufficient R161H T cells. Interestingly, IL-17A-/- R161H T cells polarized under Th17 conditions produced elevated amounts of other Th17-related cytokines, i.e. IL-17F, GM-CSF and IL-22. Supplementing these cultures with recombinant IL-17A normalized the elevated production of those cytokines. RNAseq analysis revealed that IL-17A-/- T cells displayed lower IL-24 expression compared to their IL-17 sufficient counterparts. Mechanistic studies indicated a negative feedback loop where IL-17A induces Th17 cells to produce IL-24, which suppresses production of Th17 lineage cytokines. Finally, injection of recombinant IL-24 ameliorated adoptive Th17-induced EAU, and conversely, depletion of IL-24 in Th17 cells increased their pathogenicity and elevated disease severity. These data suggest that IL-17A exerts a negative feedback on autopathogenic Th17 cells via IL-24 production, which limits the expression of other Th17 lineage cytokines that may contribute to pathogenicity.

   



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  The effect of HIV-1 infection on S1P-R1 expression and function in thymocytes and their progenitors during entry into and egress from the human thymus
  Presenter: Rachel S. Resop
  All Authors:Rachel S. Resop, Joshua Craft, Dimitrios Vatakis, Bianca Blom and Christel H. Uittenbogaart
  UCLA
   
 

Lack of adequate T cell regeneration in HIV infected individuals is likely due to a defect in the entry of hematopoietic stem cells (HSC) into and egress of naïve T cells from the thymus to the periphery. These phenomena remain understudied and not well understood. We studied the effect of HIV-1 infection on the receptors to Sphingosine-1-phosphate (S1P), a chemotactic sphingolipid mediator, during thymocyte trafficking. Our novel findings show that HIV infection changes the expression patterns of S1P-R1, which as we recently reported (Resop et al., JACI 2016) is normally expressed at the mRNA and protein levels only in the most mature CD3hi thymocytes that have lost CD69 (CD3hiCD69-) and are about to exit the thymus. We used multicolor (10+) flow cytometry and Quantitative Real-Time PCR (qRT-PCR) to examine the dynamics of S1P-R1 expression on Hematopoietic Stem Cell (HSC) progenitors and thymocytes during HIV infection. Mice implanted with human fetal thymus/liver (HIS-mice) infected with CXCR4- or CCR5-tropic HIV-1 were injected intravenously with CFSE-labeled HSC to examine the effect of HIV on HSC entry, thymocyte development and egress. We also investigated the mechanism of changes in S1P-R1 and its transcriptional regulator, Kruppel-Like factor 2 (KLF2). We found that S1P-R1 is expressed at low-moderate levels on human HSC. In infected HIS mice CFSE labeled CD34+ progenitors developed into mature thymocytes in the human thymus/liver implant and a subset expressed S1P-R1, indicating that entry into the thymus and development are functional during early HIV infection. Surprisingly, we found that S1P-R1 (as well as KLF2) was significantly upregulated in mature thymocytes post-HIV infection. Intriguingly, S1P-R1 was also upregulated by HIV within the CD3+CD69+ population, which normally does not express S1P-R1. S1P-R1 signaling as measured by pAkt was not impaired in infected thymocytes, which is interesting in the context of published data demonstrating that S1P-R1 response in HIV infection may be impaired in other cell types. Our results show that the mechanism of increased S1P-R1 in the thymus by HIV may be due to cytokines including Interferon-beta (IFN-β), which significantly increased S1P-R1 expression in both CD3hiCD69- and CD3+CD69+ thymocyte subsets treated in vitro with exogenous cytokine. Tumor Necrosis Factor alpha (TNF-α) also significantly increased S1P-R1 mean Fluorescence Intensity (MFI) on thymocytes in vitro. Additional cytokines perturbed during HIV infection are currently under investigation. S1P-R1 upregulation post-HIV-1 infection may offer insight into T cell reconstitution mechanisms during infection and provide potential alternate avenues for immunotherapy.

   



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  The Effect of HIV-1 infection on S1P-R1 expression and function during entry into and egress from the human thymus
  Presenter: Rachel S. Resop
  All Authors:Rachel S. Resop, Josh Craft, Dimitrios Vatakis, Bianca Blom, Christel H. Uittenbogaart
  University of California, Los Angeles
   
 

Lack of adequate T cell regeneration in HIV infected individuals despite adherence to antiretroviral therapy is likely due in part to a defect pertaining to entry of hematopoietic stem cells (HSC) into, and egress of mature naïve T cells from, the thymus to the periphery. Limited work has been done to further elucidate these phenomena. We studied the effect of HIV-1 infection on the receptors to Sphingosine-1-phosphate (S1P), a chemotactic sphingolipid mediator, during the processes of entry into and egress from the human thymus. Previously, we have shown that human thymocytes migrate toward S1P and that S1P receptor 1 (S1P-R1) is the main S1P receptor responsible for response to S1P humans as it has been shown to be in mice. During T cell development, S1P-R1 expression is significantly increased at the mRNA and protein level in the most mature CD3hiCD69- thymocyte subset about to exit the thymus as mature naïve T cells. We have demonstrated that thymocytes expressing S1P-R1 respond to S1P exposure in vitro with internalization of the receptor upon binding and downregulation of S1P-R1 mRNA. In our current work, we are examining the dynamics of S1P-R1 expression on both immature hematopoietic stem cells (HSC) and mature CD3hiCD69- cells within the human thymus during HIV infection, which has thus far not been described. We profiled CD34+CD38lo HSC in both fetal and postnatal thymus and found that S1P-R1 is expressed at low to moderate levels on this population. Currently, we are examining S1P-R1 expression within the fetal liver in order to correlate these levels to that observed in the thymus. In two series of NSG mice implanted with human fetal thymus/liver (thy/liv) grafts and infected with CXCR4- or CCR5-tropic HIV-1, we observed that CFSE-labeled CD34+ progenitors developed into mature thymocytes in the human thy/liv implant of infected NSG mice. A minor subset of these cells expressed S1P-R1 and likely migrated to the periphery, indicating that entry into the thymus and development are likely functional during early infection. Morever, we examined the effect of HIV on S1P-R1 expression on CD3hiCD69- cells (as well as additional populations) in the human thy/liv implant. We verified persistent infection and immune activation by demonstrating that MxA and ISG15, two Interferon-alpha secondary genes, were upregulated. Surprisingly, our results show that S1P-R1 as well as its transcriptional regulator, Kruppel-Like Factor 2 (KLF2) were both significantly upregulated in mature thymocytes 5 and 9 weeks post HIV infection. Intriguingly, S1P-R1 was not only upregulated within the CD3hiCD69- population, but was also expressed within the CD3+CD69+ population, which normally does not express S1P-R1 in healthy thymi. Moreover, this effect was observed in both intrathymic and systemic HIV-1 infection. Hence, we investigated S1P-R1 function after HIV infection of the human thy/liv implants both in vitro and ex vivo by directly measuring Akt signaling induced by S1P/ S1P-R1 binding in a flow-based pAkt detection assay. Our findings indicate that S1P-R1 signaling may not be impaired in infected thymocytes, which is an intriguing contrast to data in the literature indicating that S1P-R1 response in HIV infection may be impaired in T cells from peripheral lymphoid tissues. Finally, we investigated the mechanism of the increase in S1P-R1 and KLF2. We found that Tumor Necrosis Factor alpha (TNF-a), Interferon-alpha (IFN-a) and Interferon gamma were elevated in the infected thy/liv implant. S1P-R1 expression levels, as measured by the Mean Fluorescence Intensity, increased to a statistically significant extent on mature thymocytes upon treatment with exogenous TNF-a, but not with IFN-a, indicating that secretion of TNF-a in the thymus may contribute to the increase in S1P-R1 expression. If S1P-R1 remains upregulated and fully functional for prolonged periods post HIV-1 infection, this discovery may offer insight into T cell reconstitution mechanisms during infection as well as provide a potential alternate avenues for immunotherapy.

   



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  HIV-1 infection increases Sphingosine-1-phosphate receptor 1 expression in the human thymus
  Presenter: Rachel Resop
  All Authors:Rachel Resop, Marc Douaisi, Josh Craft, Christel Uittenbogaart
  UCLA
   
 

Lack of T cell regeneration in HIV infected individuals despite successful antiretroviral therapy is likely due in part to an impact on egress of naïve T cells from the thymus to the periphery. However, there are limited data available to elucidate the impact of HIV on these processes. We studied the effect of HIV on the receptors to Sphingosine-1-phosphate (S1P), a chemotactic sphingolipid. We have shown that human thymocytes migrate to S1P and that FTY720 inhibits migration by functioning as an agonist to S1P, thereby downregulating S1P receptor 1 (S1P-R1), the main S1P receptor responsible for response to S1P in mice and humans. During T cell development, S1P-R1 expression is significantly increased at the mRNA and protein level in the most mature CD3hiCD69- thymocyte subset about to exit the thymus as mature naïve T cells. We have demonstrated that thymocytes expressing S1P-R1 respond to S1P exposure in vitro with increased Akt phosphorylation, internalization of the receptor upon binding, and downregulation of S1P-R1 mRNA. In our current work we examined the dynamics of S1P-R1 expression in the human thymus during HIV infection, which has thus far not been described. Two series of NSG mice implanted with human fetal thymus/liver (thy/liv) grafts and infected with CXCR4- or CCR5-tropic HIV-1 were used to analyze the effect of HIV on S1P-R1 in the human thy/liv implant. Persistent infection and immune activation were verified by demonstrating that two Interferon-alpha secondary genes, MxA and ISG15, were upregulated. Surprisingly, our results show that S1P-R1 as well as its transcriptional regulator, Kruppel-Like Factor 2 (KLF2) were both significantly upregulated in mature thymocytes at two time points post HIV infection. Hence, S1P-R1 function after HIV infection of the human thy/liv implants was investigated in vitro and ex vivo by directly measuring Akt signaling induced by S1P/ S1P-R1 binding. Our findings indicate that S1P-R1 signaling is not impaired in infected thymocytes, which is in contrast to published data in other cell types that point to impaired S1P-R1 response in HIV infection. The mechanism of the increase in S1P-R1 and KLF2 was investigated and our results show that Tumor Necrosis Factor alpha (TNF-α), Interferon-alpha and Interferon gamma were elevated in the infected thy/liv implant. Upon treatment with exogenous TNF-α, S1P-R1 expression level, as measured by the Mean Fluorescence Intensity, was increased to a statistically significant extent in mature thymocytes, indicating that secretion of this cytokine in the thymus may contribute to the increase in S1P-R1 expression. Moreover, we found that CFSE-labeled CD34+ progenitors developed into mature thymocytes in the human thy/liv implant of infected NSG mice and that a minor subset expressed S1P-R1 and likely migrated to the periphery, indicating that entry into the thymus and development are likely functional during early infection. If S1P-R1 remains upregulated and fully functional at various time points post HIV-1 infection, this discovery may offer insight into T cell reconstitution mechanisms during infection as well as provide a potential alternate immunotherapy for patients.

   



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  Preeminent Role for CD103+/CD141+ Dendritic Cells bearing CCR7 for Tumor Antigen Trafficking and Priming of T cell Immunity in Melanoma.
  Presenter: Ed Roberts
  All Authors:Ed Roberts, Miranda Broz, Mikhail Binnewies, Mark Headley, Amanda Nelson, Denise Wolf, Tsuneyasu Kaisho, Matthew Krummel
  UCSF
   
 

Intratumoral dendritic cells (DC) bearing CD103 in mice or CD141 in humans drive intratumoral CD8+ T cell activation. Using multiple strategies, we identified a preeminent role for these DC in trafficking tumor antigen to lymph nodes (LN), resulting in direct CD8+ T cell stimulation and providing antigen handoff to less-stimulatory myeloid cells. CCR7 loss resulted in defective LN T cell priming and increased tumor outgrowth with the loss of CD103+ DC trafficking, as opposed to that of CD11b+ DC, being the critical determinant of tumor specific CD8+ T cell expansion. CCR7 expression levels in human tumors correlate with signatures of CD141+DC and strongly correlated with intratumoral T cells and better clinical outcomes. This work identifies an ongoing pathway to T cell priming which should be harnessed for tumor therapies.

   



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  Migratory Dendritic Cells Activated in the Tumor Microenvironment Seed Lymph Node Resident Populations with Tumor Antigens Using Distinct Pathways
  Presenter: Edward Roberts
  All Authors:Edward Roberts, Roberts EW*, Ruhland MK*, Mujal AM, Bepplar C, Marchuk K, Cai E, Binnewies M, Fries A, Krummel MF
  UCSF
   
 

Lymph node resident dendritic cells have important roles in supporting effective CD8+ T-cells priming; however, how these DC acquire antigen is controversial. The mechanism by which antigen is acquired has important consequences for the fate of that antigen influencing the efficiency of and context within it is presented to CD8+ T cells. Here we demonstrate that tumor derived antigen is actively brought into the draining lymph node and is seeded into resident DC by direct cell contact dependent hand off. This mechanism of passing does not occur in the healthy tissue as, despite migratory dendritic cells loading with the same antigen when it was present as a normal tissue antigen, no passing of antigen was observed to resident DC. Although no passing was observed in this tolerogenic context, it could be induced by damaging tissue with low doses of irradiation. In the tumor context antigen was passed by different mechanisms to different resident dendritic cell populations within the lymph node with inhibitor studies suggesting that CD8a+ dendritic cells specifically acquiring antigen through LC3 associated phagocytosis. Administration of melanin, an inhibitor of LC3 associated phagocytosis, in vivo could thus block accumulation of tumor antigen in the resident CD8a population. We now seek to understand the impact of this pathway in priming anti-tumor immune responses.

   



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  Evolutionary Origin of the Mammalian Hematopoietic System Found in a Colonial Chordate.
  Presenter: Benyamin Rosental
  All Authors:Benyamin Rosental, Benyamin Rosental, Mark A. Kowarsky, Daniel M. Corey, Katherine J. Ishizuka, Karla J. Palmeri, Shih-Yu Chen, Rahul Sinha, Jun Seita, Irving L. Weissman, Ayelet Voskoboynik.
  Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine.
   
