The experiments in the present application are directed toward resolving: 1.) The mechanism of CTL-mediated lysis. The mechanism by which cytotoxic T lymphocytes kill specific target cells in still unknown. A study of lysis in lectin-mediated cytolysis (LDCC) led us to propose a new mechanism for CTL-mediated cytolysis, in which target cell death occurs as a result of distortion of target cell MHC proteins. Leakage at the interface between the transmembrane MHC proteins and surrounding lipids is postulated to lead to irreversible osmotic damage. Experiments are included in this proposal to test our hypothesis. Antibodies to MHC proteins, other transmembrane proteins, non-transmembrane proteins, and purely lipid antigens will be coupled to the surface of inert microspheres, microtiter wells, RBC or other nonlytic cell types. These will then be incubated with 51Cr-labeled target cells, and the rate of 51Cr release monitored. 2.) The relationship of lectin-mediated polyclonal activation of CTL function to LDCC and to antigen activation of CTL function. Certain plant lectins have been used both to activate (CTL function, and to mediate lysis in cases where MHC-restricted recognition is presumed not to occur. We have recently shown that target cell lysis in LDCC is in fact MHC-restricted, and we have obtained preliminary data indicating that lectin-mediated, polyclonal generation of CTL function is also controlled by MHC proteins involved in lectin presentation. We propose experiments to explore this point further, and to analyze its significance for CTL function generally. Using specific MHC antibodies, appropriate H-2 recombinant and congenic strains, and MHC positive and negative cell lines, we will probe the involvement of MHC proteins in activation of CTL function in primary and secondary reactions, in both syngeneic and allogeneic combination. We will also clone out individual CTL generated by lectin and compare their properties will CTL generated by specific antigen.
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