Our studies are focused in three main areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, and in particular, individual TCR signal transducing subunits and signal transducing motifs in T cell development. Second, we have extended our studies to include analysis of signal transducing molecules that function downstream of the TCR or that inhibit TCR signaling.
The aim of these studies is to understand how these molecules participate in TCR mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Third, we have begun to characterize the function of chemokine receptors that are expressed on developing T cells. These cell surface proteins mediate chemotaxis in response to specific ligands that are expressed in discreet regions of the thymus. Chemokine receptors are candidates for regulating homing of progenitor cells to the thymus and for regulating intrathymic migration of thymocytes.? ? Role of T cell antigen receptor (TCR) signaling in thymocyte development.? 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. 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 demonstrated 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 potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire. ? ? Role of LAT in T cell development.? 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. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, tyr235) are necessary and sufficient for LAT activity in T cells, which includes activation of the calcium and MAP Kinase (MAPK) downstream signaling pathways. These 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. Thus, their inactivation in all cells would likely result in embryonic lethality. However, by mutating specific LAT tyrosines we have been able to uncouple the TCR from downstream signaling pathways in T cells without affecting the ability of other receptors or cells to utilize these pathways. We generated 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 exhibited normal T cell development, thereby validating the targeting strategy. Inactivation of all four distal LAT tyrosines yielded a null phenotype, demonstrating the critical role of these residues for Tcell development. Surprisingly, knock-in mutation of the first tyr residue (tyr136) resulted in a fatal lymphoproliferative disorder characterized by expansion and multi-tissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds phospholipase C-gamma, examination of the signaling response of T cells from these mice revealed a severe defect in TCR induced/phospholipase C-gamma-mediated calcium flux. However, MAP Kinase signaling was intact in these cells, indicating that the TCR was selectively uncoupled from the calcium but not the MAPK pathway. These results reveal a critical role for LAT in coordinating downstream signals initiated by TCR engagement and demonstrate that this function is essential for normal T cell homeostasis. ? ? Structure and signaling potential of the gamma/delta TCR complex.? Most vertebrate species contain two separate lineages of T cells that are distinguished by the antigen binding clonotype-specific chains contained within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the more abundant alpha/beta TCR has been extensively characterized, much less is known about the structure or function of the gamma/delta TCR which is expressed on the smaller subset of gamma-delta T cells. We found that the subunit composition of the gamma/delta TCR differs from that of the alpha/beta TCR in that a component of the alpha/beta TCR, the CD3delta chain, is not present in gamma/delta TCRs. These results revealed a major difference in the subunit structure of the alpha/beta and gamma/deltaTCRs. Interestingly, signal transduction by the gamma/delta TCR was found to be superior to the alpha/betaTCR as assessed by several criteria. Our data suggest that the structural difference between alpha/beta and gamma/delta TCRs may influence the signaling potential of the TCR complex and that this may have important functional consequences on T cell activation. Indeed, recent findings indicate that TCR signal strength plays a critical role in regulating alpha/beta vs. gamma/delta lineage choice.? ? Role of the chemokine receptor CCR9 in T cell development? The ordered progression of thymocytes through distinct stages of development is also associated with migration into and between different thymus microenvironments where they are exposed to different growth factors and signals. Chemokines are a group of small, structurally related molecules that regulate trafficking of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled receptors. The chemokine CCL25 is highly expressed in the thymus and small intestine, the two known sites of T lymphopoesis. The receptor for CCL25, CCR9, is expressed on the majority of thymocytes raising the possibility that CCR9 and it ligand may play an important role in thymocyte development. To investigate the role of CCR9 during lymphocyte development, we generated CCR9-deficient (CCR9-/-) and CCR9 transgenic mice. These studies demonstrated that lymphocyte progenitors from CCR9-/- mice had a markedly reduced capacity to repopulate the thymus when forcedforced to compete with progenitor cells from CCR9+/+ mice. In other experiments, overexpression of CCR9 in transgenic mice inhibited early thymocyte development and blocked the normal migration of immature thymocytes within the thymus. These results indicate that CCR9 participates in regulating both the migration of progenitor cells to the thymus and the migration of developing thymocytes within the thymus. However, CCR9 is not essential for normal T cell development, suggesting that the possibility of functional redundancy.
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