 

To gain insight into the evolutionary relationship between vertebrate and invertebrate hematopoietic system, we have characterized the immune system and cell populations of the colonial tunicate Botryllus schlosseri. B. schlosseri belongs to a group considered the closest living invertebrate relative of vertebrates, it has bidirectional blood cell flow through an interconnected vasculature. To isolate and characterize the Botryllus cell populations we adapted Fluorescence-Activated Cell Sorting. We used Cytof Mass Cytometry to scan 50 diverse antibodies. Antibodies that differentially bind to B. schlosseri cells, in combination with lectins and fluorescent reagents activated by enzymes, were used to isolate live B. schlosseri cell types. Additionally, we used mouse serum against the Botryllus Histocompatibility Factor and analysis of cell size, granularity and auto fluorescence to isolate 34 cell populations. We prepared libraries from these populations for RNAseq, and analyzed their gene expression. This analysis revealed cell population homolog to mammalian hematopoietic stem cells, which upon transplantation, migrated to known stem cell niche and differentiated into other cell lineages. Interestingly, we have shown that this niche is homolog to mouse bone marrow stromal cells. Using functional immunological assays for cytotoxicity and phagocytosis we characterized 3 different phagocytic cell-types. One of these demonstrated transcriptional and functional features resembling myeloid cells in vertebrates. Furthermore, we identified a B. schlosseri cytotoxic cell population originating from large granular lymphocyte-like cells. Our data suggests that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells may result from a convergent evolutionary mechanism.

   



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  CD8aa+ Intestinal Intraepithelial Lymphocytes (IEL) Arise from Two Major Thymic Precursor Subsets
  Presenter: Roland Ruscher
  All Authors:Roland Ruscher, Rebecca L. Kummer, You Yeong Lee, Stephen C. Jameson, Kristin A. Hogquist
  University of Minnesota Center for Immunology
   
 

TCRaß+ CD8aa+ intestinal intraepithelial lymphocytes (CD8aa IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRß+ thymocytes: a nascent PD-1+ population and a T-bet+ population that accumulates with age. Both gave rise to intestinal CD8aa IEL upon adoptive transfer. PD-1+ cells contained more strongly self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing a4ß7 was expressed by these IELp already at a thymic stage. The T-bet+ IELp population localized to the medulla and expressed NK1.1, CXCR3 and CD103, as well as IFN? reporter protein. The two populations further differed in TCR Va usage and MHC- restriction: While PD-1+ IELp were classical MHC class I restricted, the T-bet+ IELp were dependent on non-classical MHC class I. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells.

   



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  Leveraging Chemerin and its Receptors in Immune Defense Against Breast and Prostate Cancer
  Presenter: Nicole Salazar
  All Authors:Nicole Salazar, Nicole Salazar, Yayue Zheng, Leona Nease, Jesse Rosalez, Russel Pachynski, Eugene Butcher, Brian Zabel
  Stanford School of Medicine
   
 

Background: Breast and prostate cancer kill approximately 67,000 people every year in the US. Our lab has previously shown the significant growth inhibiting effect of chemerin in melanoma. Chemerin is a secreted protein with a complex but well-established role in immune function capable of attracting anti-tumor immune cells such as macrophages and natural killer cells. Hypothesis: We asked if chemerin and its stroma-expressed presenting receptor CCRL2 are downregulated by tumors as part of a conserved tumor immune evasion strategy, and if chemerin and CCRL2 enhance anti-tumor immunity in mouse models of breast (BrCa) and prostate cancer (PrCa). Methods: We generated chemerin over-expressing cancer cell lines and chemerin and receptor knock-out mice to 1) determine whether tumor-expressed chemerin inhibits the establishment or growth of tumors and 2) define the independent roles of chemerin and CCRL2 in tumor suppression. We evaluated the in vivo anti-tumor effects of chemerin and CCRL2 in transplantable orthotopic EMT6 and 4T1.2 mammary carcinoma models, and a transplantable ectopic TRAMP-C2 prostate adenocarcinoma model. At the conclusion of the study, we resected the tumors and assessed the composition of the tumor infiltrating leukocyte population by flow cytometry. To extend the translation relevance we assessed gene expression levels of chemerin and CCRL2 in human tumor vs normal tissues. Results: While over-expression of chemerin did not affect the growth rate of mouse BrCa or PrCa cell lines in vitro, when implanted in vivo chemerin significantly suppressed tumor growth. The results were most striking in ectopic PrCa, where no mice developed tumors. In BrCa models, the effect was significant yet less-pronounced. When the TIL populations were plotted as a mean Log2 frequency ratio (chemerin vs. control tumors), there was an increase in anti-tumor leukocytes (NK, CD8+ T cells, total T cells, and DC) and a decrease in immune suppressive, pro-tumor myeloid-derived suppressor cells (MDSC, CD45+CD11b+Gr1+). To ask if host-expressed CCRL2 impacted tumor growth, PrCa TRAMP-C2 cells (which do not express chemerin or CCRL2) were implanted s.c. into WT or CCRL2 KO mice. CCRL2 KO mice developed significantly fewer and smaller tumors than their WT counterparts. This led us to hypothesize that 1) CCRL2 expressed by non-neoplastic stroma normally serves to sequester chemerin away from growing tumors, and 2) tumors down-regulate CCRL2 to limit intra-tumor chemerin accumulation and evade immune surveillance. Indeed, using publicly available human gene expression data, chemerin and CCRL2 were downregulated in many human cancers, and this downregulation correlated with poor survival outcomes, and is consistent with tumor immune evasion. Conclusions: These results demonstrate that chemerin and its receptors can be leveraged to disrupt the growth of breast and prostate tumors, and shift the tumor microenvironment to favor anti-tumor immune defense. These studies may lead to novel chemerin-dependent approaches to engage host immune defenses to slow or reverse cancer progression.

   



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  From T Cell Receptor to antigen, systems approach to discovering T cell antigen(s) in Multiple sclerosis and Experimental autoimmune encephalomyelitis
  Presenter: Naresha Saligrama
  All Authors:Naresha Saligrama, Naresha Saligrama, Fan Zhao, William Stuart Serratelli, Ricardo Fernandes, David M Louis, Xuhuai Ji, Vamsee Aditya Mallajosyula, Murad R Mamedov, Arnold S Han, Yueh-hsiu Chien, Christopher K Garcia, Jorge Oksenberg, and Mark M Davis
  Stanford University
   
 

The role of T cells in Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is well established. However, long-standing quest in MS is to determine key T cell antigen(s), which drive the initial response. There is a need for comprehensive understanding of the T cell receptor (TCR) repertoire in MS with a focus on identifying and targeting dominant T cell clones. By using single cell paired TCR sequencing and mass spectrometry based cytometry (CyToF), we have analyzed both MS patients and in mice with EAE with respect to their overall immune system and the specific T cell types. We have analyzed the TCR repertoire of brain homing, activated CD4, CD8, and gd T cells from the blood of 18 recent onset untreated MS patients and found significant T cell clonal expansion which is not seen in healthy controls. Using yeast displayed peptide-HLA (p-HLA) library which contains ~ 5 X 108 peptide antigens, we have screened expanded CD4 TCRs from HLA-DR*1501 homozygous MS patients and found ligands which are not myelin. These peptides from yeast library, which resembles peptides from pathogen can activate CD4 T cell clones from MS patients. Similarly, in mice upon EAE induction there is co-mobilization and oligoclonal expansion of CD4, CD8, and gd T cells day 10 post-immunization both in blood and in the central nervous system (CNS) as determined by single cell paired TCR sequencing. Moreover, these expanded T cell clones are similar and shared between mice and tissue (blood and the CNS), indicating shared specificities. Many of the expanded CD4 and CD8 T cell clones do not respond to myelin indicating initiation of broad antigen specificities upon EAE immunization. We have determined antigen specificities for some of these expanded CD8 TCRs from EAE using yeast displayed mouse class I peptide-MHC (p-MHC) library. We show that CD8 specific peptides derived from yeast library can significantly abrogate MOG35-55 induced EAE. Moreover, CD8 T cells isolated from class I peptide immunized mice can suppress the proliferative capacity of MOG35-55 specific CD4 T cells in vitro. Overall, our results suggest that there is coordinated and focused antigen specificity of the T cell responses involving all cell types, both in MS and in EAE. Moreover, we have developed a systematic approach in which one can employ single cell paired TCR sequencing and yeast displayed p-HLA/MHC library to find disease triggers not in only MS but also in other neurological disorders.

   



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  Gut microbiota as a source of signals that trigger spontaneous ocular autoimmunity
  Presenter: Ryan Salvador1
  All Authors:Ryan Salvador1, R Horai1, CR Zárate-Bladés1, K Itoh2, Y Jittayasothorn1, Y Umesaki3, RR Caspi1
  1Laboratory of Immunology, NEI, National Institutes of Health, Bethesda, MD, USA; 2Bio-Technical Center, Japan SLC, Inc., Hamamatsu, Shizuoka, Japan; 3Yakult Central Institute, Kunitachi, Tokyo, Japan
   
 

Autoimmune uveitis is a major cause of blindness. The disease is thought to be driven by T cells specific to unique retinal antigens that have become activated and acquired the ability to cross the blood-retinal barrier (BRB). However, it is unknown where and how these autoreactive T cells become activated, as their specific antigens are sequestered behind the BRB. We developed an interphotoreceptor retinoid binding protein (IRBP)-specific T cell receptor (TCR) transgenic (R161H) mouse as a model of spontaneous uveitis that permits to study natural triggers of the disease. Using this model, we demonstrated that elimination of gut commensals by oral antibiotic treatment (ABX) or by rearing under germ-free conditions (GF), significantly attenuated uveitis and reduced Th17 cells in the gut lamina propria. Retina-specific R161H T cells were activated in the intestine of specific-pathogen-free (SPF) but not GF mice, through their clonotypic TCR. Furthermore, upon exposure to bacteria-rich extracts of intestinal contents from SPF, but not from GF or ABX mice, retina-specific T cells upregulated activation markers and transferred uveitis to naïve recipients. These results suggested a role for gut microbiota as a source of antigen for stimulating retina-specific T cells, but did not identify a specific commensal or antigen mimic, and did not dissect the relative importance of adaptive vs. innate microbial signals. Towards that end, we performed gnotobiotic studies and 16S rRNA sequencing. Exposure of GF R161H mice to a SPF environment restored full development of uveitis, but mono-colonization with segmented filamentous bacteria (SFB, a Th17-inducing organism) or Turicibacter strain H121 (T. H121, isolated from mono-contaminated GF R161H mice that had uveitis) only partially restored disease. Unlike intestinal content extracts from SPF mice, extracts from mice monocolonized with SFB or T. H121 failed to activate R161H cells in vitro, indicating lack of “antigen” activity; monocolonization with SFB (not T. H121) restored gut Th17-producing cells, indicating presence of innate “adjuvant” activity. We conclude that microbial-derived adaptive “antigen” and innate “adjuvant” activities are both required, and combine to trigger full development of uveitis.

   



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  Using the Collaborative Cross to study adaptive immunity to Toxoplasma gondii
  Presenter: Juan Camilo Sánchez-Arcila
  All Authors:Juan Camilo Sánchez-Arcila, Darian Galvez, Jennifer Eggleston, Scott P Souza, Kirk DC Jensen
  Department of Molecular and Cell Biology, University of California Merced
   
 

Toxoplasma gondii, the causative agent of human toxoplasmosis, is distributed worldwide and infects billions of people. Similar to other parasitic infections, immunological responses induced by T. gondii are not sterilizing, leading to the possibility of multiple re-infections during one’s lifetime. Additionally, there are no fully protective vaccines for any human parasitic pathogen. In this project, we employ an unbiased genetic screen to find novel immunological responses required to protect against a highly virulent strain of T. gondii. The Collaborative Cross (CC) is a panel of multi-parental recombinant inbred mouse lines derived from eight founders of laboratory and wild-derived origin that span the three major Mus musculus subspecies (M. domesticus, M. musculus, M. castaneus). The CC panel captures ~90% of the genetic variation within the Mus musculus species, affording discovery of novel biological mechanisms. To determine new requirements for immunity to T. gondii, the immune responses of 53 CC lines are currently being interrogated. Our model consists of challenging mice with a highly virulent French Guyana strain, GUY-DOS, capable of evading immunological memory responses generated in some but not all founder CC lines. Quantitative Trait Locus (QTL) analysis of loci associated with susceptibility to low virulent natural infections, weight loss (as a marker of pathogenicity), antibody reactivity to T. gondii, and survival to GUY-DOS secondary infections in vaccinated or naturally infected animals are being mapped. In a more advanced stage of the project, we will also perform gene expression-QTL analysis to map variables obtained after transcriptomic analysis of the studied mice. We hypothesize that the CC system will reveal new immunological insights that will aid in vaccine design for T. gondii. Ongoing results from this project will be discussed.

   



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  A Common Mechanism of NLRP1B Inflammasome Activation by Two Distinct Bacterial Effectors
  Presenter: Andrew Sandstrom
  All Authors:Andrew Sandstrom, Patrick S. Mitchell, Lisa Goers, Edward W. Mu, Cammie F. Lesser, Russell E. Vance
  University of California Berkeley
   
 

Many immune responses are initiated through the recognition of pathogen-associated molecular patterns (PAMPs), such as flagellin or bacterial lipopolysaccharide, by germline encoded receptors. While the mechanisms by which these receptors recognize PAMPs are well established, there is mounting evidence that the immune system is also able to recognize pathogen-associate activities, such as pathogen-induced potassium efflux or pathogen-associated enzymatic activities. However, the molecular mechanisms by which the immune system can detect these pathogen-associated activities are unclear. To uncover such mechanisms, we investigated how the NLRP1B inflammasome is able to sense the enzymatic activity of the Bacillus anthracis lethal factor (LF) protease. Unexpectedly, we found that cleavage of NLRP1B by LF induces proteasomal degradation of NLRP1B. Degradation of NLRP1B releases an active fragment of NLRP1B which self-oligomerizes, and recruits and activates caspase-1 to initiate a downstream inflammatory response. This model of activation further led us to identify a novel activator of NLRP1B, the secreted E3 ligase IpaH7.8 from Shigella flexneri. These results identify a novel unified mechanism through which the NLRP1B inflammasome is able to detect pathogen-associated enzymatic activities.

   



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  A Common Mechanism of NLRP1B Inflammasome Activation by Two Distinct Bacterial Effectors
  Presenter: Andrew Sandstrom
  All Authors:Andrew Sandstrom, Patrick S. Mitchell, Lisa Goers, Edward W. Mu, Cammie F. Lesser, Russell E. Vance
  University of California Berkeley
   
 

Inflammasomes are large multi-protein complexes that respond to a range of stimuli to recruit and activate Caspase-1 (CASP1). Activation of CASP1 leads to the maturation and release of pro-inflammatory cytokines and pyroptosis, a lytic form of cell death. Some inflammasomes are activated through the recognition and binding of pathogen-associated ligands. However, others respond to pathogen-associated activities, such as potassium efflux or the enzymatic activity of pathogen-secreted effectors. Yet, the molecular mechanisms by which pathogen-associated activities lead to inflammasome activation have remained unclear. To uncover how inflammasomes respond to pathogen-associated activities we investigated how the NLRP1B inflammasome senses pathogen-associated protease activity. NLRP1B is activated after proteolytic cleavage by the Lethal Factor (LF) protease secreted by Bacillus anthracis. In a mechanism we term ‘functional degradation’ we found that cleavage of NLRP1B by LF leads to the proteasomal degradation of NLRP1B. Degradation liberates a processed C-terminal fragment of NLRP1. This C-terminal fragment then oligomerizes to recruit and activate CASP1. This model of activation further led us to identify IpaH7.8, an E3 ligase secreted by Shigella flexneri, as a novel activator of NLRP1. These results provide insight into how a single protein can sense and respond to diverse pathogen-associated activities.

