Natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and mononuclear phagocytes represent three major systems of cell-mediated cytotoxicity against tumors. In order to dissect cell surface events important in the cytolytic mechanism, monoclonal antibodies (MABs) against killer cell surfaces have been screened for the ability to block cell-mediated cytotoxicity in the absence of complement. One MAB (RH1-38) specifically blocks cytotoxicity mediated by at least three different effector cells, including NK cells, CTLs, and a monocyte-like cell (phorbal myristate acetate-stimulated HL-60). The antibody immunoprecipitates from PMS-stimulated HL-60 a bimolecular complex (195,000 and 125,000 daltons) which is biochemically similar to the LFA-1 antigen, a previously described cell surface antigen found on CTLs. However, RH1-38, unlike previously described anti-LFA-1 antibodies, does not inhibit effector-target binding. In addition, kinetic studies and single cell cytotoxicity assays have demonstrated that RH1-38 blocks a late step in the cytolytic mechanism. Thus, RH1-38 recognizes either a functionally different epitope on the LFA-1 molecule or alternatively a distinct, functionally important cell surface molecule. Using already developed techniques to determine molecular weight, subunit structure, and surface antigen density, the principal investigator proposes to test the hypothesis that differences in molecular structure and/or surface antigen density are related to cytotoxic activity. In addition, using the HL-60 cell line as a source of antigen, the molecule recognized by RH1-38 will be further characterized biochemically. A variety of other, noncytotoxic immunologic functions will be examined with respect to inhibition by this monoclonal antibody, in order to survey its effect on the immune response. As time permits, it is further proposed to: 1) examine molecular structure and antigen density on lymphocytes from select NK-deficient disease states; 2) develop methods to insert the antigen into the surface on noncytotoxic cells using antigen reconstituted into phosphatidyl choline vesicles; and 3) develop methods for exogenous cell-free translation of the mRNA that encodes for the relevant antigen. Characterization of this and other MABs and the functionally important cell surface molecules they recognize is likely to yield complementary information leading to a dissection of cell surface events important in the cytotoxic response. This will lead to a better understanding of host defense against infectious and malignant disease. (CS)
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