PROGRAMMING FOR LYSIS STAGE IN NK CYTOTOXICITY

我们研究的总体目标是调查 NK细胞启动其细胞毒作用的潜在机制 针对敏感目标细胞的活性。有传闻称， NK细胞的细胞溶解是一个分泌过程，最近 我们和其他人已经报道了可溶性细胞毒介质。 并被推测参与了致命的打击阶段 解体。我们已经制定了一个模型，它占到了 NK CMC的已知特征以及在哪些自然杀手中 细胞毒因子(NKCF)起一定作用。我们建议研究 靶细胞(TC)激活或触发NK的阶段导致 靶细胞细胞毒性。我们的初步调查结果表明 涉及多聚磷肌醇水解级联反应的机理 和蛋白激酶的激活。因此，这项提案将 研究NK细胞活化途径，启动对NK细胞活性的研究 触发因素所涉及的NK结构的性质。我们的研究 将在NK CMC反应和NKCF中同时进行 制作。将对提纯的亚群进行研究 NK和单细胞水平。 我们的具体目标是(1)表征最初的途径 NK靶细胞对NK细胞的激活作用这将包括 GTP结合蛋白的作用，磷酸二酯酶的激活， 磷脂酰肌醇途径、钙动员、蛋白激酶C 激活和磷酸化，以及膜去极化。 这些研究将与NKCF或 NKCF中抗原性的肿瘤坏死因子样分子；(2)定义 参与活化的NK效应细胞膜的性质 确定仅识别/绑定是否足以 激活。这将通过使用非Lytic 结合NK细胞、靶细胞变异体和阻断单抗 抗体。 上述研究将使用我们开发的方法进行，以 流动分选法分离纯化NK杀伤细胞和非杀伤细胞 细胞仪。此外，激活将与 灵敏生物测定法和放射免疫测定法测定分泌物 治疗NKCF和肿瘤坏死因子的技术。

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.