Previous studies of the natural killer (NK) lytic mechanism have provided evidence for the stimulus secretion model and led to the postulation that lytic mediators are transferred from the effector to the target cell. Subsequent work in our laboratory has demonstrated that soluble natural killer cytotoxic factors (NKCF) can be detected in the supernatant of effector cells stimulated with NK targets. These factors are NK specific and lyse only NK sensitive targets. Further studies have shown that NKCF conform with known characteristics of the NK CMC reaction and we have postulated that NKCF plays a role in NK CMC cytotoxicity function. The following model for NK CMC was then proposed by which, following effector target cell binding, the target stimulates the effector to release NKCF. These factors then bind to and lyse the target cells. Preliminary studies have shown that NKCF are glycoproteins and can be fractionated on HPLC gel-filtration into cytotoxic fractions of molecular weight 20-30 kilodaltons. Furthermore, we have been able to generate xeno and monoclonal antibodies that can inhibit NK CMC sctivity and in a few instances neutralize NKCF activity. In the proposal we plan to: 1) further characterize and biochemically purify NKCF to homogeneity, 2) characterize antibodies (poly or monoclonal) specific for NKCF, and examine their role in NK CMC reactions, and 3) in collaboration with other investigators, compare NKCF with recently described cytolytic granules or cytotoxins and lymphotoxins derived from NK and CTL lines. We will use conventional methods of protein purification and structure analysis. Conventional and monoclonal antibodies will be generated against NKCF and will be characterized for their role in blocking NK CMC reactions. The studies proposed are significant and unique in the field of CMC. They are also clinically important since they describe naturally derived antitumor cytolytic molecules that may be relevant in immunotherapy. (LB)
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