   



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  Enhancer connectome in primary human T cells reveals target genes of autoimmune disease-associated DNA elements
  Presenter: Ansuman Satpathy
  All Authors:Ansuman Satpathy, Maxwell Mumbach, Evan Boyle, Chao Dai, William Greenleaf, Howard Chang
  Stanford University
   
 

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in primary human T cell subtypes. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune disease.

   



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  Development of Faux-Biotics to prevent and manage antibiotic-resistant infections
  Presenter: Hannah Savage
  All Authors:Hannah Savage, Hannah P. Savage, Erin E. Olsan, Eric M. Velazquez, Stephanie A. Cevallos, Henry Nguyen, and Andreas Bäumler
  UC Davis
   
 

Antibiotic-resistant bacterial infections are a significant concern for both human and animal health, and the occurrence of antibiotic-resistant infections is expected to continue to rise. After a single antibiotic dose, a fecal microbiota transplant (FMT) from a healthy donor provides colonization resistance against antibiotic-resistant infections. However, in the face on ongoing antibiotic treatment, Faux-Biotics, or non-biotic replacements, for FMTs are needed to confer protection. Using E. coli KPC, a carbapenem-resistant Enterobacteriaceae (CRE), we have found that the combination of Clostridia and endogenous Enterobacteriaceae can replicate the protective ability of a full FMT in mice. Secretion of butyrate by Clostridia alters host epithelial cell metabolism to reduce oxygen and nitrate in the intestinal lumen. This function can be replicated by the drug 5-aminosalicylic acid, which provides full protection against CRE in combination with Enterobacteriaceae or partial protection when used alone. We hypothesize that endogenous Enterobacteriaceae contribute to protection by directly competing with CRE for electron acceptors. Investigations into replacements for endogenous Enterobacteriaceae are ongoing. Further studies of the mechanisms behind colonization resistance will provide insights into potential prevention and treatment options for patients on antibiotics to protect against antibiotic-resistant infections.

   



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  A wave of regulatory T cells into neonatal skin mediates tolerance to commensal microbes
  Presenter: Tiffany C. Scharschmidt
  All Authors:Tiffany C. Scharschmidt, Kimberly S. Vasquez, Hong-An Truong, Sofia V. Gearty, Mariela L. Pauli, Audrey Nosbaum, Iris K. Gratz, Michael Otto, James J. Moon, Jan Liese, Abul K. Abbas, Michael A. Fischbach, Michael D. Rosenblum
  University of California, San Francisco
   
 

Each centimeter of our skin contains over a million lymphocytes and a million commensal bacteria. Very little is known about how the cutaneous immune system continuously senses antigens from commensal bacteria without causing destructive inflammation. To elucidate how adaptive immune tolerance is established and maintained to skin commensal microbes, we engineered a prototypical skin commensal (S. epidermidis) to express the model antigen 2W (Epi-2W), allowing us to comprehensively analyze commensal-specific CD4+ T cell responses in the context of both a polyclonal T cell repertoire and a complex microbiome. Using this system, we observed that skin colonization during a defined period of neonatal life was required to establish tolerance to Epi-2W. This window of time is characterized by an abrupt wave of highly activated regulatory T cells (Tregs) that accumulate in skin during the first weeks of life. Inhibition of Treg migration into skin during this period completely abrogated tolerance to Epi-2W. Our results demonstrate that the host-commensal relationship in skin relies on a unique population of Tregs that control antigen-specific responses directed at skin microbes, and that there is a critical developmental window in which this tolerance is established. This suggests that composition of the cutaneous microbiome in the neonatal period has formative effects that shape the adaptive immune response to bacterial antigens and that disrupting the cutaneous host-commensal relationship early in life may have enduring health implications.

   



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  Regulation of the antiviral modality of human DNA-PK
  Presenter: Emily Schutsky
  All Authors:Emily Schutsky, Nathan Camp, Katelyn Burleigh, Joanna H. Maltbaek, S. Cambier, Richard C. James, Daniel B. Stetson
  University of Washington
   
 

Detection of intracellular DNA triggers a potent antiviral response that protects from virus infection and can be harnessed to enhance immunity to cancer. cGAS and STING are thought to comprise the principal pathway that mediates this response. We recently discovered that humans – but not laboratory mice – have a second, STING-independent DNA sensing pathway (SIDSP) that potently activates antiviral immunity. We have identified the DNA damage-activated kinase DNA-PK as the sensor of the SIDSP, and we have found that DNA damage and foreign DNA trigger distinct modalities of DNA-PK activity: DNA damage triggers the well-known repair pathway, but only foreign DNA activates the antiviral signaling pathway. We seek to understand how DNA-PK can trigger these distinct outcomes. To this end, we have performed global phosphoproteomics to compare responses to DNA damage versus foreign DNA. We have identified several inducible, DNA-PK-dependent phosphorylation targets that are only triggered by foreign DNA, and we are now characterizing these targets for their role in antiviral immunity. A deeper understanding of the modalities of DNA-PK activity will allow us to reprogram this pathway to trigger innate immunity in tumors, perhaps by converting DNA damage into a potent antiviral stimulus or by targeting DNA-PK with potent synthetic agonists.

   



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  A novel endocytic mechanism used for antigen transfer from peripheral to immune cells
  Presenter: Nina Serwas
  All Authors:Nina Serwas, Kyle Marchuk, Rulan Yi, John Ngo, Andrew Oberst, Matthew Krummel
  UCSF
   
 

Beside protecting an organism from dangerous ‘foreign’ pathogens, one of the most crucial roles of the immune system is to tolerate ‘self’ and prevent the development of autoimmunity. Central tolerance mechanisms during cell development prevent outgrowth of autoreactive lymphocytes. However, a significant number of autoreactive cells escape these mechanisms, thus, peripheral systems developed to keep immune responses under control. These include the presentation of ‘self’ by specialized antigen presenting cells (APCs) in a non-inflammatory context. It is not fully understood how APCs endocytose self-antigen and how they are able to distinguish non-harmful ‘self’-antigen from potential dangerous ‘foreign’. To elucidate this mechanism, we developed a co-culture system of immune cells with malignant or primary cells which have been modified to constitutively express cytoplasmic ZsGreen, a highly stable fluorescent protein. We applied high resolution lattice light sheet live microscopy (LLSM) and flow cytometry to understand how antigen from the cytosol of these cells is transferred to immune cells and how this antigen then is intracellularly sorted after uptake. Our data reveals that immune cells acquire antigen from various malignant and primary cells in a contact-dependent manner. With LLSM we were able to capture the actual moment of transfer. Self-antigens from living cells are transferred to immune cells in numerous small particles (1-5 μm). These packages of antigen are actively nibbled off from protrusions of the antigen-providing cells. The process is dependent on actin polymerization and the activity of different classes of phosphoinositide 3-kinases. Importantly, the transfer is clearly distinguishable from uptake of particles from cells undergoing programmed cell death which represent potential harmful antigens. Those particles are larger in size and separate from dying cells also in the absence of immune cells. Once separated, particles bind to the surface of immune cells until they are eventually invaginated. In summary, we provide detailed insides in how antigens from live cells enter into cells of the immune system and discriminate this mechanism from uptake of dying cells. Future work will address how perturbation of this antigen trading will affect immune regulation and provide a potential future therapeutic angle for modulations of immune tolerance.

   



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  Antigen transfer in immune homeostasis
  Presenter: Nina K Serwas
  All Authors:Nina K Serwas, Matthew F Krummel
  UCSF, Department of Pathology, San Francisco, CA
   
 

The ability to tolerate an evolving definition of ‘self’ is crucial for every living being, however, the way in which 'self' is tolerated as we develop is barely understood. Extracellular vesicles (EVs) can promote tolerance in cancer, and antigen-presenting cells (APCs) can transport EVs to lymph nodes to generate immune responses. As tumors rarely invent pathways but rather hijack available mechanisms, we hypothesize that the immune system uses the flow of EVs from ‘true-self’ to determine the evolving ‘self’. Thus, the role and mechanism of EVs in disseminating ‘self’-material deserves considerable attention. Here, we aimed to analyze the first contact of peripheral cells with immune cells. To this end, we co-cultured cancer or primary cells expressing the stable GFP-derivate ZsGreen with bone marrow-derived macrophages. Strikingly, macrophages acquired fluorescent antigen from both, cancer and primary cells which suggested the transfer of self-antigens. We then visualized antigen transfer with high resolution lattice light sheet fluorescent microscopy. Unexpectedly, we observed that, besides large chunks of material, numerous small particles with sizes below 1um were transferred to the macrophages within two hours of co-culture. The transfer mechanism involves close membrane contact of both cells whereas the antigen of the donating cell drips of from protrusion into the macrophages. Future work will address the mechanisms involved in the antigen transfer and relate this process to tolerance maintenance in the immune system.

   



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  SUPPRESSION OF SYSTEMIC AUTOIMMUNITY BY THE INNATE IMMUNE ADAPTOR STING
  Presenter: Shruti Sharma
  All Authors:Shruti Sharma, Allison M. Campbell*2, Jennie Chan*1, Stefan A. Schattgen*1, Gregory M. Orlowski*1, Ribhu Nayar3, Annie H. Huyler1, Kerstin Nündel4, Chandra Mohan5, Leslie J. Berg3, Mark J. Shlomchik6, Ann Marshak-Rothstein†4 and Katherine A. Fitzgerald†1, 7(* and † contributed equally)
  University of Massachusetts Medical School
   
 

Cytosolic DNA-sensing pathways that signal via STING mediate immunity to pathogens and also promote autoimmune pathology in DNAseII/III (Trex1)-deficient mice. In contrast, we report here that STING potently suppresses inflammation in a model of systemic lupus erythematosus (SLE). Lymphoid hypertrophy, autoantibody production, serum cytokine levels, and other indicators of immune activation were markedly increased in STING-deficient autoimmune-prone mice compared to STING-sufficient littermates. As a result, STING-deficient autoimmune-prone mice had significantly shorter lifespans than controls. Importantly, TLR-dependent systemic inflammation during TMPD (pristane)-mediated peritonitis was similarly aggravated in STING-deficient mice. Mechanistically, STING-deficient macrophages failed to express negative regulators of immune activation, including IDO-1, and thus were hyper-responsive to TLR ligands, producing abnormally high levels of pro-inflammatory cytokines. This hyper-reactivity corresponds to dramatically elevated numbers of inflammatory macrophages and granulocytes in vivo. Collectively these findings reveal an unexpected negative regulatory role for STING, having important implications for STING-directed therapies.

   



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  Mechanisms of Maternal Suppression of Neonatal Anti-Commensal Immune Responses
  Presenter: Meera Shenoy
  All Authors:Meera Shenoy, Meghan Koch
  Fred Hutchinson Cancer Research Center
   
 

To ensure host health, the immune system must be able to discriminate between pathogenic and beneficial microbes, but potential pathogens and commensals can share many of the same traits, making this an incredibly difficult task. This process is further complicated within early life, when the immune system is still developing and is first encountering microbes. These early interactions may play a fundamental role in shaping the immune system’s ability to distinguish friend from foe. As the majority of early-life microbes are provided to the neonate by the mother, we hypothesize that microbial and immune signals passed from the mother to the neonate following birth serve to educate the neonatal immune system. Recent work from our group demonstrated that breast milk derived T-independent maternal antibodies suppress the formation of intestinal T-dependent antibody responses in the neonate. Using mouse models lacking specific immunoglobulin isotypes, we demonstrate that maternal IgG2b and IgG3 are necessary for suppressing neonatal immune responses. These maternal antibodies are required post-birth up to day 14 of life in order to prevent T-dependent antibody responses, and in their absence, there is significant accumulation of commensal microbes within the mesenteric lymph nodes. Mice lacking maternal antibodies demonstrate no obvious defects in global barrier function as measured by translocation of antigens they have not previously experienced. Based on these results, we propose that maternal IgG is suppressing neonatal antibody responses through Fc receptors in an antigen-specific manner. As such, we have obtained mouse models deficient in activating and inhibitory Fc receptors to investigate whether these receptors mediate maternal IgG effects. Through these studies, we are establishing the factors which prevent the development of anti-commensal antibody responses, which is the first step in being able to predict and prevent the development of anti-commensal associated chronic diseases such as inflammatory bowel disease.

   



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  Reversing the pro-tumorous functions of tumor-associated macrophages using siRNA-loaded lipid nanoparticles
  Presenter: Nour Shobaki1,2
  All Authors:Nour Shobaki1,2, Yusuke Sato1, Hideyoshi Harashima1
  Laboratory for Molecular Design of Pharmaceutics, Hokkaido University, Japan 1, Fred Hutchinson Cancer Research Center, Seattle, WA 2
   
 

Tumor-associated macrophages (TAMs) are highly present in the tumor-microenvironment (TME), they generally have pro-tumorous functions and enhance tumor survival and progression [1,2]. Targeting TAMs to reprogram their phenotype and function via siRNA-based gene silencing could be a promising approach for cancer immunotherapy. In this study, a lipid nanoparticle (LNP) formulation composed primarily of an original and a pH-sensitive cationic lipid (CL4H6 lipid), referred to as CL4H6-LNP [3], was optimized and used to target and deliver siRNA to TAMs. The CL4H6-LNP induced strong gene silencing in murine bone marrow-derived macrophages (BMDM) with a superior efficiency compared to the Invitrogen™ Lipofectamine™ RNAiMAX transfection reagent, measured by qRT-PCR at the mRNA level of the model target gene (CD45). The CL4H6-LNP was optimized for high stability in the blood circulation, and it induced high and selective uptake and gene silencing in TAMs in BALB/c Ajcl-nu/nu mice carrying human tumor xenograft ((OS-RC-2); renal cell carcinoma (RCC)). After intravenous administration of the LNPs at two doses of 2 mg siRNA/kg, they induced ~70% gene silencing of the model target gene (CD45) at the protein level, measured by flow cytometry. Furthermore, an anti-tumor therapeutic response was obtained by targeting TAMs using the siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of two targeted genes responsible for the pro-tumorous functions of TAMs at three doses of 1 mg siRNA/kg. The treatment increased macrophage (CD11b+ cells) infiltration to TME by 59% and increased the proportion of CD169+ cells by 50%, compared to PBS(-)-treated mice as measured by qRT-PCR. The treatment also reversed the pro-tumorous functions of TAMs, mainly angiogenesis and tumor cell activation, evaluated by the decrease of expression of CD31, vascular endothelial growth factor receptor 2 (VEGFR2), and transforming growth factor β (TGF-β) by 37%, 12%, and 43%, respectively. This research has promising clinical and pharmaceutical applications as novel macrophage-based cancer immunotherapies for human patients. [1] Komohara Y., et al., Adv. Drug Deliv. Rev., 99(Pt B),180-185 (2016) [2] Li X., et al., Mol. Cancer, 18, 177 (2019) [3] Sato Y., et al., J. Control. Release, 295, 140-52 (2019)

