<p><p>LAT is a transmembrane adaptor protein that is critical for the emanation of signals downstream of the TCR. Following TCR engagement, LAT is phosphorylated on multiple tyrosine residues, allowing it to serve as a scaffold for a multi-protein signaling complex. Mutation of tyrosine 136 on LAT abrogates binding of PLC-&#947;1. The disruption of this interaction has severe consequences on TCR-mediated calcium signaling and MAPK activation. Mice harboring a mutation at this tyrosine, LATY136F (LAT<super>m/m</super>) mice, have drastically impaired thymocyte development; however, CD4<super>+</super> T cells in the periphery rapidly expand and instigate a fatal lymphoproliferative syndrome. In order to bypass the severe developmental defects exhibited in LAT<super>m/m</super> mice, our laboratory previously developed a conditional knock-in mouse line in which the mutated LAT allele is expressed in mature T cells following deletion of a floxed wildtype LAT allele (ERCre<super>+</super>LAT<super>f/m</super> mice). LAT<super>f/m</super> mice develop a similar lymphoproliferative syndrome as LAT<super>m/m</super> mice. We used both of these mouse models to analyze the contribution of two other proteins that are essential for TCR-mediated signaling, RasGRP1 and Gads, in LAT-mediated autoimmunity. </p><p><p>Analysis of LAT<super>m/m</super>RasGRP1<super>-/-</super> mice demonstrated that the additional deletion of RasGRP1 increased the thymocyte development block and, as a result, young mice contained markedly reduced T cell populations. However, by four months of age, a lymphoproliferative disease had developed in these mice. To bypass the severe developmental block, we analyzed LAT<super>f/m</super>RasGRP1<super>-/-</super> mice and observed that they developed disease similarly to LAT<super>f/m</super> mice. We also assessed the effect of Gads deletion in both mouse models of LAT disease. LAT<super>m/m</super>Gads<super>-/-</super> mice had an even more dramatic block in the DN stage of thymocyte development compared to LAT<super>m/m</super> controls, although by four months of age CD4<super>+</super> T cells had expanded. Following deletion of the wildtype LAT allele, LAT<super>f/m</super>Gads<super>-/-</super> mice also developed disease. Our results indicated that LAT-mediated autoimmunity can occur independently of the critical T cell signaling components RasGRP1 and Gads. </p><p><p>In addition, we more closely examined RasGRP1-mediated Erk activation in T cells. RasGRP1 is a Ras-guanyl nucleotide exchange factor that is required for positive selection of thymocytes, activation of T cells, and control of T cell mediated-autoimmunity. While the importance of various RasGRP1 structural domains has previously been explored, RasGRP1 also contains a tail domain of unknown function. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain, RasGRP1<super>d/d</super> mice. Analysis of these mice demonstrated that deletion of the tail domain led to impaired T cell development but, with age, CD4<super>+</super> T cells expanded and auto-antibodies were produced. RasGRP1<super>d/d</super> thymocytes were unable to activate Erk and underwent aberrant thymic selection processes. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, our data revealed the physiological relevance of the tail domain of RasGRP1 in the control of Erk signaling.</p> / Dissertation

In T cells, signalling events initiated at the immunological synapse by T cell receptor (TCR) have been already thoroughly studied. In contrast, signalling pathways involved in MHCII-mediated signal transduction on the APC side of this structure are still rather poorly understood. Transmembrane adaptor proteins (TRAPs) are a class of proteins with an essential role in signalling triggered by TCR and other immunoreceptors. So far no TRAPs involved in MHCII signalling have been identified. In this work a new TRAP, highly enriched in the immunological synapse in APCs is described and termed Nvl. Nvl is expressed exclusively in professional antigen presenting cells - B cells, monocytes/macrophages and dendritic cells, and the level of its expression positively correlates with the expression of MHCII. Nvl is present in tetraspanin microdomains and together with tetraspanins it is localized to the polarized structures of the cell, including uropod, cleavage furrow, and, most interestingly, the immunological synapse. The presence in the immunological synapse indicates possible role for Nvl in MHCII- mediated signal transduction. Indeed, Nvl becomes tyrosine-phosphorylated after stimulation via MHCII, although strong phosphorylation has also been observed after FcγR stimulation or treatment with...