PROGRAMMING FOR LYSIS STAGE IN NK CYTOTOXICITY

NK 细胞毒性裂解阶段的编程

• 批准号: 3185061
• 负责人: BENJAMIN BONAVIDA
• 金额: $ 21.65万
• 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/3185061
• 关键词: calcium; flow cytometry; human tissue; leukocyte activation /transformation; membrane activity; monoclonal antibody; neutrophil; phosphatidylinositols; protein kinase C; radiotracer; 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.

我们研究的总体目的是调查 NK细胞启动其细胞毒性的基本机制 针对敏感靶细胞的活性。 这意味着 NK细胞的细胞解析是一个分泌过程，最近 美国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的热门阶段 裂解。 我们已经制定了一个模型，该模型是大多数的 NK CMC的已知特征和自然杀手 细胞毒性因子（NKCF）起作用。 我们建议检查 靶细胞（TC）激活或触发NK的触发阶段，导致 靶细胞细胞毒性。 我们的初步发现已经确定 一种涉及多磷酸肌醇水解级联反应的机制 和蛋白激酶激活。 因此，该提议将 检查NK激活的途径并开始研究 触发涉及的NK结构的性质。 我们的研究 将在NK CMC反应和NKCF中并行完成 生产。 研究将通过纯化的子选集进行 NK和单细胞水平。 我们的具体目的是（1）表征 NK靶细胞激活NK细胞。 这将包括 GTP结合蛋白的作用，磷酸二酯酶激活， 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）定义 NK效应细胞膜的性质参与激活和 确定单独识别/约束是否足以 激活。 这将通过使用非乐谱来实现 结合NK细胞，靶细胞变异和阻断单克隆 抗体。 上面的研究将以我们开发的方法来完成 通过对流进行排序，净化NK杀手和非杀手细胞 细胞仪。 此外，激活将与 使用敏感的生物测定和放射免疫分泌物的分泌 NKCF和TNF的技术。