The overall objective of the proposed research is to contribute to the better understanding of the function of lymphocytes expressing the gammadelta T-cell receptor (TCR), particularly subsets found in ectodermally-derived tissues, the epidermis and the adjacent mammary glands which are exposed to the external environment. How these T cells function in relation to cell-mediated immunity as a whole is unknown.
The specific aims of this proposal are: (1) To study the reactivity of murine gammadelta TCR+ epidermal T cells (ETC) and mammary gland T cells (MTC) to various normal and tumor cells with and without cell stress. Taking advantage of collections of non-clonal and clonal cell lines and hybridomas derived from the murine epidermis and lactating mammary gland and transgenic mouse spleen cells, expressing TCR identical to ETC, we will optimize the conditions necessary for these cells to be activated by normal cells, e.g. keratinocytes, melanocytes, and mammary stromal cells, and by tumor cells, e.g. squamous cell carcinoma, melanoma, mammary carcinoma, and embryonic cell lines. Attempts to increase the lymphocyte reactivity to these target cells will be performed by stressing the cells with heat shock, ultraviolet light, and heavy metals. (2) To isolate and characterize in molecular terms naturally processed endogenous self antigens that are recognized specifically by epidermal and mammary gland gammadelta TCR+ cells. We plan to purify endogenous small molecular weight (<3000 D) acid-eluted cell material from a variety of cell sources that specifically stimulate ETC and MTC and obtain general characteristics using mass spectrometry and obtain sequence information using microsequencing methods should the material prove to be a peptide. (3) To determine if MHC class I, class I-related, or class II molecules present endogenous self antigens to epidermal and mammary gland gammadelta TCR+ cells. The application of several methods, such as blocking class I and class II molecules with monoclonal antibodies, assessing reactivity to cells from various mouse strains expressing different sets of presenting molecules, and using cell lines transgenic for various antigen presenting molecules, will provide the means to characterize the purported class I or class II molecules recognized by ETC and MTC. (4) To develop an assay system for epidermal and mammary gammadelta T cell function using partially reconstituted severe combined immunodeficiency (scid) mice. Taking advantage of the availability of large numbers of relatively homogeneous gammadelta T cells from T cell clones and from gammadelta TCR+ transgenic mice, we will attempt to generate mice with defined gammadelta reconstitution patterns and then attempt to study immune responses of gammadelta T cell subsets to specific antigens in vivo as well as to analyze the possible effects to gammadelta T cells on other cell populations.
