The objective of this proposal is to clarify factors influencing T cell differentiation in the skin nd in the thymus, through analysis of a normal resident of mouse epidermis called Thy-1+ dendritic epidermal cells (DEC). Thy-1+ DEC express T cell antigen receptors (TCRs) similar to those on minor populations of adult human and mouse T cells, i.e., heterodimeric products of two rearranging genes named gamma and delta. All AKR/J Thy-1+ DEC clones examined to date display remarkable homogeneity in the composition of their gamma/delta TCRs, and these TCRs are indistinguishable from those on a large portion of fetal thymocytes at d15 of gestation but not at later times. A major hypothesis being tested is that the similarities between Thy-1+ DEC and d15 thymocytes reflect similarities in the microenvironments in which these populations differentiate.
The specific aims are: 1) Clarify the extent of and reason(s) for restricted heterogeneity of gamma/delta TCRs in Thy-1+ DEC. Strategies include use of monoclonal antibodies (Abs) to epitopes on the gamma/delta TCR to stain cells in situ and in suspension, and analysis of TCR gene rearrangement/expression in hybridomas made by fusing Thy-1+ DEC to a thymoma line. 2) Define factors affecting the differentiation of Thy-1+ DEC in order to clarify the relationships of gamma/delta+ cells in skin and thymus to each other and to other T cells. Strategies include chronic in vivo inoculation of Abs to antigens (e.g., Ia or TCR gamma/delta epitopes) whose expression may be critical for normal differentiation, and analysis of the staining profiles of skin of normal and nude mice with Abs to cell surface and cytoskeletal components in order to clarify the epithelial defect in nude mice manifested by thymic dysgenesis and abnormal skin which does not support the development of normal Thy-1+ DEC. 3) Investigate functional heterogeneity in freshly isolated and cloned Thy-1+ DEC. Strategies include examining their secretion of, and responsiveness to lymphokines known to affect other T cells, whether their non-MHC-restricted cytotoxic activity correlates with their activation state and whether it can be replaced by alloantigen-specific killing by altering the conditions used in their growth/selection. The proposed studies should provide insights into the heterogeneity of Thy-1+ DEC, their relationships to other T cells, factors that affect their differentiation, and the ligands for their gamma/delta receptors. In the process, the proposed studies should contribute to a better understanding of the factors that regulate the growth and differentiation of T cells, including the role(s) of epithelial cells in that process.
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