Research is directed at understanding the cellular and genetic events that control T lymphocyte development. Current studies center on the signal transduction molecules and pathways that regulate immature T lymphocyte (thymocyte) maturation and mature T lymphocyte function. 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. 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 pathways 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 a critical role for LAT in T cell homeostasis through its ability to integrate signals downstream of the TCR. CCR9: T cell development continues into adulthood and requires the periodic migration of T-progenitor cells from the bone marrow to the thymus. The ordered progression of thymocytes through distinct stages of development is also associated with migration into and between different thymic 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. Surprisingly, both T cell and B cell development appeared normal in CCR9-/- mice. However, competitive bone marrow transplantation experiments demonstrated that CCR9-/- bone marrow cells had a markedly reduced capacity to repopulate the thymus compared to bone marrow cells from CCR9+/+ mice. Overexpression of CCR9 in transgenic mice inhibited early thymocyte development and blocked the normal migration of immature thymocytes within the thymus. These results demonstrate that CCR9 participates in regulating the migration of progenitor cells to the thymus and in regulating the migration of developing thymocytes within the thymus. Gamma/delta TCR. Most vertebrate species contain two separate lineages of T cells that are distinguished by the clonotype-specific chains contained within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the alpha/betaTCR has been extensively characterized, much less is known about the structure or function of the gamma/deltaTCR. We found that the subunit composition of the gamma/deltaTCR differs fundmentally from that of the alpha/betaTCR in that a major subunit of the alpha/beta TCR, the CD3delta chain, is not a component of the gamma/deltaTCR. Interestingly, signal transduction by the gamma/deltaTCR was consistently superior to the alpha/betaTCR as assessed by several criteria. These results demonstrate a major difference in the subunit structure of the alpha/beta and gamma/deltaTCRs. Moreover, our data suggest that this structural difference may influence the signaling potential of the TCR complex and have important functional consequences on T cell activation.
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