The proposed research will delineate the detailed cellular and molecular mechanisms by which tumor-induced suppressor T (Ts) lymphocytes subvert the host immune response to tumors. We have developed a model using the in vitro generation of syngeneic cytolytic T lymphocytes specific for the P815 mastocytoma from the spleens of immune and """"""""early"""""""" tumor-bearing host (TBH) mice. We found that spleen T cells from mice with advanced tumors specifically suppress the response of immune but not of """"""""early"""""""" TBH spleen cells. The proposed studies will expand this model to: 1) Characterize the surface phenotypes of these Ts cells. We will use monoclonal antibodies against Thyl and Lyt markers and the I-J subregion of the histocompatibility complex to eliminate lymphocyte populations by complement-mediated lysis or to select them with """"""""panning"""""""" techniques. 2) Determine whether a Ts cascade mediates Ts function. By combining this phenotyping data with cell separation and mixing experiments, we will determine whether suppression is mediated by primed suppressor-effector cells. 3) Test the sensitivity (in vitro and in vivo) of activated tumor-specific Ts cells to manipulations such as irradiation, treatment with alkylating agents or steroids, reduction of tumor load, or addition of exogenous IL-2. 4) Determine the role of secreted soluble mediators (TsF's) in tumor-specific suppression. Ts cells will be cultured with or without tumor cells and the supernatant fluids tested for suppression of CTL generation to determine whether secretion of TsF occurs constitutively or needs to be stimulated by antigen. The specificity of TsF will be tested by adsorption to tumor cell membranes, and the presence of I-J determinants will be tested by adsorption to affinity columns. 5) Characterize tumor-specific Ts hybridomas that secrete monoclonal TsF. After fusion of Ts cells from TBH's with the HAT-sensitive, T-cell lymphoma BW5147.G.1.4 and selection of hybrid cell lines, supernatant fluids will be screened for TsF in the in vitro CTL induction model. Positive cell lines will be cloned by limiting dilution. Ts hybridomas and monoclonal TsF will be characterized and compared to TsF secreted by TBH spleen cells. This material will greatly facilitate much of the research described above and in future investigations could be used as probes to determine the nature of suppressogenic epitopes on tumor cells or to produce monoclonal anti-TsF antibodies. The proposed research, by delineating the details of tumor-specific Ts cell function, should lead to more rational methods for modulating host immune responses to tumors.
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