Lymphocytes from one individual will stimulate those of another into mitosis when the cells are incubated together in culture. This mixed lymphocyte response (MLR) may result in the generation of cytotoxic effector T cells, and is controlled by genes in the major histocompatibility complex (MHC). Products of the HLA-D locus have been postulated to induce mitosis, while HLA-A, B, and C gene products are proposed to serve as target antigens for cytotoxic cells. The object of the work proposed here is to determine the true binding specificity of responder lymphocytes before or during the MLR, using specific probes to detect antigen binding cells. The probes will be purified cell membrane molecules solubilized by detergents or by proteolysis with papain, and anti-idiotypic sera directed against HLA antibodies. Probe molecules will be labeled externally with fluorescein isothiocyanate or 125I-Bolton-Hunter reagent, and membrane molecules will also be internally labeled with 14C- or 3H-amino acids. Ultimate objectives include quantitating antigen binding T and B-cells, determining their specificity for different membrane molecules, including products of the MHC such as HLA antigens and Ia-like B-cell alloantigens, purifying such cell populations using anti-fluorescein affinity columns and the fluorescence-activated cell sorter, and characterizing the membrane receptors of the antigen-binding cells. We will also investigate the antigen specificity of cytotoxic cells generated to hapten-modified self-antigens using similar techniques. The overall aim is to more clearly understand the structure and function of gene products of the MHC and their role in tumor and transplant rejection.