2014 In the last year, our work has covered several major areas: I. 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. In the last year, we published work demonstrating that Itk helps regulate the balance between Th17 cells, CD4+ effector T cells that respond to extracellular bacteria (but which also 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 inflammatory conditions that normally generate Th17 cells. We link this to impaired repression of the lipid phosphatase, Pten, impaired activation of mTOR and alterations in T cell metabolism and in responses to IL-2. Our results show that Itk is part of a positive feedback loop that normally represses Pten expression upon TCR activation, thereby integrating T cell responses to multiple signaling pathways and suggest Itk as a potential therapeutic target for autoimmunity (Gomez-Rodriguez et al, J. Exp Med 2014). II. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we helped describe and characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific member of the PI3K catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets. We further found that these CD8+ cells exhibited increased expression of granzyme B, increased anti-TCR (CD3)-induced degranulation and normal to increased killing of P815 murine mastocytoma cells coated with anti-CD3 antibodies, a measure of general cytotoxicity. However, P110elta CD8 cells also expressed high levels of CD57, a marker associated with T cell senescence (Lucas et al, Nature Immunol. 2014). Our work has provided new insight into the requirements for PI3K in immune homeostasis and function and demonstrated a new immunodeficiency caused by activating mutations in PI3K. III. SAP: Another major focus of our work 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) resulting from a strong negative signal that alters the immunological synapse formed between T cells and B cell targets, thereby inhibiting cell interactions and cytolysis. 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. Over the last year, our work on SAP has converged with studies of the Tec kinase Itk and their effects on innate T lymphocyte development. These rare innate-like subsets of T cells develop effector function within the thymus and help set the tone of early immune responses. The roles of these rare lymphocytes are increasingly being recognized. We previously found that development of innate T cells including NKT cells is dependent on SAP and SLAM family both in mice and humans (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. submitted) 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 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). In recent work, we are investigating how Itk-deficiency increases the generation of PLZF+ cells and affects the balance of signaling from the TCR and SLAM family members (Kraus et al submitted). Together, these studies have provided insight into these rare but immunologically important cells and whether they contribute to phenotypes of XLP and other immunological disorders. 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 and long-term humoral immunity, the heart of protective responses to most immunizations. In the last year, we also contributed to collaborative studies further characterizing requirements for the differentiation of Tfh cells (Nakayama et al J. Immunol, 2014;Hu et al Immunity 2013). 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).

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17
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2014
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Human Genome Research
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Panchal, Neelam; Booth, Claire; Cannons, Jennifer L et al. (2018) X-Linked Lymphoproliferative Disease Type 1: A Clinical and Molecular Perspective. Front Immunol 9:666
Cannons, Jennifer L; Preite, Silvia; Kapnick, Senta M et al. (2018) Genetic Defects in Phosphoinositide 3-Kinase ? Influence CD8+ T Cell Survival, Differentiation, and Function. Front Immunol 9:1758
Preite, Silvia; Cannons, Jennifer L; Radtke, Andrea J et al. (2018) Hyperactivated PI3K? promotes self and commensal reactivity at the expense of optimal humoral immunity. Nat Immunol 19:986-1000
Kapnick, Senta M; Stinchcombe, Jane C; Griffiths, Gillian M et al. (2017) Inducible T Cell Kinase Regulates the Acquisition of Cytolytic Capacity and Degranulation in CD8+ CTLs. J Immunol 198:2699-2711
Ritter, Alex T; Kapnick, Senta M; Murugesan, Sricharan et al. (2017) Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 114:E6585-E6594
Cannons, Jennifer L; Schwartzberg, Pamela L (2017) SAP and Lessons Learned from a Primary Immunodeficiency. J Immunol 199:1531-1533
Gomez-Rodriguez, Julio; Meylan, Françoise; Handon, Robin et al. (2016) Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4. Nat Commun 7:10857
Ruffo, Elisa; Malacarne, Valeria; Larsen, Sasha E et al. (2016) Inhibition of diacylglycerol kinase ? restores restimulation-induced cell death and reduces immunopathology in XLP-1. Sci Transl Med 8:321ra7
Olnes, Matthew J; Kotliarov, Yuri; Biancotto, Angélique et al. (2016) Effects of Systemically Administered Hydrocortisone on the Human Immunome. Sci Rep 6:23002
Marom, A; Barak, A F; Kramer, M P et al. (2016) CD84 mediates CLL-microenvironment interactions. Oncogene :

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