Research is directed at understanding the cellular and genetic events that control T cell development. Current studies center on the signal transduction molecules and pathways that regulate thymocyte maturation and mature T cell function. The T cell antigen receptor (TCR). A major focus of research in the lab has been investigating TCR signal transduction in thymocyte development. TCR signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. Though conserved, ITAM sequences are nonidentical, raising the possibility that the diverse developmental and functional responses controlled by the TCR may be regulated, in part, by distinct ITAMs. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies demonstrate that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was found to be critical for thymocyte selection (the process by which auto-reactive cells are deleted in the thymus). CD5. Other cell surface structures can influence the TCR signaling response. The analysis of one such molecule, CD5, which has been shown to negatively regulate TCR signaling and to participate in thymocyte selection, constitutes another area of investigation in the laboratory. Examination of CD5 expression during T cell development revealed that surface levels of CD5 are regulated by TCR signal intensity and by the affinity of the TCR for selecting ligands. To determine if the ability to regulate CD5 expression is important for thymocyte selection, we generated transgenic mice that constitutively express high levels of CD5 throughout development. Over-expression of CD5 significantly impaired positive selection of some thymocytes (those that would normally express low levels of CD5) but not others (those that would normally express high levels of CD5). These findings support a role for CD5 in modulating TCR signal transduction and thereby influencing the outcome of thymocyte selection. The ability of individual thymocytes to regulate CD5 expression represents a mechanism for """"""""fine tuning"""""""" of the TCR signaling response during development. The current results indicate that this potential for signal modulation may be particularly useful for generating the maximum possible TCR diversity in the mature T cell repertoire. Since a probable mechanism for CD5 function is via the activation-induced binding of regulatory molecule(s) to sequences within the CD5 cytoplasmic domain, transgenic mice that express a tail-less form of CD5 (mCD5) were generated. Both the intact and mCD5 transgenes were then used to reconstitute CD5 surface expression in CD5-/- mice. These experiments revealed a critical function for the cytoplasmic domain in CD5 signaling. The laboratory is currently attempting to identify molecules that interact with CD5 and that may be involved in regulating signal transduction by the TCR. LAT. Linker for Activation of T cells (LAT) is an integral membrane protein that functions as a critical adaptor linking the T cell antigen receptor (TCR) to multiple downstream signaling pathways required for T cell activation. LAT-deficient cell lines exhibit defects in activation of the two major signaling patways in T cells: the PLC-gamma mediated calcium pathway and the Ras/MAP Kinase (MAPK) pathway. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, tyr235) are necessary and sufficient for LAT activity in T cells and function by recruiting downstream effectors. The calcium and MAPK signaling pathways are also activated by a large number of other receptors and are required for the development and function of many different cell types. Because these signaling pathways function to regulate cellular events unrelated to TCR signaling, their inactivation would likely result in embryonic lethality or pleiotropy. Significantly, the four LAT tyrosines exhibit preferential binding to specific effector molecules, and mutation of different residues in cell lines results in distinct biochemical and signaling consequences. These observations suggested that by mutating specific LAT tyrosines it may be possible to uncouple the TCR from downstream signaling pathways in T cells without effecting the ability of other receptors to utilize these pathways. To explore the role of LAT-coupled signaling pathways in T cell development, we generated in collaboration with Dr. L. Samelson's group (NCI)""""""""knock-in"""""""" mutant mice that express LAT proteins containing single or multiple tyrosine-phenylalanine mutations of the four critical tyrosine residues. Knock-in mice that express the wild-type version of the protein exhibit normal T cell development thereby validating the targeting strategy. Conversely, inactivation of all four distal LAT tyrosines yielded a null phenotype, demonstrating the critical role of these residues for T cell development. Surprisingly, knock-in mutation of the first tyr residue (tyr136) resulted in a profound fatal lymphoproliferative disorder characterized by expansion and multi-tissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds PLC-gamma, examination of the signaling response of T cells from these mice revealed a severe defect in TCR induced calcium flux. However, MAPK signaling was intact in these cells, indicating that the TCR was specifically uncoupled from the calcium pathway. These results reveal an inhibitory role for calcium signaling in T cell homeostasis.
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