Our laboratory studies signal transduction involving tyrosine phosphorylation and non-receptor tyrosine kinases, molecules required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth and development involved in the formation and progression of cancer. Mutations affecting such signaling pathways have been found to give rise to a number of human disorders including several primary immunodeficiencies. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how these molecules contribute to normal function of cells in the immune system. Our goals include understanding how manipulation of these pathways can help in the development of therapeutics for disease. A large portion of our work is focused on studies of the Tec family of tyrosine kinases, the prototypical member of which, Btk, is responsible for the human genetic disorder X-linked agammmaglobulimemia and is required for normal B cell development and function. We have shown that mutation of Tec family kinases expressed in T cells can also severely impair T lymphocyte function in mice and influence responses to infections in vivo. Previous studies have demonstrated that the Tec kinases are critical for antigen receptor induced activation of phospholipase-c gamma, a key enzyme required for Ca++ mobilization. In the last year, we have concentrated on the newly discovered role forTec kinases in regulation of the actin cytoskeleton in T lymphocytes. We have recently reported altered activation of Cdc42, a Rho family GTPase that is an upstream activator of the Wiskott Aldrich Syndrome Protein in cells deficient for the Tec kinase Itk. and altered subcellular localization of the guanine nucleotide exchange factor Vav. Moreover, we have recently published that cells deficient in Tec kinases also show abnormal responses to chemokines, small molecules that direct cell polarization and cell trafficking in the immune system. Our results place the Tec kinases as critical regulators of the actin cytoskeleton, cell adhesion and migration and suggest that cytoskeletal defects may contribute to the phenotypes associated with Tec kinase deficiency including those seen in X-linked agammaglobulinemia. In continuing work, we are also examining the effects of mutation of Tec kinases on T cell responses in vivo. We had previously found that mutation of the Tec kinases alters the balance of T helper cell differentiation and cytokine production. We are now extending these studies using a combination of transgenic and gene-targeted mice to alter expression of the Tec kinases in T lymphocytes. As an extension of these studies, we are examining other signaling molecules involved in T helper cell differentiation including SAP, a small SH2 containing adaptor protein, mutations of which are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We had previously generated mice deficient in SAP and have found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. We further showed that the impaired antibody responses in these mice was secondary to a defect in CD4+ T cells, ie SAP deficient T cells fail to provide an essential signal to B cells for generating long-term antibody responses, a critical step for the development of successful immunization and immune responses, the hallmark of successful vaccine development. Understanding the cellular interactions and signals that are defective in these mice is therefore of high importance or understanding the requirements for successful vaccine development. To understand the defect in T cells in the SAP-deficient mice, we have continuedd to examine T cell function and biochemistry of T cell activation in cells from SAP-deficient mice. We have found that T cells from SAP deficient mice show increased Th1 cytokine production (IFN-g) and dramatic defects in Th2 cytokine production (IL-4, 5, and 10) in response to TCR stimulation. Given the role of Th2 cytokines in promoting B cell help for antibody production, the defect in Th2 cytokine production provides potential new insights into the mechanism of disease in XLP. In the last year, we have made significant progress in understanding these phenotypes. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca++ mobilization as well as ERK, JNK and Vav activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation and nuclear NF-kappaB1/p50 levels. Nonetheless, nuclear levels of c-Rel, another NF-kappaB subunit, are normal. Similar defects were observed in Fyn-deficient cells. Re-expression of wildtype (WT), but not a Fyn-binding mutant of SAP rescued the defects in both PKC-theta recruitment and IL-4 production in SAP-deficient cells. Moreover, SLAM engagement increased TCR-mediated PKC-theta recruitment, nuclear p50 levels and IL-4 production in WT but not SAP-deficient T cells, suggesting a potential new role for SLAM in T cell signaling. Our data indicates that a SAP/Fyn pathway is required for the efficient recruitment of PKC-theta/Bcl-10 as well as proper patterns of activation of NF-kappaB, and suggests a potentially novel pathway of T helper 2 (IL-4) cytokine regulation. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder.
| Linterman, Michelle A; Rigby, Robert J; Wong, Raphael K et al. (2009) Follicular helper T cells are required for systemic autoimmunity. J Exp Med 206:561-76 |
| Chiang, Y Jeffrey; Jordan, Martha S; Horai, Reiko et al. (2009) Cbl enforces an SLP76-dependent signaling pathway for T cell differentiation. J Biol Chem 284:4429-38 |
| Schwartzberg, Pamela L; Mueller, Kristen L; Qi, Hai et al. (2009) SLAM receptors and SAP influence lymphocyte interactions, development and function. Nat Rev Immunol 9:39-46 |
| Steinfelder, Svenja; Andersen, John F; Cannons, Jennifer L et al. (2009) The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1). J Exp Med 206:1681-90 |
| Sahu, Nisebita; Venegas, Ana M; Jankovic, Dragana et al. (2008) Selective expression rather than specific function of Txk and Itk regulate Th1 and Th2 responses. J Immunol 181:6125-31 |
| Readinger, Julie A; Schiralli, Gillian M; Jiang, Jian-Kang et al. (2008) Selective targeting of ITK blocks multiple steps of HIV replication. Proc Natl Acad Sci U S A 105:6684-9 |
| Schwartzberg, Pamela L (2007) Formin the way. Immunity 26:139-41 |
| Konno, Akihiro; Kirby, Martha; Anderson, Stacie A et al. (2007) The expression of Wiskott-Aldrich syndrome protein (WASP) is dependent on WASP-interacting protein (WIP). Int Immunol 19:185-92 |
| Zhao, Ling; Cannons, Jennifer L; Anderson, Stacie et al. (2007) CBFB-MYH11 hinders early T-cell development and induces massive cell death in the thymus. Blood 109:3432-40 |
| Gomez-Rodriguez, Julio; Readinger, Julie A; Viorritto, Irene C et al. (2007) Tec kinases, actin, and cell adhesion. Immunol Rev 218:45-64 |
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