   



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  Role of TWEAK/Fn14 in chronic inflammatory disorders of the skin
  Presenter: Daniel Sidler
  All Authors:Daniel Sidler, Rana Herro, Yuko Kawakami, Toshi Kawakami, Linda Burkly, Michael Croft
  La Jolla Institute for Allergy and Immunology
   
 

Atopic dermatitis and Psoriasis are chronic inflammatory disorders of the skin with significant morbidity and characteristic extra-cutaneous manifestations. Despite particular differences in respect of etiology, pathogenesis and systemic manifestations, these diseases share characteristic histological features such as epidermal hyperplasia, dermal fibrosis and hypervascularization. Discovering molecules that might contribute to the development or maintenance of both diseases could provide new opportunities for therapeutic intervention. It is widely accepted that keratinocytes and dermal fibroblasts play a pivotal role in both diseases by amplifying and maintaining inflammatory and fibrotic responses. TNF superfamily members, including TWEAK (TNFSF12), have been implicated in control of these cell types, but experimental and mechanistic insights are scarce. Employing TWEAK-deficient mice, we demonstrated that the TWEAK/Fn14 axis is indispensable for the development of experimental atopic dermatitis (Der f/SEB model) and psoriasis (Imiquimod model). Furthermore, we found that subcutaneous injection of recombinant TWEAK into naïve mice is sufficient to induce robust cutaneous inflammation with histological and molecular signatures of both atopic dermatitis and psoriasis. Moreover, in vitro experiments revealed that keratinocytes and dermal fibroblasts produced key inflammatory chemokines and cytokines in response to TWEAK that are characteristic of both atopic dermatitis and psoriasis, and these molecules were amplified when TWEAK was combined with the signature cytokines of these diseases, IL-13 and IL-17. Similarly, subcutaneous injection of TWEAK into naïve mice with recombinant IL-13 or IL-17 strongly synergized with respect to infiltration of cells into the skin and intra cutaneous expression of keratinocyte and fibroblast-derived inflammatory cytokines and chemokines. These studies help to understand the complex network of contributing factors active in the pathogenesis of chronic skin inflammatory disorders, and highlight TWEAK as a central regulator of both atopic dermatitis and psoriasis.

   



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  Identification of tumor-specific CD8+ T cells in EBV driven lymphoepihtelioma-like carcinoma: a case report
  Presenter: Yannick Simoni
  All Authors:Yannick Simoni, Yannick SIMONI, Etienne BECHT, Chiew Yee LOH, Joe Poh Sheng YEONG, Tony Kiat Hon LIM, Angela TAKANO, Daniel S.W. TAN, NEWELL W. Evan
  Fred Hutch Cancer Research Center
   
 

Lymphoepithelioma-like carcinoma (LELC) of the lung is an uncommon lung cancer, typically observed in young, non-smoking Asians populations. LELC is associated with the presence of Epstein-Barr virus (EBV) in lung tumor tissue, suggesting the carcinogenic role of EBV as observed in nasopharyngeal carcinoma. Here, we studied the antigen specificity and phenotype of tumor infiltrating (TIL) CD8+ T cells in one LELC patient positive for EBV infection in lung tumor cells. Using an MHC class I tetramer, we detected two populations of EBV-specific CD8+ TILs, which can be considered as tumor-specific CD8+ T cells, in the tumor of this patient. Transcriptomic analyses of these two populations reveal their distinct exhausted phenotypes and their polyclonal TCR repertoire. High dimensional analyses at protein level utilizing mass cytometry show the heterogeneity of each tumor-specific CD8+ TIL in the tumor. More importantly, it has been found that LELC tumor cells express PD-L1; however, tumor-specific CD8+ T cells do not necessarily express PD-1, suggesting that immunotherapy (i.e. anti-PD-1 treatment) may not be an appropriate strategy for treating LELC patients.

   



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  Control of T cell proliferation, differentiation, and effector function by the adaptor protein BCAP
  Presenter: Mark Singh
  All Authors:Mark Singh, Shivani Srivastava, Jeffrey Duggan, Jessica Hamerman, Daniel J. Campbell
  Benaroya Research Institute at Virginia Mason
   
 

One of the central signaling networks required for a robust effector T cell response is the phosphoinositide 3-kinase (PI3K) network. While much is known about the role of the PI3K pathway in the T cell response, the molecular mechanisms involved in regulating PI3K function during T cell activation are poorly understood. The adaptor protein B Cell Adaptor for PI3K (BCAP) has previously been detected in B cells and myeloid cells, and there is evidence suggesting that BCAP is involved in the regulation of PI3K signaling pathways in these cell populations. We have generated novel data demonstrating the role of BCAP in ensuring optimal T cell activation, proliferation and differentiation. We have determined that although BCAP is not expressed in naïve T cells, BCAP expression is detected in effector memory T cell populations, and can be rapidly induced in naïve T cells upon T cell activation in vitro. In addition, BCAP deficient T cells are hypo-proliferative, and have attenuated signaling cascades downstream of PI3K in vitro. To study the role of BCAP in T cells in vivo, we infected wild type and BCAP deficient mice with the pathogen Lymphocytic Choriomeningitis Virus (LCMV). At 7 days post infection, notable skewing in the effector CD8+ T cell populations was observed, with an increased frequency of KLRG1loCD127hi memory precursor effector cells (MPECs) at the expense of KLRG1hiCD127lo short lived effector cells (SLECs). In addition, use of mixed bone marrow chimeras showed that wild type T cells have a significant competitive advantage over BCAP deficient T cells in vivo. Collectively, these data suggest that BCAP may have a non-redundant role in determining T cell fate in vivo.

   



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  The fate of CD8+ T cells during infection is linked to their developmental origin
  Presenter: Norah L. Smith
  All Authors:Norah L. Smith, Arnold Reynaldi, Jocelyn Wang, Neva Watson, Kito Nzingha, Kristel YeeMon, Seth Peng, Jennifer Grenier, Andrew Grimson, Miles P. Davenport, Brian D. Rudd
  Cornell University
   
 

Intracellular pathogens drive a CD8+ T cell response typified by robust clonal expansion and phenotypic diversification into multiple subsets of effector cells. For example, following infection, some cells display markers for terminal differentiation (KLRG1), are efficient at clearing infected cells but are short-lived while others upregulate cytokine receptors (IL-7R), important for long term cell survival and the establishment of immunological memory. While we know that these subsets of effector cells exist, the mechanisms that regulate the fate of naïve CD8+ T cells after microbial challenge remain poorly understood. Previous studies suggest heterogeneity in the effector pool is driven by differences in TCR avidity, asymmetric cell division, and environmental cues. However, although the pool of naïve CD8+ T cells is comprised of clonotypes that were produced during various stages of life, one variable that has not been closely examined is developmental origin. In fetal life, the first wave of hematopoietic stem cells (HSCs) to colonize the thymus come from fetal liver precursors. Later in life, HSCs come from the bone marrow. To determine whether the developmental origin of naïve CD8+ T cells plays a deterministic role in their fate after infection, we developed a system to ‘timestamp’ CD8+ T cells from various stages of development (1 day, 7 days, and 28 days) and examined their phenotype and behavior in 8-week-old adult mice. We found naïve CD8+ T cells made early in life express higher starting levels of effector molecules (T-bet) and predominantly display a CD44hi CD122hi memory phenotype whereas cells made later in life are CD44lo CD122lo. RNA sequencing reveals that cells from differing developmental origins have unique transcriptional profiles. In vitro stimulation of stamped populations shows that fetal- and neonatal-derived CD8+ T cells are hypersensitive to IL12/IL18, proliferate and make effector molecules more rapidly than cells made in older animals. In response to infection in adulthood, both polyclonal and monoclonal CD8+ T cells produced in early life expand rapidly and skew towards a short-lived effector phenotype. These data indicate that the spectrum of CD8+ T cell differentiation observed after infection is influenced by when the responding cells were initially made.

   



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  GM-CSF plays a pathogenic role in autoimmunity to the neuroretina in the absence of Th1 and Th17 lineage cytokines
  Presenter: Bing So Jin
  All Authors:Bing So Jin, Phyllis B. Silver, Reiko Horai, Yingyos Jittayasothorn, Rachel R. Caspi
  Laboratory of Immunology, NEI, National Institutes of Health, Bethesda, MD, USA
   
 

Autoimmune uveitis is a complex group of sight-threatening diseases caused by activated retina-specific Th1 or Th17 cells that acquired the ability to cross the blood-retinal barrier, but their respective contribution to autoimmune uveitis is not fully understood. In this study, we used mice deficient in IL-17A or IFN-? (GKO), or both IL-17 and IFN-? (DKO) to dissect the role of Th1 and Th17 lineage-specific cytokines in pathogenesis of uveitis. Experimental autoimmune uveitis (EAU) was induced by active immunization with the retinal autoantigen IRBP in complete Freund’s adjuvant. IL-17A-/- mice were protected from EAU, whereas GKO mice had exacerbated disease. Surprisingly, DKO mice were fully susceptible to EAU, with scores similar to WT controls. To test whether other lineage-specific proinflammatory cytokines compensate for lack of IL-17A and IFN-?, EAU-challenged DKO mice were treated with blocking antibodies to IL-17F, IL-22, TNF-a, or GM-CSF. Blockade of IL-17F, IL-22, or TNF-a in DKO mice did not affect the severity of EAU. By contrast, treatment of DKO mice with anti-GM-CSF antibody, either as prevention or as a reversal paradigm, significantly suppressed EAU. These results suggest that IL-17F, IL-22 or TNF-a individually are dispensable, but GM-CSF appears to play a major and nonredundant role in development of EAU when IFN-? and IL-17A are both absent. To examine whether GM-CSF-secreting T cells (ThGM) are pathogenic effectors, IRBP-specific T cells (from R161H mice) were polarized in vitro to ThGM, Th1 or Th17 phenotypes, and were adoptively transferred to naïve WT mice. A high proportion of ThGM-polarized cells produced GM-CSF, and many co-produced IFN-?, Notably, these cells induced a more severe disease than did Th17 cells, (but less severe than Th1 cells), supporting the notion that ThGM cells are also uveitogenic. Together, these results indicate that ‘classic’ Th1 and Th17 cells are not the only pathogenic effectors in uveitis, and that other inflammatory cytokine(s), such as GM-CSF, can also drive disease.

   



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  Therapeutic modulation of immune homeostasis through structure-informed design of interleukin-2-targeted antibodies
  Presenter: Jamie Spangler
  All Authors:Jamie Spangler, Jakub Tomala, Vincent C. Luca, Kevin M. Jude, Marek Kovar, & K. Christopher Garcia
  Stanford University
   
 

Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell differentiation, growth, and activity by signaling through interleukn-2 receptor-β (IL-2Rβ)/IL-2Rγ heterodimers on both IL-2Rα-high regulatory T (TReg) cells and IL-2Rα-low effector cells (such as memory-phenotype [MP] CD8+ T and natural killer [NK] cells). Its essential role in orchestrating immune homeostasis has made IL-2 an attractive therapeutic target for a wide range of immune-linked disorders such as autoimmune disease, cancer, and chronic infection. Unfortunately, efforts to develop IL-2 as a therapeutic have been limited by its concurrent promotion of both regulatory and effector immune cells, which leads to harmful off-target effects and dose-limiting toxicity. It would therefore be of tremendous therapeutic value to isolate and selectively modulate the immunosuppressive and immunostimulatory effects of IL-2 in order to bias immune cell subset proliferation for particular disease applications. Certain IL-2-directed antibodies have been shown to skew the balance of immune cells to favor either TReg cells (JES6-1) or effector cells (S4B6), but the molecular mechanisms underlying their phenotypic behavior was unknown. Through structural, biophysical, and functional studies, we elucidated the unique and intricate mechanisms through which these antibodies achieve immune cell bias by modulating cytokine-receptor interactions. We found that JES6-1 sterically blocks the IL-2:IL-2Rβ and IL-2:IL-2Rγ interactions, but also allosterically lowers the IL-2:IL-2Rα affinity through a “triggered exchange” mechanism favoring IL-2Rα-high TReg cells. Conversely, S4B6 sterically blocks the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rβ interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rβ-high effector cells. We harnessed these structural insights to engineer the affinity and species reactivity of JES6-1 to enhance its therapeutic activity and translational significance. Our work provides unprecedented insight into the actions and tunability of anti-cytokine antibodies, presenting a major advance in IL-2 therapeutic development and providing a molecular blueprint for the design of other cytokine-targeted drugs.

   



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  Dysregulation of the cytokine GM-CSF induces spontaneous phagocyte invasion and immunopathology in the CNS
  Presenter: Sabine Spath
  All Authors:Sabine Spath, Juliana Komuczki, Pawel Pelczar, Bettina Schreiner, Burkhard Becher
  Benaroya Research Institute, Seattle, USA and Experimental Immunology, Zurich, Switzerland
   
 

Chronic inflammatory diseases are influenced by dysregulation of cytokines. Among them, granulocyte macrophage colony stimulating factor (GM-CSF) is crucial for the pathogenic function of T cells in preclinical models of autoimmunity. To study the impact of dysregulated GM-CSF expression in vivo, we generated a transgenic mouse line allowing the induction of GM-CSF expression in mature, peripheral helper T (Th) cells. Antigen-independent GM-CSF release led to the invasion of inflammatory myeloid cells into the central nervous system (CNS), which was accompanied by the spontaneous development of severe neurological deficits. CNS-invading phagocytes produced reactive oxygen species and exhibited a distinct genetic signature compared to myeloid cells invading other organs. We propose that the CNS is particularly vulnerable to the attack of monocyte-derived phagocytes and that the effector functions of GM-CSF-expanded myeloid cells are in turn guided by the tissue microenvironment.

