LEU-2 T/8 T CELL DIFFERENTIATION ANTIGEN GENE

本申请的主要目的是进一步阐明 CD 8在免疫应答中的作用，并确定其分子机制 参与调节这种蛋白质的表达。 细胞-细胞 结合试验将用于分析人CD 8 和I类主要组织相容性复合体（MHC）蛋白。 的 CD 8 α β异二聚体结合I类分子的能力将 与CD 8 Alpha同二聚体进行比较。 I类的结合位点 将比较CD 8 α β异二聚体和 CD 8AlphaAlpha同源二聚体。 不同形式的相对能力 CD 8（AlphaAlpha，Alpha'Alpha，AlphaBeta，Alpha'Beta）刺激T细胞 通过测量白细胞介素-2的释放来评估激活 （IL-2）和CD 3 ζ链的磷酸化，以响应 抗原刺激，以及激活诱导的结合类 I MHC蛋白。 这些研究将在I类特异性T 细胞杂交瘤，其中CD 8和T细胞受体可以结合到 相同的MHC蛋白，和II类限制性T细胞杂交瘤，其中 CD 8和T细胞受体只能与单独的MHC蛋白结合。 嵌合CD 8分子，其中配体结合部分由以下组成： 将构建CD 8 β二聚体，并测定其 在功能上与I类MHC蛋白相互作用。 小鼠CD 8 α和 已知CD 8 β基因在小鼠基因组中相距36 kb。 将尝试通过以下方式连接人CD 8 α和CD 8 β基因： 染色体步移 正确的组织特异性所需的序列， 人CD 8亚群特异性和阶段特异性表达将 使用天然人CD 8 α和CD 8 β基因鉴定， 转基因小鼠和组织培养细胞中的报告基因系统。

The main goals of this application are to further elucidate the function of CD8 in immune responses and to define the molecular mechanisms involved in the regulation of expression of this protein. A cell-cell binding assay will be used to analyze the interaction between human CD8 and class I major histocompatibility complex (MHC) proteins. The ability of CD8AlphaBeta heterodimers to bind to class I molecules will be compared to that of CD8Alpha homodimers. The binding site on class I MHC proteins for CD8 will be compared for CD8AlphaBeta heterodimers and CD8AlphaAlpha homodimers. The relative abilities of distinct forms of CD8 (AlphaAlpha, Alpha'Alpha, AlphaBeta, Alpha'Beta) to stimulate T cell activation will be assessed by measuring the release of interleukin-2 (IL-2) and the phosphorylation of the CD3 zeta chain in response to antigen stimulation, as well as the activation-induced binding to class I MHC proteins. These studies will be done both in a class I-specific T cell hybridoma, in which CD8 and the T cell receptor can bind to the same MHC protein, and in a class IIrestricted T cell hybridoma, in which CD8 and the T cell receptor can only bind to separate MHC proteins. Chimeric CD8 molecules in which the ligand binding portion consists of CD8Beta dimers will be constructed and assayed for their ability to interact functionally with class I MHC proteins. The mouse CD8Alpha and CD8Beta genes are known to be located 36 kb apart in the mouse genome. An attempt will be made to link the human CD8Alpha and CD8Beta genes by chromosomal walking. The sequences required for proper tissue-specific, subset specific and stage-specific expression of human CD8 will be identified using the native human CD8Alpha and CD8Beta genes in transgenic mice and a reporter gene system in tissue culture cells.