We have identified the prototype of a previously unrecognized sublineage of T cells. These cells emerge from the fetal thymus in a succession of waves of cels bearing different V gamma TCR, migrate to certain epithelial tissue based on V gene expression, and express essentially invariant gamma delta antigen receptors. These properties suggest that the cells are generated by different mechanisms and may have different functions than those of T cells with clonally variable TCR. We have proposed that the repertoire may be directed, rather than selected, and that these cells may function in a unique form of trauma signal surveillance for evidence of cellular damage. We have directed our efforts largely towards the dendritic epidermal T cells (DEC) of the skin, which bear invariant Vgamma3/Vdelta1 TCR. We have shown that these cells undergo phenotypic maturation in the fetal thymus, but that this process does not seem to associated with repertoire selection. We have shown that these cells recognize self antigens expressed by keratinocytes, their neighbors in the skin. Our proposed studies will deal with mechanisms of generation and function of these cells. I. Specificity and function of the DEC TCR. A. We will determine whether the DEC cells mediate cytolysis of damaged keratinocytes and will develop more sensitive assays of DEC activation to facilitate aims B and C. B. We will attempt to identify the origin of the peptide recognized by the DEC by: (1) Purification and sequence analysis of a stimulatory peptide we have identified in keratinocytes; (2) Expression cloning of the gene encoding the keratinocyte antigen; and (3) Determine the relevance of murine Hsp65 genes to the recognition of stressed keratinocytes by gene transfection using a panel of murine Hsp65 CDNAS that we have cloned. Once the antigen has been identified, we will determine the sequence motifs responsible for recognition by the DEC TCR and for presentation. C. We will attempt to identify the structures involved in presentation of the antigen by; (1) Assessing the role in antigen presentation of MHC class I gene products using a combination of genetic, gene transfer, and biochemical approaches; (2) Expression cloning of the gene encoding the presenting molecule; or (30 Production of monoclonal antibodies to antigen presenting molecules. II. Intrathymic development and origin the invariant repertoire. A. We will determine the point at which RAG gene expression is extinguished during intrathymic development of Vgamma3 thymocytes. B. We will examine the effect of transgenic terminal transferase on the development of Vgamma3+ cells and on the junctional diversity of their TCR. C. We will determine the role played by selection and by sequence-specific signals in generation of the invariant repertoire by analysis of rearrangements of non-selectable recombination substrates containing wild type and mutant TCR gamma gene sequences in Vgamma3+ fetal thymocytes and DEC cells from transgenic mice.
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