   



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  Immunogenic chemotherapy markedly enhances the efficacy of ROR1 CAR T cells in an autochthonous model of lung adenocarcinoma
  Presenter: Shivani Srivastava
  All Authors:Shivani Srivastava, Sushma Yechan-Gunja, Megha Sarvothama, Stanley R. Riddell
  Fred Hutchinson Cancer Research Center
   
 

Immunotherapy using chimeric antigen receptor (CAR)-modified T cells has impressive efficacy in hematological malignancies but has been less effective in solid tumors such as lung cancer, which is the leading cause of cancer mortality worldwide. Prior studies of CAR-T cells for solid tumors relied primarily on transplantable and xenogeneic models, which do not replicate the complex tumor microenvironment of human disease. To develop a CAR-T cell therapy model for lung cancer, we adapted the KrasLSL-G12D/+p53flox/flox (KP) mouse model to express the tumor-associated antigen ROR1, which is highly expressed in human lung adenocarcinoma but absent from vital adult tissues. We infected KP mice with Cre-expressing lentivirus to induce deletion of p53 and activation of oncogenic KrasG12D in lung epithelia, mimicking the two most common mutations in non-small cell lung cancer. The lentivirus also encoded ROR1, resulting in expression of ROR1 in nascent tumors. This model replicates initiation and progression of human lung adenocarcinoma, including infiltration by myeloid cells and CD4+ Tregs. Adoptive transfer of ROR1 CAR-T cells into mice bearing established lung tumors modestly controlled tumor growth for the first 6 weeks of treatment but did not improve survival. While CAR-T cells were present in some tumors at 5-fold higher number relative to control T cells, they only infiltrated and induced regression of a small fraction of tumors. Bioluminescence imaging of luciferase-labeled CAR-T cells revealed that most CAR-T cells resided in spleen and lymph nodes rather than the lung, were PD-1-, and most likely ignorant of tumor antigen. The inability of most CAR-T cells to infiltrate lung tumors may be due to the minimal production of T cell-attracting chemokines like CXCL9 and CXCL10 and the fact that a large fraction of circulating CAR-T cells in vivo showed a CD62L-KLRG1+ phenotype that has been associated with poorer ability to traffic to non-lymphoid tissues. Consistent with the impairment of tumor infiltration by CAR-T cells, combination therapy with anti-PD-1 did not enhance antitumor activity despite the expression of PD-1 on CAR-T cells and PD-L1 on tumor-associated myeloid cells. We next tested whether combination therapy of anti-PD-L1 with oxaliplatin and cyclophosphamide (Ox/Cy), which induces immunogenic cell death and may locally induce inflammation, could improve CAR-T cell recruitment to tumors. RNAseq analysis of tumors demonstrated that Ox/Cy upregulated genes involved in immune cell chemotaxis, including Cxcl9, Cxcl10, and Sele. Additionally, a larger fraction of circulating CAR-T cells were CD62L+CD44hi and capable of binding E/P-selectin, enabling extravasation into inflamed tissue, after Ox/Cy treatment. Consequently, Ox/Cy pre-treatment significantly improved CAR-T cell infiltration and induced dramatic regression of a larger fraction of tumors, but did not enhance the activity of control T cells. These data indicate that immunogenic chemotherapy can enhance CAR T cell activity against solid tumors, suggesting this model will be useful to identify combinations for clinical translation.

   



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  Identifying new human pathways involved in bacterial toxin resistance using a novel forward genetic screen
  Presenter: caroline stefani
  All Authors:caroline stefani, Ky Sha, Adam Lacy-Hulbert, Lynda Stuart
  Benaroya research institute
   
 

Bacterial toxins are essentials virulence factors allowing bacterial entry, persistence, and dissemination. Toxins are key players in host-pathogen interaction; therefore identifying the mechanisms of cellular resistance will be of fundamental importance to fight against numerous human pathogens. Furthermore, because of their highly specific action in cells, through their co-evolution with host, bacterial toxins are also selective and efficient tools to characterize cell biological pathways. For some toxins, even low amounts of toxin are sufficient to recapitulate the deadly symptoms of the infection. As part of our host defense response, cells are able to counteract toxins effects by sensing them or their effects and inducing biological responses and repair. However these mechanisms of cell resistance remain unknown. To answer this intrinsic question, we undertaken a forward genetic screening approach using transposon mutagenesis as an unbiased in vitro strategy to understand the mechanisms of cells resistance to toxins and the mechanisms of cellular repair. Specifically, this strategy relies on piggyBac transposon mutagenesis to generate a library of mutagenized cells presenting gain and lost-of function profile. The selection of resistant mutant clones and NextGen sequencing let us identify transposon insertions sites and candidate genes that contribute to protection. By combining the analyses of toxins sharing the same entry, the same trafficking or the same cellular target, we were able to identify candidate resistance genes that counteract multiple steps of bacterial toxin pathogenesis. We were also able to identify resistance genes specific to each toxin.

   



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  Ontogeny and function of “Trm-like” CD8 T cells within the mediastinal lymph node following influenza virus infection.
  Presenter: Michael Stolley
  All Authors:Michael Stolley, David Masopust
  University of Minnesota
   
 

Following influenza virus infection, we unexpectedly observed a sizable population of memory T cells within the lung-draining lymph node (medLN) expressing canonical markers of tissue-residency including CD69, CD103, and granzyme B; closely resembling tissue resident memory (Trm) T cells from the upstream tissue. Moreover, these “Trm-like” cells were greatly enriched in the medLN compared to other non-draining lymph nodes suggesting disequilibrium with the circulation. Among its nuanced functions, CD69 is transiently upregulated upon TCR stimulation. Accordingly, previous reports have attributed the presence of CD69+ memory T cells in the medLN after pulmonary infection to tonic TCR signaling from a “depot” of residual antigen. Arguing against this interpretation, we failed to observe proliferation or conversion to a “Trm-like” phenotype of congenically distinct antigen-specific T cells adoptively transferred into influenza-memory mice 30 days post-infection. Additionally, inflammation alone was insufficient to drive a “Trm-like” phenotype on bystander LCMV-specific memory T cells when mice were infected with influenza virus expressing an irrelevant antigen. Given these observations, we instead favored the hypothesis that “Trm-like” cells populating the medLN emigrated directly from the lung following resolution of pulmonary infection. To test this hypothesis, a depletion strategy was utilized by which we could eliminate circulating influenza-specific memory CD8 T cells while preserving those within the airways and lung parenchyma. At an early timepoint post-depletion, flu-specific memory T cells were efficiently removed from blood and secondary lymphoid tissues including the medLN. Strikingly, a population of memory T cells re-emerged within the medLN after 30+ days post-depletion where they phenocopied cells from the lung parenchyma. Functionally, “Trm-like” memory T cells from the medLN more rapidly reacquired a Trm phenotype upon entry into the lungs and gut after secondary challenge with influenza virus (but not LCMV) infection. Teleologically, the lung is a sensitive tissue primarily involved in gas exchange where immunity is known to rapidly wane. We propose a model by which pulmonary immunosurveillance is achieved, while avoiding potentially deleterious consequences of persistent lung memory, through the stockpiling of resident memory T cells in the draining lymph node.

   



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  Communicating Viral Immunity from the Inside Out – How sensing through RIG-I like Receptors leads to Macrophage polarization and Viral control in West Nile Virus infection
  Presenter: Amy Stone
  All Authors:Amy Stone, Courtney Wilkins, Richard Green, Michael J Gale, Jr.
  University of Washington
   
 

RIG-I-Like Receptors (RLRs) include RIG-I, MDA5, and LGP2. RLRs are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) is an emerging, neurotropic flavivirus that infects the central nervous system to cause disease and death. RIG-I and MDA5 work in tandem to initiate innate immune responses against WNV infection, though the role of LGP2 in this process is not well defined. To gain a deeper understanding of the contributions of each RLR in the innate immune response and immunity to WNV, we conducted a systems biology approach to assess the role of each RLR in immune programming. We conducted genome-wide RNAseq and bioinformatics analysis of WNV infection in bone marrow derived macrophages from wild type mice and from specific RLR-deficient mice. Here we show that the RLRs drive distinct immune gene activation and polarization of the immune response. In our data, the RLR-dependent, WNV-induced immune response polarization overshadows the classical drivers of viral innate immune responses, interferon I (IFN) and IFN-stimulated genes, thus underscoring the importance of innate immune activation for channeling the adaptive immune system into specific effector pathways. Characterization of the genes expressed demonstrated a strong induction of a pro-inflammatory macrophage signature and a suppression of a wound healing macrophage signature in a functional anti-viral response that was lost upon removal of RIG-I and MDA5. These analyses reveal that distinct RLR-driven transcriptional programs lead to differential functional output in immune response initiation against WNV infection. Our study demonstrates a clear, defined role for RIG-I, MDA5, and LGP2 in immune programming against virus infection. Supported by NIH/NIAID 1R01AI104002-01A1 (Gale), T32 AI007509 Diseases of Public Importance Fellowship and F32 AI124520-01 NRSA Individual Postdoctoral Fellowship (Stone).

   



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  Identification of a suppressor of autoimmune disease that affects deoxynucleotide synthesis in T cells
  Presenter: Gautam Sule
  All Authors:Gautam Sule, Tanya Tarasenko, Wenjie Zheng, Bethany Scott and Silvia Bolland
  NIAID, NIH
   
 

FcγRIIB-deficient mice represent a well-characterized animal model of systemic lupus erythematosus. They develop spontaneous anti-nuclear antibodies (ANA) and fatal glomerulonephritis when on the C57BL/6 (B6) background (B6.FcγRIIB -/- mice). In contrast, the same mutation on the BALB/c background (BALB.FcγRIIB -/- mice) is phenotypically benign, indicating differences in lupus susceptibility between the BALB/c and B6 strains. After extensive backcrossing, we narrowed down the genomic interval to a 151KB genomic fragment on chromosome 12 responsible for the suppressive/protective effect in BALB/c mice. We generated a transgenic mouse line expressing this BALB/c genomic region directly in the C57BL/6 background (A12 Tg) and subsequently crossed to B6.FcγRIIB -/- mice to test the putative protective effect. The BALB/c-derived A12 transgene was able to suppress the spontaneous disease that normally develops in B6.FcγRIIB -/- mice: it reduced spontaneous germinal center formation, serum autoantibody titers and proteinuria. A12 transgene also reduced the number of activated and memory T cells, as well as the number of follicular helper T cells when compared to the B6.FcγRIIB -/- mice. Additional studies suggest that reduction in the autoimmune phenotype is T cell-intrinsic. Rrm2 is the only protein-coding gene present in the A12 transgene. RRM2 forms one of the subunits for ribonucleotide reductase, which catalyzes the rate-limiting step for the production of deoxyribonucleotides from ribonucleotides. We are in process of characterizing the mechanism by which Rrm2 is able to have this protective effect. Finally we will test the ability of Rrm2 to suppress autoimmunity in other susceptible strains/lupus models to identify the translatability of RRM2 as a target for therapy.

   



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  Increased expression of TLR3 in a transgenic model uncovers differential competition among endocytic TLRs
  Presenter: Wenxiang Sun
  All Authors:Wenxiang Sun, Wenjie Zheng, Silvia Bolland
  NIH
   
 

Toll-like receptors (TLRs) are important for the identification and clearance of invading pathogens. TLR activation results in the production of inflammatory cytokines and type I interferon, which are involved in the initiation and/or perpetuation of many inflammatory autoimmune diseases. TLRs have been suggested as susceptibility factors in autoimmune disease. We have established that increased expression of TLR7 in a transgenic mouse model (TLR7tg) leads to spontaneous and lethal inflammatory autoimmune disease. We have explored the possibility that TLRs other than TLR7 might be susceptibility factors for autoimmune disease and might induce pathology in overexpression conditions. We generated a TLR3 transgenic line (TLR3tg) containing 20 copies of the endogenous mouse TLR3 gene. These mice had normal life span and no obvious changes in immunological characteristics, interferon secretion or spontaneous pathology. We observed that increased expression of TLR3 had consequences in RNA-sensing sensitivity, as TLR3tg bone marrow-derived macrophages showed enhanced polyI:C-induced type I interferon responses compared to wild type cells. We confirmed this result by in vivo polyI:C treatment and analysis of peritoneal macrophages and serum cytokines. Interestingly, TLR3 overexpression greatly enhanced the synergistic effect of a TLR7 agonist and polyI:C co-treatment, but it did not enhance the synergy with a TLR9 agonist co-treatment. On the other hand, TLR3 overexpression resulted in lower sensitivity to TLR9 agonists both in vitro and in vivo. Further experiments will determine whether TLR3 overexpression affects the receptor intracellular localization with consequences in overall sensitivity of the pathway.

   



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  Re-polarizing tumor-derived macrophages to achieve immune control of breast cancer
  Presenter: Lijuan Sun
  All Authors:Lijuan Sun, Ana Santos Almeida, Tim Kees, Xue-Yan He, Jean Albrengues, David Ng, Sylvia Adams, Mikala Egeblad
  Cold Spring Harbor Laboratory
   
 

Bone marrow-derived macrophages can be activated in culture to become tumoricidal macrophages using interferon-γ (IFN-γ) and toll like receptor (TLR) agonists. Nevertheless, in hormone receptor (HR)-positive breast cancer, macrophage infiltration correlates with poor prognosis, in part because these macrophages suppress T cell activation. To test whether tumor-associated macrophages can be activated or reprogrammed to become tumoricidal, we isolated macrophages from the bone marrow or primary tumors of genetically engineered MMTV-PyMT mice (a mouse model of luminal B breast cancer), and applied toll-like receptor (TLR) agonists and IFNγ to these macrophages in culture. We found that macrophages activated with IFNγ and either lipopolysaccharide (LPS) or monophosphoryl Lipid A (MPLA) – two TLR4 agonists – killed about 90% of cancer cells in <48 hours, but largely left non-transformed cells unharmed. However, using the TLR4 agonists alone, IFNγ alone, or the TLR3 agonist poly I:C with IFNγ?did not induce tumoricidal effects of the tumor derived macrophages. Only the combination of IFNγ with either LPS or MPLA activated iNOS expression in macrophages, and this enzymes was required for the killing of the cancer cells. The tumoricidal-activated macrophages expressed co-stimulatory molecules CD86 and CD40, which are necessary for T cell activation. Furthermore, their expression of IL12 and TNFα?were increased while expression of TGFβ?were decreased. Importantly, IFNγ combined with MPLA could also activate macrophages isolated from patient blood or pleural effusions to kill 80-90% of breast cancer cells isolated from the same patient in culture. Whereas stimulation macrophages with LPS or MPLA resulted in tumoricidal cells, stimulation of neutrophils with either LPS or MPLA together with IFNγ stimulated neutrophils to form neutrophil extracellular traps (NETs). NETs can promote breast cancer metastasis, possibly hindering the use of the TLR4 agonists in patients. However, inhibitors of NET formation (PAD4 inhibitors and DNase) could be added to the cultures without reducing the tumoricidal activity of the macrophages. LPS has severe side effect, but MPLA is a commonly used vaccine adjuvant. Therefore, the combination of IFNγ, MPLA and PAD4 inhibitor might allow activation of macrophages to control breast cancer.

