The long-term objectives of this proposal are to evaluate the ontogeny and differentiation of murine dendritic Thy-1+ cells and to evaluate the possible role of skin as an organ of extrathymic T cell maturation and/or differentiation, and finally attempt to identify and propagate cells of equivalent phenotype and genotype from human skin.
The specific aims are to: 1) Evaluate the ontogeny of Thy-1+ dEC in mouse skin, and determine whether these cells are thymic-dependent or independent, and whether the Thy-1+ EC population is comprised of cells of both NK and T cell lineages; 2) Evaluate the capacities of Thy-1+ EC subpopulations to differentiate into functionally, phenotypically, and genotypically more mature cells after in vitro culture, and when placed into thymic microenvironments; 3) Determine whether the epidermal microenvironment can function as a site of extrathymic T cell maturation; and 4) Attempt to propagate and phenotypically and genotypically identify CD3+, delta/delta+ cells from human skin. A variety of techniques will be employed to address these issues, including intrathymic injection of scid mice, thymectomies of scid mice, skin grafting, in vitro thymic organ culture, epidermal explant organ culture, proliferation assays, cytotoxicity assay, FACS analysis using single and two color analysis, Northern and Southern blot analysis, and if the need arises utilize immunoprecipitation of cell-surface proteins. These studies are designed to provide further insight into the significance and immunologic role of murine Thy-1+ EC and their phenotypic, genotypic, and functional counterparts (i.e., CD3+, CD4-, CD8-, TCR alpha/beta-, delta/delta+) known to be present in human peripheral blood and thymus, and speculated to be present in human epidermis. These studies will provide additional insights into the nature of the interactions between the skin and the immune system in both normal and disease states, and may provide exciting new insights into the role of skin in both acquired and congenital immunodeficiency states.
