ROLE OF SLP-76 IN THYMIC DEVELOPMENT AND FCR FUNCTION

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

The adapter protein SLP-76 is expressed in T (but not B) lymphocytes and in myeloid cells and is a substrate for the CD3zeta associated protein tyrosine kinase ZAP-70 and Syk. SLP- 76 interacts with Vav, Grb2, the Fyn binding protein FYB130 and may link surface receptors that contain ITAM motifs (TCR, FcgammaR, FcepsilonRI) to the Ras, JNK and Rho GTPases signaling pathways. We recently generated SLP-76 -/- mice. They exhibit impaired viability, hemorrhaging and a profound block in thymic development with complete absence of double positive (DP) CD4+8+ thymocytes and of peripheral T cells, despite ongoing TCRbeta rearrangement. This block could not be overcome by in vivo treatment with anti-CD3 mAb, indicating that SLP-76 collects all pre-TCR signals that drive the development and expansion of DP thymocytes. Monocytes, NK cells, mast cells and platelets were present in SLP-76-/- indicating that SLP-76 is not essential for the development of these lineages. To better understand the role of SLP-76 in T cell development and in signaling via the TCR and related receptors that use the ITAM containing FcR-gamma chain, we propose to 1. Analyze the role of SLP-76 in thymocyte development. We will a) examine the role of SLP-76 in TCRbeta allelic exclusion by assessing the effect of introduction of a TCRbeta transgene on rearrangement of the endogenous TCRbeta locus in SLP-76-/- mice and assessing endogenous TCRbeta locus rearrangement in SLP-76-/- mice by single cell PCR analysis; b) analyze the role of SLP-76 in thymocyte development. We will a) determine which of the domains and motifs of SLP-76 are essential for thymocyte development by reconstituting SLP-76-/- mice with transgenes that encode WT SLP-76 and SLP-76 deletion and point mutants; b) determine the role of discrete SLP-76 domains and motifs in thymocyte development by assessing maturation, expansion, allelic exclusion, negative and positive selection in SLP-76-/- mice reconstituted with transgenes that code for SLP-76 mutants. 2. Determine the role of the SLP-76 linked signaling pathways in thymocyte development by reconstituting SLP-76-/- mice with transgenes that encode activated forms of Ras, Raf calcineurin and Vav. 3. Determine the role of SLP-76 in signal transduction by receptors that use the FcRgamma chain. We will examine a) FcepsilonnRI dependent anaphylaxis and FcepsilonRI mediated activation of mast cells from SLP-76-/- mice; b) FcgammaR function in macrophages and NK cells from SLP-76-/- mice; c) Glycoprotein VI collagen receptor triggering of platelet activation in SLP-76-/- mice. The results of these studies should lead to a better understanding of T cell development and signaling via TCRs and FcRgamma chain-containing receptors in hematopoietic cells and have important implications for the pathogenesis and treatment of autoimmune, allergic and neoplastic diseases.