Research in the Section on Cellular and Developmental Biology 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, thymocyte selection, and mature T cell function.

Role of T Cell Antigen Receptor (TCR) Signaling in Thymocyte Development
Frolova, El-Khoury, Love in collaboration with E.W. Shores (FDA)
A major focus of our research has been an investigation of the role of 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, -delta, and -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. We genetically reconstituted the 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 the alterations on TCR signaling. The results of the 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 potentially useful T cells are instructed to survive and differentiate further and by which potentially auto-reactive cells are deleted in the thymus).

Mechanism of CD5-Mediated TCR Signal Inhibition
Park, Love
CD5 has been shown to regulate TCR signaling 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 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. Overexpression 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 signaling response during development. The current 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 occurs 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.

Role of LAT in T Cell Development
Park, Love in collaboration with L. Samelson and C. Sommers (NCI)
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 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 would likely result in embryonic lethality. However, by mutating specific LAT tyrosines, it may be possible to uncouple the TCR from downstream signaling pathways in T cells without affecting the ability of other receptors to use these pathways. To explore the role of LAT-coupled signaling pathways in T cell development, 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 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 multitissue 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 the mice revealed a severe defect in TCR-induced calcium flux. However, MAP kinase signaling was intact in these cells, indicating that the TCR was specifically uncoupled from the calcium pathway. The results reveal an inhibitory role for calcium signaling in T cell homeostasis.

Structure and Signaling Potential of the Gamma/Delta TCR Complex
Hayes, Love
Most vertebrate species contain two lineages of T cells that are distinguished by the clonotypic chains contained within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the alpha/betaTCR has been well characterized, much less is known about the structure or function of the gamma/deltaTCR. We found that the subunit composition of the majority of gamma/deltaTCRs expressed on ex vivo T cells differs from that of the alpha/betaTCR in that the CD3delta chain is not a component of the gamma/deltaTCR. Nevertheless, signal transduction by the gamma/deltaTCR was at least as proficient as that by the alpha/betaTCR. Following activation and expansion of gamma/delta T cells, the gamma/deltaTCR was found to contain CD3delta. The results reveal a fundamental difference in the structure of the alpha/betaTCR and gamma/deltaTCR expressed on naïve T cells. The results also demonstrate the potential for flexibility in the subunit composition of the gamma/deltaTCR and raise the possibility that the structure and signaling capabilities of the TCR may change depending on the activation state of the T cell.

Role of CCR9 in T Cell Development
Uehara, Love in collaboration with J. Farber (NIAID)
T cell development continues into adulthood and requires the periodic migration of progenitor cells from the bone marrow to the thymus. The ordered progression of thymocytes through distinct stages of development is also associated with migration of cells into and between different thymic microenvironments. 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, thus raising the possibility that CCR9 and its ligand may be involved in regulating T cell migration within the thymus. To investigate the role of CCR9 during lymphocyte development, we generated CCR9-deficient (CCR9^-/-) mice by homologous recombination. 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 reduced capacity to repopulate the thymus compared with bone marrow cells from CCR9^+/+ mice. In addition, CCR9^-/- mice contained reduced numbers of gamma/delta T cells in the intestine. These results indicate that CCR9 is involved in regulating the migration of progenitor cells to the thymus as well as the migration of progenitor or mature gamma/delta T cells to the intestine.