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: 3734660
• 负责人: C REYNOLD VERRET
• 金额: --
• 依托单位: CLARK ATLANTA UNIVERSITY
• 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/3734660
• 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 细胞毒性T淋巴细胞（CTL）通过其自身的孔形成 蛋白质穿孔素 穿孔蛋白是主要的裂解效应分子， 高度激活的CTL和自然杀伤细胞 CTL高度 抗自身溶细胞颗粒或分离的 穿孔素。 调解这种阻力的因素的性质尚未得到证实。 定义了 其主要目的是表征表达的基因和蛋白质， 保护它们免受穿孔素介导的裂解的CTL。 小鼠肿瘤细胞 已经用CTL衍生的cDNA转染，并且已经被选择用于 对穿孔素的抗性。 来自CTL衍生的cDNA文库的基因， 保护受体细胞免受穿孔素的侵害将被鉴定、测序和 分析了 为了产生抗保护性的单克隆抗体， 蛋白质，仓鼠细胞系将类似地用鼠 CTL cDNA，并针对穿孔素抗性进行选择。 细胞系将 用于免疫同类仓鼠。 作为另一种方法，来自CTL膜的蛋白质将被分级分离， 重组为脂质体。 保护脂质体的蛋白质组分 将进行进一步的分级分离。 因此，将纯化保护性蛋白。 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 保护性CTL，尤其是那些特异性 在CTL膜中表达。 穿孔素与敏感细胞系和耐药CTL的结合将 被衡量。 穿孔素异构化并插入靶膜。 的 插入率和通道形成率将比较 敏感和抗性细胞。 这种测量方法对于 了解抵抗机制。 细胞毒性T淋巴细胞对于根除病毒感染至关重要。 感染的细胞和可能的许多瘤形成。 CTL的作用机制 介导的细胞溶解和细胞可以抵抗CTL攻击的手段是 这一努力的广泛目标。