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.

我们研究的总体目标是调查 NK细胞启动细胞毒性的潜在机制 针对敏感靶细胞的活性。 据称， 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性介质 并被认为参与了致命打击阶段 裂解。 我们制定了一个模型，该模型占大多数 NK CMC 的已知特征以及其中自然杀手 细胞毒因子（NKCF）发挥作用。 我们建议审查 靶细胞 (TC) 激活或触发 NK 阶段，导致 靶细胞的细胞毒性。 我们的初步调查结果已确定 涉及多磷酸肌醇水解级联的机制 和蛋白激酶激活。 因此，本提案将 检查 NK 激活途径并启动相关研究 参与触发的 NK 结构的性质。 我们的研究 将在 NK CMC 反应和 NKCF 中​​并行进行 生产。 研究将用纯化的亚群进行 NK 和单细胞水平。 我们的具体目标是（1）描述初始路径 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 激活和磷酸化，以及膜去极化。 这些研究将与 NKCF 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 确定仅识别/结合是否足以 激活。 这将通过使用非裂解来完成 结合 NK 细胞、靶细胞变体并阻断单克隆 抗体。 上述研究将采用我们开发的方法来完成 通过流式分选分离纯化 NK 杀伤细胞和非杀伤细胞 细胞仪。 此外，激活将与 使用灵敏的生物测定和放射免疫测定进行分泌 NKCF 和 TNF 技术。