   



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  The role of KAP1 in regulatory T cells
  Presenter: Shigeru Tanaka
  All Authors:Shigeru Tanaka, Steven F Ziegler
  Benaroya Research Institute
   
 

Regulatory T cells (Tregs) are indispensable for immune tolerance and Foxp3 is a master transcription factor of Tregs. We previously reported that KAP1 (KRAB-associated protein 1) and Foxp3 form a complex in human Tregs and work together to repress the transcription of inflammatory cytokines. However, the mechanisms of how KAP1 affects the Tregs suppression ability are still largely unknown. To address this, we generated Treg specific KAP1 deficient mice by crossing Foxp3-cre mice with KAP1-flox mice. Interestingly, Treg specific KAP1 deficient mice develop spontaneous lung inflammation and lymphadenopathy. We also see the increased levels of inflammatory cytokines such as IFNg and IL-13 in KAP1 deficient animals. Consistent with these, KAP1 deficient Tregs failed to suppress the expansion of responder T cells in vitro. We also found some of the effector molecules (e.g. IL-10 and KLRG1) and the transcription factors (Foxp3, RORg, T-bet, and Blimp-1) which are important for suppression function are decreased in KAP1 deficient Tregs. These results suggest that KAP1 has important roles in Tregs by positively regulating the transcription of Treg signature genes. We are now studying the direct targets of KAP1 in Tregs by ChIP-seq. We are also addressing the impacts on histone modification status in KAP1 deficient Tregs because KAP1 is also known as a component of chromatin remodeling complex.

   



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  Enrichment of diabetogenic T cells in vivo using implantable scaffolds
  Presenter: Martin A Thelin
  All Authors:Martin A Thelin, Stephan Kissler, Frederic Vigneault, Alexander L. Watters, Des White, Sandeep T. Koshy, Sarah A. Vermillion, David J. Mooney, Omar A. Ali*, Thomas Serwold*
  Joslin Diabetes Center at Harvard Medical School, Wyss Institute for Biologically Inspired Engineering at Harvard University, and University of California, San Francisco
   
 

Type 1 diabetes (T1D) is the most common chronic autoimmune disease in children and its prevalence is increasing globally. When a child is diagnosed with T1D, the immune system has already eradicated a great number of the insulin-producing cells and subsequently, a life-long insulin replacement therapy is required. T cells are known to play an essential role in the development of T1D both in humans and non-obese diabetic (NOD) mice. A major roadblock in the study of T1D is that the T cells that promote T1D, while abundant in the pancreas, are exceedingly rare in the blood. Currently, there is no efficient way of capturing T cells during the development of T1D without removing the pancreas. In order to enable the study of rare ß-cell specific T cells, we developed an implantable scaffold to enrich for diabetogenic T cells. To determine whether scaffolds recruit populations of T cells that are similar to the T cell populations found in the pancreas, we loaded the scaffolds with beta cell lysates and implanted them subcutaneously onto the backs NOD mice. After 2 weeks, the scaffolds were harvested and autoimmune T cells were analyzed. We found that antigen-specific T cells become enriched within scaffolds containing their cognate antigens. These T cells induced diabetes after adoptive transfer, indicating their pathogenicity. Furthermore, T-cell receptor (TCR) sequencing identified many expanded TCRs within the ß-cell scaffolds that were also expanded within the pancreata of NOD mice. These data demonstrate the utility of biomaterial scaffolds loaded with disease-specific antigens to identify and study rare, therapeutically important T cells.

   



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  Peripheral Reprogrammed CD4+ Cytotoxic T lymphocytes Efficiently Control Tumor Growth.
  Presenter: Nicolas Thiault
  All Authors:Nicolas Thiault, Nicolas Thiault, Mohd. Mushtaq Husain, Hitoshi Iwaya, Hilde Cheroutre
  La Jolla Institute for Allergy and Immunology
   
 

The initial commitment to either the CTL- or Th-lineage is made during thymic development of T cells, where thymocytes expressing an MHC class II–reactive TCR commit to the CD4+ helper T cell lineage, whereas thymocytes with specificity for MHC class I differentiate into the CD8+ CTL lineage. The molecular regulation is controlled by the action and counteraction of key transcription factors, such as the Th cell transcription factor, ThPOK, which promotes the Th fate and Runx3 which drives the differentiation of MHC class I restricted thymocytes into the CD8+ CTL-lineage. This dichotomy persists in the periphery for mature T cells, where ThPOK continues to suppress the cytotoxic fate of MHC class II–restricted CD4+ Th cells even as they differentiate into effector Th subsets. Recently, our team made a major breakthrough and showed that the thymic lineage commitment of CD4 Th cells is not fixed and that mature peripheral CD4+ T cells have the plasticity to lose ThPOK expression. We demonstrated that the loss of Thpok expression and the Th fate coincides with the re-expression of CD8? and the induction of a typical CTL phenotype, including 2B4 and Granzyme B expression and IFN-? and TNF-? cytokine production. Using an in vitro cytotoxic assay, our lab found that these reprogrammed ThPOK-CD4+ T cells display killer capacities. We also demonstrated that this mechanism is mediated in part by continuous Ag exposure of CD4 T cells that leads to the down-regulation of ThPOK and the induction of the Runx3-controlled cytolytic machinery allowing reprogramming of CD4 Th cells to CTL. Moreover, our data also indicated that peripheral Ag-induced reprogramming of conventional CD4 T cells is not a rare but rather widespread and common phenomenon, which occurs in various conditions such as in response to chronic viral infections (including chronic CMV and LCMV infections). In a new study we now investigate the role of CD4 CTL in anti-tumor immune surveillance. Using the well-described B16-melanoma tumor model, we were able to demonstrate that in vitro differentiated OVA-specific CD4 CTL display anti-tumor capacity and efficiently controlled/killed OVA-expressing B16 tumor cells. Extend studies on CD4 CTL in the cancer field will not only greatly expand our basic understanding of anti-tumor immunity but it will undoubtedly have major implications for translational research to design new cell therapies to prevent and/or treat devastating cancers.

   



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  The source of leukotriene in initiating pulmonary innate type 2 inflammation
  Presenter: Hung-An Ting
  All Authors:Hung-An Ting, John W. McGinty, Jakob von Moltke
  University of Washington
   
 

Helminth, allergen, and individual particles (e.g., chitin) induce rapid innate type 2 inflammation in the lung with the signature of group 2 innate lymphoid cells (ILC2) activation. As the primary source of type 2 cytokines IL-5 and IL-13, ILC2 are activated by multiple endogenous signals, including leukotrienes(LT). LTs are a family of eicosanoid lipid mediators that required 5-lipoxygenase (5-LO or Alox5) for biosynthesis, and Alox5 is required for pulmonary ILC2 activation. But the cellular source of leukotriene in pulmonary type 2 inflammation is still elusive. Here we dissect the source of Alox5-mediated LT production for type 2 inflammation. Deletion of Alox5 diminished ILC2 activation in the lung during helminth infection and chitin challenges. Reconstitution of wild type bone marrow in Alox5 knockout recipient reversed the defect of ILC2 activation, suggesting that Alox5 from hematopoietic compartment was required for ILC2 activation. While our data excluded the Alox5-expressing hematopoietic cells—DC, alveolar macrophage, neutrophil and mast cells as the major source of LTs in pulmonary ILC2 activation in vivo, recent study reported the lung as the main organ for biogenesis of platelet. Indeed megakaryocyte and platelet expressed Alox5, and deletion of Alox5 in megakaryocyte/platelet using Platelet factor 4 (Pf4)-Cre abolished ILC2 activation. Our study added a new player to the squad as a novel tissue-resident sentinel to initiate pulmonary type 2 responses.

   



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  PSGL-1, an immune checkpoint regulator that promotes T cell exhaustion in chronic viral infection and cancer
  Presenter: Roberto Tinoco
  All Authors:Roberto Tinoco, Florent Carrette, Monique L Barraza, Jonathan Magaña, Marcus W Bosenberg, Susan Swain, Linda M. Bradley
  Sanford|Burnham Medical Research Institute; UC San Diego
   
 

Chronic viruses, including HIV and hepatitis B and C, can survive in humans by inducing T cell dysfunction. Using a murine chronic virus that models human infections, we investigated the function of the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1) that is upregulated on responding T cells. PSGL-1-deficient mice unexpectedly cleared the virus due to dramatic increases in the intrinsic survival of multifunctional effector T cells that had downregulated PD-1 and other inhibitory receptors. Notably, this response resulted in immunopathology requiring CD4+ T cells. Mechanistically, PSGL-1 ligation on exhausted CD8+ T cells sustained PD-1 expression and diminished their survival during TCR stimulation. In a model of malignant melanoma where T cell dysfunction also occurs, PSGL-1 deficiency led to PD-1 downregulation, improved T cell responses and tumor control. Thus, PSGL-1 not only plays a fundamental role in balancing viral control and immunopathology, but also functions as a checkpoint that regulates T cell function in the tumor microenvironment.

   



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  Depletion of T regulatory cells without hampering anti-tumor immunity for improved tumor therapy
  Presenter: Jakub Tomala
  All Authors:Jakub Tomala, Barbora Tomalova, Blanka Rihova, Tomas Etrych, Libor Kostka, Karel Ulbrich, 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
   
 

Regulatory T (Treg) cells are one of the most important populations of immune cells with suppressor/regulatory activity. However, they also play critical role in impeding of immunosurveillance against autologous tumors. This is not so surprising as tumor cells mostly originate from normal cells of self origin thus are protected by Treg cells against anti-tumor reactions. Elimination of Treg cells from organism via administration of aCD25 mAb was repeatedly reported to be beneficial in tumor treatment. However, long-term persistence of aCD25 mAb in circulation together with its inhibitory effect on activated effector cells (CD25+) presents serious disadvantages. In order to overcome above mentioned problems, we developed a novel approach based on avidin-biotin system. We applied biotinylated aCD25 mAb (aCD25-BIO) and at selected time point, we eliminated it from the organism via administration of avidin covalently bound to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier (HPMA-avidin conjugate). Initially, we tested in vivo biological activity of aCD25-BIO in comparison to native aCD25 mAb and observed no significant differences. Its application led to decrease of Treg cells in organism and the optimal time for aCD25 mAb elimination from organism was determined to be 5 days after injection. We administered HPMA-avidin conjugate and saw steep decrease of anti-CD25-BIO within 4-8 hours afterwards and the amount of anti-CD25-BIO in sera was reduced to non detectable concentrations. Comparison of free avidin and HPMA-avidin conjugate showed that free avidin is not as effective as its conjugated counterpart. In conclusion, we assume our system is effective in terms of Treg cell depletion and subsequent anti-CD25 mAb elimination from organism, thus not hampering anti-tumor immunity.

   



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  Contrasting effects of wild type and detoxified adenylate cyclase toxin of Bordetella pertussis on proliferation and expansion of CD8+ and CD4+ T lymphocytes
  Presenter: Jakub Tomala
  All Authors:Jakub Tomala, Irena Adkins, Martina Svedova, Radek Spisek, Peter Sebo, Marek Kovar
  Institute of Microbiology ASCR, Videnska 1083, Prague 4, Czech Republic
   
 

The adenylate cyclase toxin (CyaA) is a key virulence factor of B. pertussis that subverts host defense. CyaA targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase enzymatic domain (~400 residues) that subverts cellular signaling by increasing cAMP levels. In parallel, the ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. In this study, we show that CyaA-mediated cAMP signalling of the wild type toxin decreases the capacity of LPS-stimulated dendritic cells (DCs) to induce CD4+ and CD8+ T cell proliferation and expansion in vivo and limits the induction of IFN-? producing CD8+ T cells while enhancing IL-10 and IL-17 production. In contrast, ablation of the adenylate cyclase enzymatic activity of the toxin (CyaA-AC-) converts the molecule into an efficient antigen delivery tool for stimulation of adaptive T cell immune responses. We identified here that CyaA-AC- by its pore-forming activity and potassium efflux enhances the capacity of DCs to stimulate CD8+ and CD4+ T cell expansion in vivo. This revealed a novel self-adjuvanting capacity of the CyaA-AC- toxoid that is exploited as a tool for delivery of immunotherapeutic anti-cancer CD8+ T cell vaccines into DCs. Acknowledgement: This work was supported by Czech Science Foundation grant 13-12885S and Institutional Research Concept RVO 61388971.

   



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  Design and production of IL-2-antibody single-chain fusions for cancer immunotherapy
  Presenter: Jakub Tomala*+
  All Authors:Jakub Tomala*+, Seth Ludwig+, Huilin Yang+, Michael Leff+, Marek Kovar*, Jamie Spangler+
  *Institute of Microbiology CAS; + Biomedical Engineering, Johns Hopkins University
   
 

Interleukin-2 (IL-2) is a multifunctional cytokine that plays a critical role in immune cell differentiation, growth, and activation. Due to its ability to potently stimulate immune effector cells (i.e. CD8+ T cells and natural killer [NK] cells), IL-2 has been used extensively in cancer immunotherapy.Unfortunately, its concurrent promotion of regulatory T cells (Tregs) and harmful off-target effects have limited its clinical efficacy. Moreover, the serum half-life of IL-2 is vanishingly short ( < 8 min), further complicating its use as a drug. Boyman and colleagues demonstrated that complexes of mouse IL-2 (mIL-2) with the anti-mIL-2 monoclonal antibody (mAb) S4B6 dramatically enhanced proliferation of immune effector cells compared to free IL-2 or S4B6 administration (1), presenting the exciting possibility of using antibody-cytokine complexes as therapeutic tools. However, therapeutic administration of these complexes is limited by the need for optimization of the cytokine:antibody dosing ratio and concerns of complex dissociation in vivo. To overcome these limitations, we designed, engineered and produced protein chimera consisting of mIL-2 linked to light chain of anti-IL-2 mAb S4B6 through a flexible oligopeptide spacer (2). In addition to improving upon the stability and therapeutic potential of cytokine-antibody complexes, our single-chain fusions have the potential to exhibit superior biological activity by circumventing cytokine dissociation. We showed the single-chain fusions we produced contain both IL-2 and S4B6 mAb in a single molecule and IL-2 interacts with binding site of S4B6 mAb in cis (1). We are now building on this powerful result to develop other translationally relevant cytokine-antibody fusions that could be used to address a wide range of disease challenges. 1.Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 2006;311(5769):1924-7. 2.Tomala J, Kovarova J, Kabesova M, Votavova P, Chmelova H, Dvorakova B, et al. Chimera of IL-2 linked to light chain of anti-IL-2 mAb mimics IL-2/anti-IL-2 mAb complexes both structurally and functionally. ACS Chem Biol 2013;8(5):871-6.

