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.

Signaling molecules that function downstream of the TCR or that function to "fine-tune" the TCR signal

Our results with TCR-ITAM mutant mice suggested that other signaling molecules can compensate for the reduction in TCR signal strength. An initial FACS-based search for candidate compensatory molecules led us to CD5, a TCR associated trans-membrane protein that inhibits TCR signaling. Importantly, we found that CD5 surface expression is regulated by and parallels TCR signal intensity. Thus, rather than simply functioning as a co-receptor, CD5 acts to fine-tune TCR signals during thymocyte selection since its level of surface expression depends upon the intensity of TCR signaling. An obvious benefit of such fine-tuning of the TCR signaling response would be to enable the generation of a T cell repertoire with the maximum possible diversity since it would allow a broader range of TCRs to pass through the signaling window of positive selection. Since little was known about how CD5 regulates TCR signaling, we initiated a project to characterize CD5 function, both genetically and biochemically. The results of these experiments suggest a mechanism for CD5 mediated TCR signal inhibition that we are currently testing experimentally. We have also begun a search for additional tuning molecules using a microarray based screen. The identification of such molecules may have importance for the diagnosis and treatment of human autoimmune diseases. In another study, we identified a novel T-lineage restricted putative adaptor protein, designated Themis. Biochemical studies indicate that Themis functions at a distal point in the TCR signaling pathway and may have an important role is helping to sustain TCR signaling. Themis-/- mice have been generated and their phenotype reveals an important role for this protein in late thymocyte development and selection. Current and projected experiments are directed at elucidating the mechanism by which Themis functions in T cell signaling and development.

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 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 that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells. Consistent with this, a genome-wide ChIP-seq screen identified Ldb1 binding sites within the promoter/gene body of a high percentage of genes known to be essential for HSC maintenance. Deletion of Ldb1 also resulted in decreased expression of these genes suggesting that Ldb1 complexes function as "master regulators" of the transcriptional program regulating HSC maintenance/self-renewal.

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