PROGRAMMING FOR LYSIS STAGE IN NK CYTOTOXICITY

NK 细胞毒性裂解阶段的编程

• 批准号: 3185063
• 负责人: BENJAMIN BONAVIDA
• 金额: $ 14.27万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1991-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3185063
• 关键词: G protein; antibody dependent killer cell; calcium; calmodulin; flow cytometry; human tissue; laboratory mouse; laboratory rat; leukocyte activation /transformation; membrane activity; monoclonal antibody; neutrophil; phosphatidylinositols; protein kinase C; radiotracer; receptor; single cell analysis; surface antigens; tissue /cell culture; tumor necrosis factor alpha

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.

我们研究的总体目标是调查 NK细胞启动其细胞毒性的潜在机制 对敏感靶细胞的活性。 据推测， NK细胞的细胞溶解是一种分泌过程， 我们和其他人已经报道了可溶性细胞毒性介质 并被认为参与了致命打击阶段 溶解 我们制定了一个模型， NK CMC的已知特征，其中自然杀伤细胞 细胞毒性因子（NKCF）发挥作用。 我们建议研究 NK被靶细胞（TC）激活或触发阶段， 靶细胞毒性。 我们的初步调查发现 涉及多磷酸肌醇水解级联的机制 和蛋白激酶活化。 因此，本提案将 检查NK激活的途径，并启动对 NK的结构性质参与触发。 我们的研究 将在NK CMC反应和NKCF中平行进行 生产 研究将使用纯化的 NK和单细胞水平。 我们的具体目标是：（1）描述 NK细胞被NK靶细胞激活。 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，Ca++动员，蛋白激酶C 活化和磷酸化以及膜去极化。 这些研究将与NKCF的分泌或 NKCF中的抗原性TNF样分子;（2）为了定义NKCF中的抗原性TNF样分子， 参与活化的NK效应细胞膜的性质， 确定仅承认/约束是否足以 activation. 这将通过使用非溶解性 结合NK细胞、靶细胞变体和阻断性单克隆抗体 抗体的 上述研究将采用我们开发的方法进行， 纯化通过在流上分选分离的NK杀伤细胞和非杀伤细胞 细胞计数器。 此外，激活将与 使用灵敏的生物测定和放射免疫测定的分泌 NKCF和TNF的技术。