Our laboratory studies signal transduction in the immune system with a focus on tyrosine phosphorylation-based signaling involved in T cell responses. These pathways are required for normal cellular growth and differentiation but also cause abnormal growth, differentiation and development in multiple human disorders including primary immunodeficiencies, autoimmunity and cancer. Using multidisciplinary approaches of genetics, genomics, protein biochemistry and cell biology, our goals are to understand how signaling molecules contribute to normal function of immune cells and their dysfunction in disease, and how manipulating signaling pathways affects responses to infection and immunization. Research: Tec Kinases: Mutations affecting the Tec kinase, Btk, cause the genetic disorder X-linked Agammmaglobulimemia, characterized by abnormal B cell development and function. Over the last 15 years, we showed that the Tec kinases expressed in T lymphocytes, Itk and Rlk, are important modulators of T cell signaling: mutations of Itk and Rlk do not prevent T cell development and function, but alter outcomes by affecting T cell receptor signaling strength. We helped describe their roles in TCR-induced activation of PLC-g and Ca++ mobilization, defined kinase-independent functions of Itk in regulating the actin cytoskeleton and cell adhesion, (required for effector T cell functions) and showed these kinases affect patterns of cytokine production by CD4+ T helper cells and the ability of mice to respond to distinct types of infection. Confirming their importance, mutations of Itk have now been described in a profound EBV-induced lethal immunodeficiency. Research: In recent work, we examined how Itk contributes to regulating the balance between Th17 cells, effector T cells that respond to extracellular bacteria (and which contribute to autoimmune and inflammatory disorders), and regulatory T cells (Tregs), a subset of cells responsible for keeping immune responses in check. We found that Itk mutation increases Tregs differentiation under conditions that normally generate Th17 cells. We link this to impaired repression of Pten, leading to altered inositide phosphate regulation, impaired activation of mTOR and alterations in T cell metabolism. Our results show that Itk is part of a positive feedback loop that normally represses Pten upon TCR activation, thereby integrating T cell responses to multiple signaling pathways (Gomez-Rodriguez et al, under revision). We are also evaluating the role of Itk in CD8+ T cell cytolytic responses to viral infection to understand how mutations affecting Itk leads to fatal responses to EBV. II. SAP: A second major focus is SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1), characterized by fatal EBV-infection, lymphomas, and antibody defects. SAP is a small SH2 containing adaptor that binds phosphorylated tyrosine residues in the intracellular tails of SLAM family co-stimulatory receptors. We previously generated SAP-deficient mice and found these mice recapitulate features of XLP, including increased T cell activation and decreased antibody production upon infection (Czar et al PNAS). Notably, SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination. These defects have been confirmed in humans with XLP. We showed that SAP-/- T cells have a selective defect in adhesion to B cells but not other cells, preventing them from delivering contact-dependent signals required for B cells to form germinal centers (Qi et al, Nature, 2008;Cannons et al, Immunity, 2010), leading us to propose that defective T cell help for B cell antibody responses and defective T and NK cell killing of EBV-infected B cells in XLP, resulted from impaired interactions with B cells (Schwartzberg, Nat Rev. Immunol 2009). Confirming this idea, we found that SAP-/- CD8 cytotoxic lymphocytes show selective defects in killing B cell targets, despite normal killing of other cells (Zhao et al 2012, Immunity). We showed the SHP-1 phosphatase was recruited to SLAM family members in the absence of SAP, leading to a strong negative signal that alters the immunological synapse formed between T cells and B cell targets, thereby inhibiting cell interactions and cytolysis. Importantly, this work demonstrated that XLP phenotypes require the upstream SLAM family members (Ly108 and 2B4) that deliver negative signals. Furthermore, we found similar results in CD4 cells (Kageyama et al, 2012 Immunity) providing common mechanistic insight into the pathophysiology of XLP and suggesting potential therapeutic approaches to XLP via blocking SLAM family members. We are now examining whether similar mechanisms are involved in other XLP phenotypes (lymphoproliferation) and whether similar defects are observed in other immunodeficiencies associated with increased EBV sensitivity. Finally, our work on SAP has helped define the importance of SLAM members in immunity, including CD84 and Ly108 in germinal