LEU 2 T/8 T CELL DIFFERENTIATION ANTIGEN GENE

LEU 2 T/8 T 细胞分化抗原基因

• 批准号: 2177689
• 负责人: JANE R PARNES
• 金额: $ 22.1万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177689
• 关键词: CD3 molecule; CD8 molecule; MHC class I antigen; MHC class II antigen; beta globulin; cell cell interaction; chemical binding; chimeric proteins; chromosome walking; cytotoxic T lymphocyte; fusion gene; gene expression; genetically modified animals; histocompatibility gene; human genetic material tag; hybridomas; interleukin 2; laboratory mouse; leukocyte activation /transformation; phosphorylation; reporter genes; site directed mutagenesis; tissue /cell culture

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.

本申请的主要目的是进一步阐明其功能 CD8在免疫应答中的作用并确定其分子机制 参与了该蛋白的表达调控。细胞-细胞-细胞 结合实验将用于分析人CD8之间的相互作用 和I类主要组织相容性复合体(MHC)蛋白。这个 CD8AlphaBeta异二聚体与I类分子结合的能力将 与CD8Alpha同源二聚体比较。I类上的结合位点 CD8的MHC蛋白将与CD8AlphaBeta异二聚体和 CD8AlphaAlpha同源二聚体。不同形式的人的相对能力 CD8(AlphaAlpha、Alpha‘Alpha、AlphaBeta、Alpha’Beta)刺激T细胞 将通过测量白细胞介素2的释放来评估激活 (IL-2)和CD3 Zeta链的磷酸化 抗原刺激以及激活诱导的与类的结合 I MHC蛋白。这些研究将在I类特定的测试中进行 细胞杂交瘤，其中CD8和T细胞受体可与 相同的MHC蛋白，在II类限制性T细胞杂交瘤中，其中 CD8和T细胞受体只能与不同的MHC蛋白结合。 配体结合部分由以下组成的嵌合CD8分子 CD8Beta二聚体将被构建并检测它们的能力 与I类MHC蛋白在功能上相互作用。鼠标CD8Alpha和 已知CD8Beta基因在小鼠基因组中相距36kb。 将尝试通过以下方式将人类CD8Alpha和CD8Beta基因连接起来 染色体行走。适当的组织特异性所需的序列， 人CD8的亚集特异性和阶段特异性表达 利用天然人CD8Alpha和CD8Beta基因鉴定 转基因小鼠和组织培养细胞中的报告基因系统。