   



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  MOTO-CARs™: Driving Immunotherapy Forward
  Presenter: Michelle Townsend
  All Authors:Michelle Townsend, Kelsey A. Bennion, Guoying Wang, Zachary D. Ewell, David Lum, Michael Boyer, and Kim L. O’Neill
  Brigham Young University
   
 

Immunotherapy is becoming a new standard for cancer treatment. By engineering patient immune cells to target tumors, researchers are harnessing the immune system to combat cancer. Utilizing vectors designed to introduce an scFv fused to a signaling domain, these engineered cells have the ability to specifically identify and target cancer cells. While a majority of the field is focused on T-cell chimeric antigen receptors (CAR T-Cells), these cells have several inherent problems within solid tumors, as they are unable to traffic to the tumor site effectively. We investigated the use of an engineered macrophage toll-like receptor chimeric antigen receptor (MOTO-CAR™) designed to target mesothelin. We found that upon exposure to mesothelin-positive tumor cells (HCC-1806), murine MOTO-CARs™ are not only effective at eliminating the cancer cells, but also secrete significant levels of TNF-a when compared to mock controls (p-value = 0.0093), indicating activation and polarization to an M1 phenotype. When MOTO-CARs™ are co-incubated with cancer cells both in vitro and in vivo there is a significant decrease in tumor growth (p-value < 0.0001 in vitro; p-value = 0.0006 in vivo). In NSG mouse models, the tail vein injected MOTO-CARs™ were able to traffic to the tumor and elicit an anti-tumor response. These engineered cells are most effective within the first 48 hours post transfection. Furthermore, we found that MOTO-CARs™ are stably transfected for over seven days. Our results show that macrophages are a viable alternative to CAR T-cells and can be engineered to specifically target cancer cells via a cancer-specific scFv. We anticipate increased MOTO-CAR™ efficacy within an immunocompetent mouse model as the secreted TNF-a will aid in polarizing the tumor microenvironment towards an inflammatory, anti-tumor state. Additionally, the MOTO-CARs™ will present cancer-specific antigen to the host adaptive immune response to elicit a cancer-specific response as opposed to a target-specific response.

   



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  Phenotype and function of mucosal-associated invariant T (MAIT) cells in experimental and clinical sepsis.
  Presenter: Shubhanshi Trivedi
  All Authors:Shubhanshi Trivedi, Daniel Labuz *, Cole Anderson *, Claudia Araujo^, Toni Blair^, Elizabeth Middleton$, Alex Tran #, Matthew A. Mulvey #, Robert A. Campbell^, J. Scott Hale#, Matthew T. Rondina &,^, Daniel T. Leung *,#
  *Division of Infectious Disease, #Division of Microbiology and Immunology, $Division of Pulmonary and Critical Care, University of Utah, School of Medicine, &George E. Wahlen VAMC Department of Internal Medicine and GRECC, Salt Lake City, Utah, ^Molecular Medicine Program, University of Utah, Salt Lake City, Utah
   
 

Sepsis is an acute systemic inflammatory response to infection associated with high morbidity and mortality. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands and are activated during infections. Here, we investigated the function and phenotype of MAIT cells during clinical and experimental sepsis, and the mechanisms by which MAIT cells either contribute to or protect against sepsis pathology. We found that, compared to age- and sex-matched healthy donors and paired 3-month convalescence samples, MAIT cells in acutely septic patients were significantly reduced in frequency and displayed an increase in activation markers. When stimulated in vitro, MAIT cells from acutely septic patients had decreased IFN-γ production, changes which are reversed at convalescence. In a murine model of sepsis, MAIT cells expressed lower levels of IFN-γ and IL-17α, whereas these changes were not seen in non-MAIT T cells. Finally, MAIT-deficient (MR1-/-) mice had significantly increased sepsis-induced mortality and bacterial load compared to wild type, associated with reduced tissue-specific cytokine responses. Our data suggest that MAIT cells are highly activated and dysfunctional during clinical and experimental sepsis, and that MAIT cells may contribute to tissue-specific cytokine responses that are protective against mortality due to acute sepsis.

   



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  Increased susceptibility to Klebsiella pneumonia and impaired lung innate immunity after Salmonella Typhimurium intestinal infection.
  Presenter: Shubhanshi Trivedi
  All Authors:Shubhanshi Trivedi, Allie Grossmann2, Owen Jensen1 and Daniel T. Leung1,3.
  1Division of Infectious Diseases, 2Division of Anatomic Pathology, 3Division of Microbiology and Immunology, University of Utah, Salt Lake City, UT, USA.
   
 

Pneumonia and diarrhea are the two leading causes of death in children under age 5 worldwide. Epidemiologic studies in low-resource settings have suggested that a prior episode of diarrhea is a direct risk factor for subsequent respiratory infection. Our aim was to determine the impact of intestinal infection on innate immune responses in the lung and the mechanisms behind lung-gut immunological crosstalk. Using a mouse model of intestinal infection with Salmonella enterica serovar Typhimurium (S. Typhimurium), we investigated how infection in the gut compartment can modulate immunity in the lungs and impact susceptibility to respiratory bacterial (Klebsiella pneumoniae) challenge. Using flow cytometry, we found higher frequencies of plasmacytoid dendritic cells (pDCs) and lower frequencies of monocytic dendritic cells (moDCs) in lungs of S. Typhimurium infected mice compared to uninfected mice. Furthermore, on subsequent challenge with Klebsiella pneumoniae, we found that mice with prior intestinal infection have higher lung bacterial burden and responded with lower frequencies of lung moDCs and neutrophils compared to mice without prior intestinal infection. Hematoxylin and eosin staining demonstrated increased microthrombosis, intravascular polymorphonuclear neutrophils and margination in lungs of mice with prior intestinal infection compared to mice without prior intestinal infection. In conclusion, this study reveals potential role of neutrophils and dendritic cells in immunological crosstalk between the lung and the gut during enteric infections that may affect susceptibility to subsequent respiratory infection. Our findings have the potential to uncover novel therapeutic strategies targeting the gut-lung axis during intestinal infections.

   



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  The T cell memory phenotype is actively maintained by the expression of FOXO1
  Presenter: Daniel Utzschneider
  All Authors:Daniel Utzschneider, Stephen Hedrick
  Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0377, USA
   
 

Functional immune memory governed by CD8 T cells is indispensable for protection against bacterial and viral re-infection. In order to provide such long-term protection, memory T cells must survive over longer periods in the absence of antigenic stimulation by undergoing homeostatic self-renewal. By using a Tamoxifen-dependent Cre recombinase to deplete Foxo1 in differentiated memory CD8 T cells, we delineate the role of the transcription factor in sustaining a memory population. Interestingly, the sudden depletion of Foxo1 immediately impacted multiple functional and molecular properties of a memory population such as expression of IL7Ra, CD62L, Tcf1, and Eomes. In line with the deterioration of these molecules, we observed a gradual contraction of the memory population over time. This impaired maintenance derived from a reduced number of cells undergoing homeostatic proliferation and, more strikingly, these fewer proliferating cells were unable to regenerate themselves in the absence of FOXO1 and instead gave rise to progeny lacking hallmarks of conventional memory T cells. Despite this inability to renew, FOXO1-depleted memory cells sustained the ability to mount a robust recall response following antigen re-challenge – irrespective if they were challenged immediately after the FOXO1-depletion or following a longer resting period. The resulting FOXO1-deficient cells diminished in number at an accelerated rate compared with WT T cells. We show that the functional and molecular properties of a memory T cell population and thus its ability to be sustained long-term are dependent on the perduring expression of the transcription factor FOXO1. Moreover, we illustrate that resting memory T cells exhibit continuous transcriptional plasticity.

   



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  Antigen-driven specialization and differentiation of T cells in chronic infections
  Presenter: Daniel T Utzschneider
  All Authors:Daniel T Utzschneider, Francesca Alfei, Patrick Roelli, David Barras, Stephanie Darbre, Mauro Delorenzi, Daniel D. Pinschewer, and Dietmar Zehn
  Swiss Vaccine Research Institute (SVRI), 1066 Epalinges, Switzerland, & Division of Immunology and Allergy, Department of Medicine, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland
   
 

Chronic infections induce T cells showing impaired cytokine secretion and up-regulated inhibitory receptor expression such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remained vaguely understood. Using newly generated recombinant antigen variant-expressing chronic LCMV strains, we uncovered that T cell differentiation and the acquisition of a chronic “exhausted” phenotype depends critically on the frequency of TCR engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic und functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment.

   



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  Masscytometry Based Analysis of Human Lung Macrophages
  Presenter: Sreelakshmi Vasudevan
  All Authors:Sreelakshmi Vasudevan, Joshua Vasquez,Wenxuan Chen,Brandon Aguilar Rodriguez,Siyang Zeng,Erene Niemi,Mehrdad Arjomandi
  University of California, San Francisco
   
 

Background: Macrophages play an important role in many lung pathologies including Chronic Obstructive Pulmonary Disease (COPD). These cells accumulate inhaled particles and are highly auto-fluorescent especially in smokers. Characterization of lung macrophages, using flowcytometry and fluorescent microscopy, have been challenging due to this auto-fluorescence. Objective: To develop methods for identification of human lung macrophage subsets and characterization of macrophage polarization & function. Methodology: Subjects who were scheduled to undergo lung resection were recruited. They were categorized based on smoking status into current (>20 pack years) and former smokers (stopped smoking 12 months or earlier). Lung tissue samples were collected within 15 minutes of resection and were enzymatically digested for immune cell isolation. Cells were characterized using mass cytometry. A different cohort of subjects, were recruited to perform Bronchoalveolar Lavage (BAL) and were categorized as never smokers, former smokers (stopped smoking 12 months or earlier) and current smokers. Immune cells isolated from BAL were characterized using mass cytometry. Results: We developed a 45-marker mass cytometry-based panel which allows us to characterize the phenotype and polarization status of lung macrophages isolated from digested lung tissue as well as BAL, regardless of their smoking status. The panel consists of 12 lineage markers, 16 activation or inhibitory markers, 9 cell adhesion markers and 8 homing or co-stimulatory markers. We are able to analyze the polarization status of macrophages by comparing them to in-vitro differentiated M1 and M2 THP-1 macrophages. Furthermore, comparison between never smoker and current &/or former smoker lung macrophages advance the understanding of changes in phenotype and functional status of these cells with smoking. Conclusion: Diverse lung macrophage populations may be isolated and characterized for their origin, polarization, and function using the developed mass cytometry panel. This technique is helpful in studying lung macrophages with auto-fluorescence, thus superseding the limitations of traditional flowcytometry. Future studies will focus on quantifying the macrophage subsets and determining their function in samples from COPD patients.

   



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  STAT5 paralog dose governs T cell effector and regulatory function
  Presenter: Alejandro Villarino
  All Authors:Alejandro Villarino, Arian Laurence, Gertraud W. Robinson, Michael Bonelli, Barbara Dema, Han-Yu Shih, Hong-Wei Sun, Lothar Hennighausen, Yuka Kanno, John J. O'Shea
  National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH)
   
 

The transcription factor STAT5 is fundamental to the immune system. However, the relationship between its two paralogs, STAT5A and STAT5B, and the extent to which they are functionally distinct, remains controversial. We addressed this longstanding question using genetic and genomic approaches focusing on helper T cells, the key orchestrators of adaptive immunity. Our studies demonstrate that, while both control vital aspects of T cell biology, including Tfh and Treg function, STAT5B is dominant and, thus, uniquely associated with immune tolerance. DNA binding and transcriptome analyses revealed that STAT5B also dominates at the molecular level, exhibiting greater influence on both target gene selection and transcription. This disparity is largely explained by relative abundance (i.e. paralog dose) rather than primary structural variations. Collectively, our findings provide a unifying model for the unique and redundant activities of STAT5A and STAT5B, establishing that differential expression underlies functional specificity despite extensive structural homology.

   



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  Tuft cell-derived IL-25 regulates an intestinal ILC2 - epithelial response circuit
  Presenter: Jakob von Moltke
  All Authors:Jakob von Moltke, Ming Ji, Hong-Erh Liang, Richard Locksley
  UC San Francisco
   
 

Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, designated “weep and sweep”, requires IL-13 production by tissue resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (Th2). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including tuft cells, whose functions are unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria. After helminth infection, tuft cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s, and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodeling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells.

   



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  Human tonsil cultures as a model to study adaptive immune responses in vitro
  Presenter: Lisa Wagar
  All Authors:Lisa Wagar, Ameen Salahudeen, Christian Constantz, Katherine Jackson, Michael Lyons, Peter Kim, Calvin Kuo, Scott Boyd, Mark Davis
  Stanford University
   
 

The human immune system is constantly confronted by a diversity of challenges that require complex and multi-functional responses, encompassing a mixture of immune and non-immune cells types that work in concert within lymphoid structures. Despite its critical importance, our knowledge of how human immune responses are generated has been hindered by a lack of robust in vitro culture methods that reproduce these in vivo interactions. Therefore, we have created an organoid system from human tonsils that recapitulates both heterotypic immune and stromal components and enables in vitro exploration of antigen-specific cellular and humoral responses. The live attenuated influenza vaccine (LAIV) was used as a model antigen to characterize the properties of the culture system. The tonsil cultures have the ability to self-organize and cellular aggregates are enriched for responding cells (such as plasmablasts and T follicular helper cells) after LAIV exposure. We have shown that B cell differentiation and antibody production is strongly dependent on the presence of antigen presenting cells, but not pre-existing memory or germinal center B cell subsets. These organoids can successfully mount a robust humoral response to LAIV and generate microgram quantities of influenza-specific antibodies per culture. Heavy chain BCR repertoire sequencing has revealed a proportion of plasmablasts and germinal center B cells in LAIV-stimulated cultures have CDR3 motifs that are known to bind influenza. IgM responses have also been generated against HIV proteins and other naive antigens under certain cytokine and adjuvant combinations. This platform provides many opportunities to understand and manipulate the cellular interactions that occur during an adaptive immune response, and will enable translational applications such as high throughput vaccine design and adjuvant testing.

   



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  Loss of negative costimulation permits aberrant T cell differentiation
  Presenter: Spencer Wei
  All Authors:Spencer Wei, Roshan Sharma, Nana-Ama Anang, Jacob Levine, Yang Zhao, Jing Wang, Dana Pe'er, James Allison
  The University of Texas MD Anderson Cancer Center
   
 

T cell costimulation is a principal mechanism by which T cell activation is regulated, but it remains unclear whether costimulatory pathways also control T cell differentiation. This unresolved question is critical for our fundamental understanding of how T cell activity is controlled, and also has therapeutic implications in the context of checkpoint blockade. Using a mass cytometry based systems approach we sought to address the hypothesis that negative costimulation regulates T cell differentiation as well as activation. Unsupervised population identification reveals 30 distinct T cell subsets in Ctla-4-/- and littermate control mice including 6 CD8, 3 Treg, 14 CD4 effector T cell populations. Consistent with its role in attenuating T cell activation, loss of CTLA-4 led to changes in the relative frequency of specific T cell subsets. Strikingly, however, loss of CTLA-4 led to the generation of multiple non-canonical T cell populations outside of the normal boundaries of T cell phenotypes, which were not observed in control mice. Interestingly, these non-canonical populations were all contained within the CD4 T compartment, while in contrast, no knockout specific CD8 T cell subsets were observed despite significant changes in their relative frequency. These observations indicate that CTLA-4 negative costimulation constrains CD4 T cell phenotypes and moreover, suggest that CD4 and CD8 T cell differentiation are differentially regulated by costimulation. Consistent with this model, loss of CTLA-4 differentially affected the expression of individual markers in CD4 and CD8 T cells. To determine if the role of negative costimulation in differentiation may be a generalizable phenomenon, we performed similar analyses of Pdcd-1-/- mice. This profiling identified a similar range of T cell populations and surprisingly revealed that loss of PD-1 also affects T cell phenotypes in addition to modulating the relative frequency of specific subsets. However, in contrast to CTLA-4, loss of PD-1 led an expansion of CD8 T cell phenotypes observed, while CD4 T cell phenotypes were maintained despite significant changes in relative frequency. Thus, PD-1 limits CD8 T cell phenotypes while CTLA-4 limits CD4 T cell phenotypes. Together these findings indicate that in addition to attenuating T cell activation, negative costimulation acts to constrain T cell differentiation.