center formation (Cannons et al, Immunity 2010;Kageyama et al 2012, Immunity) and innate T cell development (Kageyama et al, Immunity 2012;Dutta et al, J Immunol 2012 and 2013). We also demonstrated a novel isoform of Ly108 expressed only in Lupus-resistant mouse strains, providing insight into the contribution of SLAM receptors to autoimmunity (Dutta et al, J. Immunol 2012 and 2013). III. Our work on Tec kinases and SAP has converged in studies of rare innate-like subsets of T cells that develop effector function within the thymus to help set the tone of early immune responses. We found that development of innate T cells including NKT cells is dependent on SAP and SLAM family (Nichols et al Nat. Med 2005;Horai et al Immunity 2007;Kageyama et al Immunity 2012;Dutta et al, J Immunol 2012, Kraus et al. in prep) and negatively regulated by Tec kinases (Horai et al Immunity 2007;Qiao et al PNAS 2012;Dervovic et al J Immunol 2013). Our recent work has demonstrated that Ly108 is constitutively phosphorylated in the thymus and that both positive and negative signaling through Ly108 is involved in innate lymphocyte development (Kageyama et al, Immunity 2012;Dutta et al, J Immunol 2012 and 2013). We further revealed that costimulation through the TCR and Ly108 potentially induces expression of PLZF, a critical transcription factor that confers activated phenotypes to innate CD4 lymphocytes (Dutta et al 2013, J. Immunol). We found that Ly108 engagement potentiates TCR signaling, leading to prolonged Ca2+ mobilization, increased NFAT-binding to the Egr2 promoter, and increased Egr2 expression and binding to the PLZF promoter. Moreover, Ly108 phosphorylation and signaling is increased in Lupus-prone mouse strains. This work provides insight into signaling from this potentially clinically important receptor (Dutta et al 2013, J. Immunol). IV. Tfh cells: A final area focuses on the role of SAP in the development and function of Tfh cells, the critical helper T cell population required for providing signals to B cells for germinal center formation. We had defined in vitro conditions for the differentiation of a Tfh-like cell population that can rescue defects in SAP-deficient mice and used cellular, genetic and epigenetic studies, including ChIP-Seq to help define the cross-regulation of Tfh cells with other T cell lineages both using in vitro-derived and ex vivo Tfh cells (Lu et al, Immunity 2011, and Nakayamada et al 2011, Immunity). Our work supports the idea of plasticity between T helper cell populations that can permit an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013, Poholek et al J. Immunol, under revision).

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Project End
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Budget End
Support Year
16
Fiscal Year
2013
Total Cost
$1,585,477
Indirect Cost
Name
National Human Genome Research Institute
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Type
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Prince, Amanda L; Watkin, Levi B; Yin, Catherine C et al. (2014) Innate PLZF+CD4+ ?? T cells develop and expand in the absence of Itk. J Immunol 193:673-87
Tsang, John S; Schwartzberg, Pamela L; Kotliarov, Yuri et al. (2014) Global analyses of human immune variation reveal baseline predictors of postvaccination responses. Cell 157:499-513
Prince, Amanda L; Kraus, Zachary; Carty, Shannon A et al. (2014) Development of innate CD4+ and CD8+ T cells in Itk-deficient mice is regulated by distinct pathways. J Immunol 193:688-99
Lucas, Carrie L; Kuehn, Hye Sun; Zhao, Fang et al. (2014) Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110ýý result in T cell senescence and human immunodeficiency. Nat Immunol 15:88-97
Gomez-Rodriguez, Julio; Wohlfert, Elizabeth A; Handon, Robin et al. (2014) Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells. J Exp Med 211:529-43
Nakayamada, Shingo; Poholek, Amanda C; Lu, Kristina T et al. (2014) Type I IFN induces binding of STAT1 to Bcl6: divergent roles of STAT family transcription factors in the T follicular helper cell genetic program. J Immunol 192:2156-66
Cannons, Jennifer L; Lu, Kristina T; Schwartzberg, Pamela L (2013) T follicular helper cell diversity and plasticity. Trends Immunol 34:200-7
Dervovic, Dzana D; Liang, Haydn C-Y; Cannons, Jennifer L et al. (2013) Cellular and molecular requirements for the selection of in vitro-generated CD8 T cells reveal a role for Notch. J Immunol 191:1704-15
Dickler, Howard B; McCoy, J Philip; Nussenblatt, Robert et al. (2013) The National Institutes of Health Center for Human Immunology, Autoimmunity, and Inflammation: history and progress. Ann N Y Acad Sci 1285:133-47
Rumble, Julie M; Oetjen, Karolyn A; Stein, Paul L et al. (2009) Phenotypic differences between mice deficient in XIAP and SAP, two factors targeted in X-linked lymphoproliferative syndrome (XLP). Cell Immunol 259:82-9

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