LEU 2/T8 CELL DIFFERENTIATION ANTIGEN GENE

LEU 2/T8 细胞分化抗原基因

• 批准号: 2684799
• 负责人: JANE R PARNES
• 金额: $ 19.12万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684799
• 关键词: CD3 molecule; CD8 molecule; MHC class I antigen; T cell receptor; biological signal transduction; cell differentiation; chemical binding; cytotoxic T lymphocyte; flow cytometry; fluorimetry; gene expression; gene mutation; genetically modified animals; immunoprecipitation; laboratory mouse; leukocyte activation /transformation; monoclonal antibody; site directed mutagenesis; spleen; thymus; tissue /cell culture; yeast two hybrid system

CD8 is a heterodimeric cell surface glycoprotein expressed on the mature T lymphocyte subset that recognizes foreign antigen in association with class I major histocompatibility complex (MHC) proteins and has cytotoxic function. The main goal of this proposal is to further elucidate the role of CD in T cell activation and development by analysis of the function of the CD8beta chain in these processes. First, the mechanisms by which CD8beta stimulates T cell responses will be explored in several ways: Binding studies to a recombinant, soluble for of class I linked to beta/2- microglobulin will be used to determine whether CD8beta it can bind directly to class I MHC proteins. Transfectants expressing various forms of CD8beta and/or CD8alpha will be used to assess the role of CD8beta in the activation-dependent increase in the avidity of binding of CD8alphabeta to class I MHC. The direct or indirect interaction between CD8beta and other surface proteins that might enhance signaling will be analyzed in coimmunoprecipitation and blotting studies. The interaction of the CD8beta cytoplasmic tail with proteins that may be involved in signaling through CD8beta will be analyzed using the yeast two-hybrid system. A second major aim is to determine the physiological significance of CD8alpha-independent expression of CD8beta that we have previously identified in fetal and adult thymocytes. The presence of such forms of CD8beta will be analyzed by cell staining and flow cytometric analysis of thymocytes and peripheral lymphoid cells in normal and mutant mice, both with and without activation stimuli. The biochemical nature of such complexes will be explored to determine whether they indeed represent the CD8beta/TCRbeta disulfide-linked heterodimers we have observed in transfected cells. The function of such a complex and the cells bearing it will be examined by mAb treatment of fetal thymic organ cultures, by thymic reconstitution experiments using this population isolated from day 14 fetal thymus and by evaluation of the signaling capacity of CD8beta/TCRbeta heterodimers. An attempt will be made to generate a mutant form of CD8beta that no longer dimerizes with TCRbeta but retains dimerization and functional capacity with CD8alpha. If obtained, such a mutant would be used to distinguish effects of these two forms of dimers during thymocyte that has been identified on transfectants and that appears to be present on a subset of thymocytes. These studies will aid in our understanding of how cytotoxic T cells are activated to kill virally infected or tumor cells and may additionally provide insight into the regulation of their developmental pathway.

CD 8是一种异源二聚体细胞表面糖蛋白，在成熟的 T淋巴细胞亚群，识别与 I类主要组织相容性复合体（MHC）蛋白，并具有细胞毒性 功能 本提案的主要目的是进一步阐明 CD在T细胞活化和发育中的作用 在这些过程中的CD 8 β链。 第一， CD 8 β刺激T细胞反应将以几种方式进行探索： 与β/2-连接的I类重组可溶性for的结合研究 微球蛋白将用于确定其是否可以结合CD 8 β 直接与I类MHC蛋白结合。 表达各种形式的转染子 将使用CD 8 β和/或CD 8 α来评估CD 8 β在 结合亲和力的活化依赖性增加， CD 8 α与I类MHC结合。 直接或间接的互动 CD 8 β和其他可能增强信号传导的表面蛋白质将被 在免疫共沉淀和印迹研究中分析。 的相互作用 CD 8 β胞质尾区的蛋白质可能参与了 通过CD 8 β信号将使用酵母双杂交分析 系统 第二个主要目的是确定 CD 8 β的CD 8 α非依赖性表达， 在胎儿和成人胸腺细胞中鉴定。 这种形式的存在 将通过细胞染色和流式细胞术分析CD 8 β， 正常和突变小鼠的胸腺细胞和外周淋巴细胞， 有和没有激活刺激。 这种生物化学性质 复合体将被探索，以确定它们是否确实代表了 我们已经观察到CD 8 β/TCR β二硫键连接的异源二聚体， 转染细胞 这种复合物的功能和细胞轴承 将通过胎胸腺器官培养物的mAb处理， 胸腺重建实验使用从第一天分离的该群体 14胎胸腺，并通过评估 CD 8 β/TCR β异二聚体。 将尝试生成一个 不再与TCR β二聚化，但保留 二聚化和与CD 8 α的功能能力。 如果获得， 突变体将用于区分这两种形式的二聚体的影响 在胸腺细胞中，已经在转染子上鉴定出， 似乎存在于胸腺细胞的子集上。 这些研究将有助于 在我们理解细胞毒性T细胞如何被激活 病毒感染的细胞或肿瘤细胞，并且还可以提供对 调节它们的发育途径。