The overall goal is to begin to identify and define the molecular basis of cell-mediated cytolysis. Murine allogeneic cytotoxic T lymphocytes (CTL) with high levels of activity will be used as the model system in these studies. The first approach will be to identify the molecule(s) that is susceptible to specific types of inhibitors of CTL-mediated lysis (CML). For example, we have found that inhibitors of methylation, thiol-reactive compounds and substrates and inhibitors of serine-dependent proteases block CML. CTL will be labeled intrinsically or surface iodinated and then reacted with a nonpenetrating thiol specific hapten or with a macromolecular antiprotease that binds covalently to serine-dependent proteases. The relevant molecules will then be immunoprecipitated from cell lysates using antibody to the thiolreactive hapten or to the antiprotease. Other studies will isolate the putative protease directly by preparing and using antibody to the active site of serine-dependent proteases. The molecule(s) methylated during CML will be labeled by using a radioactive methyl donor. Immunoprecipitated or methylated molecules will be anaIyzed by polyacrylamide gel electrophoresis or by chromatography if methylated phospholipids are implicated. The second approach to relate structure and function will be to attempt to define the function of molecules reactive with antieffector cell antibodies that block CML. Most antieffector antibodies do not inhibit CML, although a few such antibodies have been described. Monoclonal anti-LFA and anti-T200 antibodies, both of which block CML, will be examined to define the molecules with which they react and/or to determine the phase(s) of CML that is blocked. lt will also be determined whether these inhibitory antibodies react with any of the molecules identified in the preceding experiments examining thiols, proteases, and methylation products. (CS)
