The overall objective of our studies is to investigate the underlying mechanisms by which NK cells initiate their cytotoxic activity against sensitive target cells. It has been implicated that cytolysis by NK cell is a secretory process and more recently soluble cytotoxic mediators have been reported by us and others and have been postulated to be involved in the lethal hit stage of lysis. We have formulated a model which accounts for most known characteristics of NK CMC and in which natural killer cytotoxic factors (NKCF) play a role. We propose to examine the activation or trigger stage of NK by target cells (TC) resulting in target cell cytotoxicity. Our preliminary findings have identified a mechanism involving the polyphosphoinositol hydrolysis cascade and protein kinase activation. Accordingly, this proposal will examine pathways of NK activation and initiate studies on the nature of structures of NK involved in the trigger. Our studies will be done in parallel in both the NK CMC reaction and NKCF production. Studies will be done with purified sub-populations of NK and at the single cell level. Our specific aims are (1) To characterize the initial pathway of activation of NK cells by NK target cells. This will include the role of GTP binding proteins, phosphodiesterase activation, the phosphatidylinositol pathway, Ca++ mobilization, protein kinase C activation and phosphorylation, and membrane depolarization. These studies will be correlated with secretion of NKCF or antigenically TNF-like molecules in NKCF; (2) To define the nature of NK effector cell membranes involved in activation and determine whether recognition/binding alone is sufficient for activation. This will be accomplished by using non-lytic conjugating NK cells, target cell variants and blocking monoclonal antibodies. The studies above will be done with a method developed by us to purify NK killer and non-killer cells separated by sorting on a flow cytometer. Furthermore, activation will be correlated with secretion using sensitive bioassay and radioimmunoassay techniques for NKCF and TNF.