   



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  Epithelial club cell junction proteins regulate calcineurin signaling and allergic asthma in response to protease allergens
  Presenter: Darin Wiesner
  All Authors:Darin Wiesner, Bruce S Klein
  University of Wisconsin - Madison
   
 

Asthma will soon afflict a tenth of the world’s population. Although fungi and their proteases account for most cases of allergic asthma, the mechanisms by which they provoke allergic inflammation remain ill defined. Here, we show that epithelial club cells lining the bronchioles sense and regulate the allergic response to Aspergillus protease. Protease disrupts the junctions between club cells in the conducting airways; in particular, cleaving the extracellular domain of e-cadherin that maintains adherens junction integrity of these cells. The loss of tensile force between adjoining club cells triggers mechanotransduction via the gated calcium ion channel, TRPV4, resulting in calcium flux, calcineurin activation, and translocation of NFATc3 or NFATc4 from the cytoplasm into the nucleus. Club cells rapidly transcribe chemokine ligand 2, recruiting monocyte-derived dendritic cells and initiating Th2 cell-dependent eosinophilia in the lungs. These data reveal a new paradigm with novel molecular targets and signaling events in lung stromal cells that recognize a common allergen and incite allergic airway disease.

   



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  Jagunal-homolog 1 is a critical regulator of neutrophil function in fungal host defense
  Presenter: Gerald Wirnsberger
  All Authors:Gerald Wirnsberger, Florian Zwolanek, Johannes Stadlmann, Luigi Tortola, Shang Wan Liu, Thomas Perlot, Päivi Järvinen, Gerhard Dürnberger, Ivona Kozieradzki, Renu Sarao, Alba De Martino, Kaan Boztug, Karl Mechtler, Karl Kuchler, Christoph Klein, Ulrich Elling, and Josef M. Penninger
  Institute of Molecular Biotechnology
   
 

Neutrophils are key innate immune effector cells essential to fight bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagunal homolog 1 (Jagn1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis revealed marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity of Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection, impaired formation of cytotoxic granules, as well as defective MPO-release and killing of Candida albicans. GM-CSF treatment protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Importantly, GM-CSF also restored the defective fungicidal activity of bone marrow cells from patients with JAGN1 mutations. These data directly identify Jagn1/JAGN1 as a novel regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for human patients.

   



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  T cell-specific gp130 signaling regulates T cells and antibody responses after acute LCMV infection
  Presenter: Kurt A. Wong
  All Authors:Kurt A. Wong, James A. Harker, Aleksandr Dolgoter, Elina I. Zuniga
  University of California San Diego
   
 

T cell fates are heavily dictated by the surrounding cytokine milieu; however the specific contributions of different cytokine families in the context of an infectious environment are incompletely understood. Here we sought to evaluate the role of T cell signaling via gp130, the common co-receptor for the IL-6 family of cytokines, during an acute viral infection. For that we infected CD4-cre x gp130fl/fl mice with LCMV ARM and determined different aspects of adaptive immune responses. We observed that the quantity of gp130-deficient virus-specific CD4 T cells were reduced after the peak of infection and had an altered cytokine profile, producing less IL-21 and more IL-2. Despite unaltered differentiation of T follicular helper (Tfh) cells, Tfh cells expressed less maf, il21, and ICOS. This was accompanied with diminished germinal centers, a dramatic reduction in plasmablast numbers and compromised IgG2 switch. We also found skewing of the virus-specific CD8 T cell compartment away from the CD127+ memory precursor phenotype and towards the KLRG1+ effector phenotype, suggesting a defect in memory development. Consistently, virus-specific CD8 T cells demonstrated a clear reduced capacity to expand after secondary challenge. Overall our data show that signaling through the gp130 pathway in T cells promotes CD8 T cell memory as well as the accumulation and function of virus specific CD4 T cells, which in turn has significant effects on downstream humoral immunity.

   



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  Precision-cut human liver slice cultures as an immunological platform
  Presenter: Xia Wu
  All Authors:Xia Wu, Jessica B. Roberto1, Allison Knupp1, Heidi L. Kenerson2, Camtu D. Truong1, Sebastian Y. Yuen1, Katherine J. Brempelis1, Marianne Truffaux3, Antony Chen3, Helen Horton3, Raymond S. Yeung2 and Ian N. Crispe1
  1Department of Pathology, University of Washington, Seattle, WA, 2Department of Surgery, University of Washington, Seattle, WA, USA, 3Infectious Diseases and Vaccines, Janssen Research and Development, B-2340 Beerse, Belgium.
   
 

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. To improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Current effort is to use antibody blockade to manipulate cytokine production and modulate tissue repair process ex vivo in human liver slices.

   



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  E-box binding protein HEB promotes the maturation into germinal center TFH cells by preventing re-expression of EBI2 on pre-TFH cells through the suppression of Eomes
  Presenter: Hidehiro Yamane
  All Authors:Hidehiro Yamane, Anastassia A. Tselikova, Sundar Ganesan, Juraj Kabat, Ke Weng, Yuan Zhuang, Paul E. Love, Pamela L. Schwartzberg, and William E. Paul
  NIAID, NIH
   
 

E-box binding proteins (E-proteins) have recently been demonstrated to regulate follicular B helper T (TFH) cell differentiation, but the underlying mechanisms remain elusive. To clarify the molecular basis of E-protein-mediated TFH differentiation, we generated mouse strains with a peripheral T cell-specific deletion of either E2A (E2AcKO) or HEB (HEBcKO) or both (E2A/HEBcKO) on an OT-II TCR-transgenic background. Naïve CD4+ OT-II T cells from these mutant mice were adoptively transferred into C57BL/6 mice, and the recipients were immunized with OVA/alum on the following day of the adoptive transfer. Seven days after immunization, E2A/HEBcKO donor cells in the spleen had a substantial defect in germinal center (GC) TFH differentiation with a reduction in clonal expansion by 2 to 3-fold. E2AcKO donor cells differentiated into GC TFH cells comparably to WT counterparts. By contrast, HEBcKO donor cells underwent pre-TFH cell differentiation but failed to maturate into GC TFH cells with normal clonal expansion, implying the importance of HEB, but not E2A, in GC TFH differentiation. Gene expression profiling analysis revealed that 5 days after immunization, HEBcKO pre-TFH cells had no defect in the expression of signature Tfh-related genes but a substantial increase in the expression of the Gpr183 gene encoding Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2), a receptor for 7α,25-dihydroxycholesterol secreted by stromal cells residing in the outer T cell zone. Surface expression of EBI2 on HEBcKO donor cells was induced normally at day 2 and downregulated at day 4 post-immunization comparably to that on WT donor cells. However, at day 5 post-immunization or later, HEBcKO pre-TFH cells re-expressed high levels of EBI2, whereas WT counterparts kept downregulating it. Consistent with the data on the surface expression of EBI2, HEBcKO donor cells accumulated in the T/B border and little or no such cells were found in either B cell follicles or GCs at day 7 post-immunization. The impaired GC TFH differentiation by HEB deficiency was accompanied by transient and aberrant expression of Eomes during the first 2 days of priming. Of note, further conditional loss of Eomes prevented HEBcKO donor cells from re-expressing EBI2 and rescued them from the GC TFH deficit. In conclusion, HEB does not directly regulate the expression of the signature TFH-related genes but fine-tunes the localization of pre-TFH cells by preventing EBI2 re-expression, allowing for their entry to B cell follicles to undergo subsequent maturation into GC TFH cells. Moreover, the HEB-mediated suppression of Eomes expression during the early priming phase plays a critical role in rendering EBI2 suppressed on pre-TFH cells. This work was supported by the Intramural Research Program of NIAID, NIH.

   



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  STING controls Herpes Simplex Virus in vivo independent of type I interferon induction
  Presenter: Livia Yamashiro
  All Authors:Livia Yamashiro, Stephen C Wilson, Huntly M Morrison, Vasiliki Karalis, Jing-Yi J Chung, Katherine J Chen, Helen S. Bateup, Moriah L. Szpara, Angus Y. Lee, Jeffery S. Cox, Russell E. Vance
  UC Berkeley
   
 

The Stimulator of Interferon Genes (STING) pathway initiates potent immune responses upon recognition of DNA derived from bacteria, viruses and tumors. To signal, the C-terminal tail (CTT) of STING recruits TBK1, a kinase that phosphorylates serine 365 (S365) in the CTT. Phospho-S365 acts as a docking site for IRF3, a transcription factor that is phosphorylated and activated by TBK1, leading to transcriptional induction of type I interferons (IFNs). IFNs are essential for antiviral immunity and are widely viewed as the primary output of STING signaling in mammals. However, other more evolutionarily ancestral responses, such as induction of NF-?B or autophagy, also occur downstream of STING. The relative importance of the various outputs of STING signaling during in vivo infections is unclear. Here we report that mice harboring a serine 365-to-alanine (S365A) point mutation in STING exhibit normal susceptibility to Mycobacterium tuberculosis infection but, unexpectedly, are resistant to Herpes Simplex Virus (HSV)-1, despite lacking STING-induced type I IFN responses. Likewise, we find Irf3–/– mice exhibit resistance to HSV-1. By contrast, resistance to HSV-1 is abolished in mice lacking the STING CTT or TBK1, suggesting that STING protects against HSV-1 upon TBK1 recruitment by the STING CTT, independent of IRF3 or type I IFNs. Interestingly, we find that STING-induced autophagy is a TBK1-dependent IRF3-independent process that is conserved in the STING S365A mice, and autophagy has previously been shown to be required for resistance to HSV-1. We thus propose that autophagy and perhaps other ancestral interferon-independent functions of STING are required for STING-dependent antiviral responses in vivo.

   



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  STING controls HSV infection in a type I IFN-independent manner
  Presenter: Livia Yamashiro
  All Authors:Livia Yamashiro, Stephen C. Wilson, Vasiliki Karalis, Angus Y. Lee, Russell E. Vance
  UC Berkeley
   
 

Stimulator of interferon genes (STING) is an adaptor protein important for recognition of DNA from various pathogens. The main downstream event following STING activation is type I IFN signaling, with distinct consequences varying on bacterial or viral infections. STING can also orchestrate other downstream signaling events such as NF-kB activation and the initiation of autophagy. However, little is known of how these pathways are regulated, or whether these ancestral functions are still an important aspect of STING signaling in innate immunity against pathogens. Using mice that carry specific mutations on STING we were able to differentiate the downstream events of STING activation in the context of HSV infection. We show that the innate immune response to HSV is STING-dependent but, surprisingly, type I IFN-independent. Instead, we found that STING-dependent autophagy results in increased CD8+ T cells, suggesting augmented antigen presentation and viral clearance. Therefore, characterization of the distinct arms downstream of STING activation improves the understanding of their contribution during infections.

   



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  HIV-specific immunity derived from chimeric antigen receptor-engineered stem cells
  Presenter: Anjie Zhen
  All Authors:Anjie Zhen, Masakazu Kamata, Valerie Rezek, Jonathan Rick, Bernard Levin, Saro Kasparian, Irvin S.Y Chen, Otto O. Yang, Jerome A. Zack, and Scott G. Kitchen
  UCLA
   
 

HIV-1 specific cytotoxic T lymphocytes (CTL) are the key host immune response to HIV infection in controlling viral replication and are an important therapeutic target. We have previously engineered HIV-specific CTL responses through the use of molecularly cloned T cell receptors (TCRs). However, generation of HIV-specific CTL responses for therapy is limited by technical barriers such as varying human leukocyte antigen (HLA) restrictions between different individuals and potential for viral escape by sequence variation. The use of of a chimeric T cell receptor containing CD4 linked to the signal domain of the T cell receptor ? chain (universal T cell receptor) can circumvent these barriers. CD4 chimeric antigen receptor (CD4 CAR) modified T cells inhibit viral replication and kill HIV infected cells in vitro and were reported to have prolonged survival in vivo. However, CD4 CAR modified T cells are susceptible to HIV infection resulting in a limited anti-HIV effect from CD4 CAR modified T cells. Here we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/ progenitor cell (HSPC) based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

   



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  Time-of-Flight Cytometry and Single-Cell Analysis Uncovers Unipotent Hematopoietic Neutrophil Lineage in Adult Mouse and Human
  Presenter: Yanfang Zhu
  All Authors:Yanfang Zhu, Paola Marcovecchio1, Amy Blatchley1, Lindsey Padgett1, Runpei Wu1, Erik Ehinger1, Cheryl Kim2, Zbigniew Mikulski1, Gregory Seumois3, Ariel Madrigal3, Huy Dinh1, Pandurangan Vijayanand3, and Catherine C. Hedrick1*
  1Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 2Flow Cytometry Core Facility, La Jolla Institute for Allergy and Immunology, La Jolla, CA 3Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA
   
 

Neutrophils are short-lived immune cells that play important roles in a variety of diseases. The oligopotent Granulocyte Monocyte Progenitors (GMPs) in the bone marrow give rise to monocytes and all granulocytes; however, the unipotent neutrophil lineage hematopoiesis has not been well established. Here, we use Cytometry by Time-of-Flight (CyTOF) and Single-cell RNA-Sequencing (scRNA-Seq) tools to identify a CD117+ (c-Kit+) Ly6A/E- (Sca1-) Siglec F- FceRIa- CD16/32+ Ly6B+ CD11a+ (LFA1a+) CD162lo CD48lo Ly6Clo CD115- Ly6G- early-stage progenitor population in adult mouse and a heterogeneous hCD117+ hCD66b+ hCD34+/- progenitor population in human bone marrow with in vivo unipotent neutrophil potency. A Ly6Glo immediate downstream late-stage neutrophil precursor is also identified by viSNE high dimensional automated mapping. This discovery should allow for comprehensive understanding of neutrophil lineage hematopoiesis and assist discovery of important new therapeutic targets for granulopoiesis-related diseases such as in cancer.