CELLULAR RESISTANCE TO T CELL MEDIATED CYTOLYSIS

细胞对 T 细胞介导的细胞溶解的抵抗

• 批准号: 5211993
• 负责人: C REYNOLD VERRET
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211993
• 关键词: autoimmunity; binding proteins; cell mediated cytotoxicity; cellular immunity; crosslink; cytolysins; cytolysis; cytotoxic T lymphocyte; gene expression; hamsters; laboratory mouse; membrane proteins; nucleic acid sequence; polymerase chain reaction; pore forming protein; protein purification; protein reconstitution; radiotracer; transfection

The immediate objective of this project is to elucidate the resistance of cytotoxic T lymphocytes (CTLs) to lysis by their own pore-forming protein, perforin. Perforin is the principal lytic effector molecule of highly activated CTLs and natural killer cells. CTLs are highly resistant to lysis by their own cytolytic granules or by isolated perforin. The nature of factors mediating this resistance has not been defined. The primary objective is to characterize genes and proteins expressed by CTLs that protect them from perforin-mediated lysis. Murine tumor cells have been transfected with a CTL-derived cDNA and have been selected for resistance to perforin. Genes from the CTL-derived cDNA library that protect recipient cells from perforin will be identified, sequenced and analyzed. In order to generate monoclonal antibodies against protective proteins, hamster cell lines will be similarly transfected with murine CTL cDNA and selected for resistance to perforin. The cell lines will be used to immunize congenic hamsters. As another approach, protein from CTL membrane will be fractionated and reconstituted into liposomes. Protein fractions that protect liposomes from perforin will be subjected to further rounds of fractionation. Thus, protective protein(s) will be purified. CTL membrane proteins that associate with perforin will be characterized by their cross-linking with perforin. Protein that bind perforin may be protective of CTLs, especially those polypeptides that are specifically expressed in CTL membranes. Binding of perforin to sensitive cell lines and to resistant CTLs will be measured. Perforin isomerizes and inserts into target membrane. The rate of insertion and channel formation will be compared between sensitive and resistant cells. Such measurements are useful for understanding mechanisms of resistance. Cytotoxic T lymphocytes are essential for the eradicating of virally infected cells and possibly of many neoplasia. The mechanisms of CTL mediated cytolysis and the means by which cell can resist CTL attack are the broad objectives of this effort.

这个项目的直接目标是阐明抗药性 细胞毒性T淋巴细胞(CTL)通过自身的孔道形成而溶解 蛋白质，穿孔素。穿孔素是主要的裂解效应分子 高度激活的CTL和自然杀伤细胞。CTL是一种高度 抗自身细胞溶解颗粒或分离的裂解 穿孔素。调解这种抵抗的因素的性质并不是 已定义。 其主要目标是确定由 保护它们免受穿孔素介导的裂解的CTL。小鼠肿瘤细胞 已被CTL来源的cDNAs导入，并已被选择用于 对穿孔素的耐药性。来自CTL衍生的cDNA文库的基因 将对保护受体细胞免受穿孔素的影响进行鉴定、测序和 分析过了。为了产生抗保护性的单抗 蛋白质，仓鼠细胞系将类似的小鼠基因 CTL基因，并对穿孔素进行抗性筛选。这些细胞系将 用于免疫同源仓鼠。 作为另一种方法，CTL膜中的蛋白质将被分级和 重组成脂质体。保护脂质体的蛋白质组分 从穿孔素中分离出来的成分将经过进一步的分馏。 这样，保护蛋白(S)就得到了提纯。 与穿孔素相关的CTL膜蛋白将被表征 通过它们与穿孔素的交联。结合穿孔素的蛋白质可能是 对CTL的保护作用，尤其是那些具有特异性的多肽 在CTL膜中表达。 穿孔素与敏感细胞系和耐药CTL的结合将 被测量。穿孔素异构化并插入靶膜。这个 插入速率和通道形成速度将在以下情况下进行比较 敏感和耐药细胞。这种测量对于以下方面很有用 了解抗药性的机制。 细胞毒性T淋巴细胞是根除病毒感染的关键 被感染的细胞，可能还有许多肿瘤。细胞毒性T淋巴细胞的作用机制 介导的细胞溶解和细胞抵抗CTL攻击的手段是 这项努力的总体目标。