Our research is directed at understanding the cellular and genetic events that regulate T lymphocyte development. Current studies focus on the role of T lymphocyte signal-transducing molecules in immature T lymphocyte (thymocyte) selection, a process essential for formation of the mature T cell repertoire. To analyze the function of specific signal-transducing proteins in T lymphocyte development, we use transgenic and gene-targeting methods to create over-expression, dominant-negative, and loss-of-function mutants in mice. In addition, we are employing molecular genetic techniques to identify and characterize the function of novel genes that are expressed in T lymphocytes.

Role of T Cell Antigen Receptor Signalling in Thymocyte Development
Frolova, El-Khoury, Love; in collaboration with Shores
A major theme of our research has been the role of T cell antigen receptor (TCR) signal transduction 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, CD3-delta, CD3-epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signalling 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. We genetically reconstituted these mice with transgenes encoding wild-type or signalling-deficient (ITAM-mutant) forms of zeta and CD3epsilon and characterized the developmental and functional consequences of these alterations for TCR signalling. The results of the studies demonstrate that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification proved 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 by which 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 self-restricted T cell repertoire.

Mechanism of CD5-Mediated TCR Signal Inhibition
Park, Love
The cell surface protein CD5 has been shown to regulate TCR signalling negatively and to participate in thymocyte selection. 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 ligands in the thymus that mediate selection. 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” the TCR signalling response during development. Our results indicate that the potential for signal modulation may be particularly useful for generating the maximum possible TCR diversity in the mature T cell repertoire. Given that a probable mechanism for CD5 function involves the activation-induced binding of regulatory molecule(s) to sequences within the CD5 cytoplasmic domain, we generated transgenic mice that express a tail-less form of CD5 (mCD5). We then used both the intact and mCD5 transgenes to reconstitute CD5 surface expression in CD5-/- mice. The experiments revealed a critical function for the cytoplasmic domain in CD5 signalling. The laboratory is currently attempting to identify molecules that interact with CD5 in an effort to determine how CD5 regulates signal transduction by the TCR.

Role of LAT in T Cell Development
Park, Love; in collaboration with Samelson, Sommers, Shores
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 down-stream signalling 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) down-stream signalling pathways. These signalling 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 down-stream signalling pathways in T cells without affecting the ability of other receptors or cells to use 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 T cell development. Surpris-ingly, knock-in mutation of the first tyr residue (tyr136) resulted in a profound fatal lympho-proliferative disorder characterized by expansion and multi-tissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds to phospholipase Cgamma, examination of the signalling response of T cells from these mice revealed a severe defect in TCR-induced/phospholipase Cgamma–mediated calcium flux. However, MAP kinase signalling was intact in these cells, indicating that the TCR was specifically uncoupled from the calcium but not from the MAPK pathway. The results reveal a critical role for LAT in coordinating down-stream signals initiated by TCR engagement and demonstrate that this function is essential for normal T cell homeostasis.

Structure and Signalling Potential of the Gamma/Delta TCR Complex
Hayes, Love; in collaboration with Fowlkes, Laky
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 fundamentally from that of the alpha/betaTCR in that it lacks a major subunit of the alpha/betaTCR, the CD3delta chain. Interestingly, signal transduction by the gamma/deltaTCR was consistently superior to that by 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 the structural difference may influence the signalling potential of the TCR complex and have important functional consequences on T cell activation.

Role of the Chemokine Receptor CCR9 in T Cell Development
Uehara, Love; in collaboration with Farber
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 its ligand 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 transplan-tation experiments demonstrated that CCR9-/- bone marrow cells had a markedly reduced capacity to repopulate the thymus compared with bone marrow cells from CCR9+/+ mice. Over-expression 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 both the migration of progenitor cells to the thymus and the migration of developing thymocytes within the thymus.