Signals Regulating T Cell Development

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Our studies are focused in three 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. 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. Second, we have continued studies characterizing 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. Finally, the lab has initiated a new area of investigation of the genes controlling the generation and maintenance of Hematopoietic Stem Cells. 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 previously 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. In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development. In addition, we are using microarray and subtractive cloning to identify genes involved in T cell signaling and T cell development. 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. Genes controlling Hematopoietic Stem cell specification and maintenance. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. We have recently initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis. The results of these experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggest further that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells.