A combined biochemical and immunologic approach will be applied to the differentiation of human cytolytic T lymphocytes (CTL) in culture. A major aim is to identify a human cytokine termed cytolytic differentiation factor (CDF), which synergizes with IL-2 to induce the formation of CTL in a 2 day, polyclonal, thymocyte bioassay. Preliminary experiments have pinpointed several procedures which will be used to enrich the factor and produce an initial panel of anti-CDF polyclonal and monoclonal Ab. Human cDNA libraries have been prepared from the mRNA of mitogen-stimulated, T cells. The mRNA translates active CDF in Xenopus oocytes. The cDNA library will be screened by differential hybridization with CDF-rich and CDF-depleted mRNA probes and with polyclonal anti-CDF antibodies. Candidate cDNA's will be tested for their capacity to hybridize mRNA that is active in the oocyte assay. Active cDNA's will generate full length probes as well as DNA/amino acid sequence information. The immunologic studies will pursue initial evidence on the important accessory role of dendritic cells (DC) in stimulating alloreactive CTL. In addition to triggering CD4+ helper cells, DC seem to bind alloreactive CD8+ cells directly to generate clusters of CD8+ lymphoblasts. We will verify that an anti CD4 MAb does not block the formation of CD8+ blasts. Then we will determine the lymphokine requirements for differentiating the blasts, and memory cells derived from the blasts, into active CTL. To monitor differentiation in the MLR and in the polyclonal CDF assay (above), we will develop and test antibodies that are specific for CTL granule constituents. This laboratory has purified granule perforin and serine esterase and has shown that perforin is a pore forming protein with immunologic cross reactivity to anti-human C9. The data on the accessory and lymphokine requirements of alloreactive CD8+ cells will be applied to the generation of human immunodeficiency virus (IDV)-specific CTL. We will stimulate the CD8+ subset from patients with AIDS. We will add appropriate accessory cells, cytokines, and different sources of antigen such as infected H-9 cells and IDV particles. CTL will be tested for function in retarding productive infection of CD4+ cells in vitro. IDV-specific CTL may provide an important resistance mechanism which is compromised in AIDS when accessory and/or helper T cell function is altered.
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