LEU 2/T8 CELL DIFFERENTIATION ANTIGEN GENE

CD8是一种异源二聚体细胞表面糖蛋白，表达于成熟的 识别外源抗原的T淋巴细胞亚群与 类主要组织相容性复合体(MHC)蛋白，具有细胞毒性 功能。这项建议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分析 在这些过程中的CD8β链。首先，通过哪些机制 CD8β刺激T细胞反应将通过几种方式进行探索： 与β/2连接的I类重组可溶物的结合研究- 微球蛋白将被用来确定它是否可以结合CD8β 直接转化为I类MHC蛋白。转化子表达形式多样 CD8β和/或CD8α的作用将被用来评估CD8β在 结合亲和力的激活依赖性增加 CD8Alphabeta到I类MHC。之间的直接或间接的相互作用 CD8β和其他可能增强信号转导的表面蛋白将是 在免疫共沉淀和杂交研究中进行了分析。互动 CD8β胞浆尾部的蛋白可能参与了 通过CD8β的信号将使用酵母双杂交进行分析 系统。第二个主要目标是确定生理意义 CD8α非依赖于CD8β的表达 在胎儿和成人胸腺细胞中鉴定。这种形式的存在 CD8β将通过细胞染色和流式细胞术分析 正常和突变小鼠的胸腺细胞和外周淋巴样细胞 有和没有激活刺激。这种生物化学的本质 将探索复合体，以确定它们是否确实代表 我们观察到CD8β/TCRbeta二硫键连接的杂二聚体 转基因细胞。这种复合体的功能和细胞承载 它将通过mAb处理胎儿胸腺器官培养进行检测，通过 利用从日间分离的该种群进行胸腺重建实验 14胎儿胸腺，并通过评价信号转导能力 CD8β/TCRbeta异源二聚体。将尝试生成一个 CD8β的突变形式，不再与TCRbeta二聚化，但保留 CD8α的二聚化和功能。如果获得，这样的 突变将被用来区分这两种形式的二聚体的影响。 在已在转染体上鉴定的胸腺细胞中 似乎存在于胸腺细胞的一个子集上。这些研究将有助于 在我们对细胞毒T细胞如何被激活以杀死 病毒感染或肿瘤细胞，并可能额外提供洞察 对其发育途径的调控。

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.