GENES ENCODING T-CELL RECEPTORS

编码 T 细胞受体的基因

基本信息

批准号：
2061431
负责人：
ELLIS L REINHERZ
金额：
$ 50万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-07-01 至 1997-04-30
项目状态：
已结题

项目摘要

Cell-cell interactions are important in many aspects of immune function. Contact between T lymphocytes and their cognate partners is mediated in large part by the interaction of the T cell restricted molecule CD2 with its ubiquitous ligand LFA-3. Abrogation of CD2-LFA-3 interaction severely impairs T cell mediated immune function including that of helper T cells, cytolytic T cells and natural killer cells. Furthermore, the expression of CD2 on thymocytes and LFA-3 on thymic stromal elements suggests a role for CD2-LFA-3 interaction during T lineage development. During the last funding interval, cDNA cloning of human and murine CD2 has been completed, the genomic organization and chromosome assignment of the gene loci determined, recombinant CD2 expressed, physicochemical properties of the extracellular segment and N-terminal adhesion domain investigated, and signal transduction function of the cytoplasmic tail characterized. The importance of both CD2 mediated adhesion and signal transduction function was defined for the process of T cell receptor (TCR) mediated activation. In turn, the dependency of CD2 signalling on TCR function was also uncovered. The present proposal will provide detailed structure-function analysis of the CD2 intracellular and extracellular segments. First, coupling of CD2 signalling to CD3zeta will be investigated and any physical association between CD3zeta and the CD2 cytoplasmic region determined. Milligram quantities of the CD2 cytoplasmic domain will be produced by E. coli expression for use as an affinity ligand to detect CD2 associated cytoplasmic protein and to resolve the structure of the CD2 cytoplasmic domain using 4-D NMR. Potential associations of CD2 with cytoskeletal components, particularly following ligation of the CD2 extracellular segment by LFA-3 will be investigated. Second, detailed mapping of the LFA-3 binding region on the CD2 extracellular segment will be performed by site-directed mutagenesis in conjunction with a solution structure of human CD2. The suggestion that there may be additional extracellular ligands of CD2 will be directly addressed utilizing recombinant CD2 extracellular segment. Such ligands will be tested for their ability to bind directly to CD2 ex vivo as well as by immunohistochemical analysis utilizing labeled CD2 extracellular segment. If ligands are identified, they will be cloned and their binding site on the CD2 extracellular segment defined relative to LFA-3. Finally, homologous recombination technologies will be used to make CD2-/- mice or mice expressing altered CD2 forms. The role of CD2 in thymocyte development with emphasis on repertoire formation including positive and negative selection will be determined.

细胞与细胞的相互作用在免疫功能的许多方面都很重要。 T 淋巴细胞与其同源伙伴之间的接触是通过很大程度上是由于 T 细胞限制分子 CD2 与其普遍存在的配体LFA-3。 CD2-LFA-3 相互作用的废除严重损害 T 细胞介导的免疫功能，包括辅助免疫功能 T 细胞、溶细胞 T 细胞和自然杀伤细胞。此外，胸腺细胞上 CD2 的表达和胸腺基质成分上 LFA-3 的表达提示 CD2-LFA-3 相互作用在 T 谱系发育过程中发挥作用。在最后一个资助期间，人类和小鼠 CD2 的 cDNA 克隆已经完成，基因组组织和染色体分配基因位点确定、重组CD2表达、理化胞外段和 N 端粘附结构域的特性研究了细胞质尾部的信号转导功能特点。 CD2 介导的粘附和信号的重要性转导功能被定义为T细胞受体的过程 (TCR) 介导的激活。反过来，CD2 信号传导对 TCR功能也被揭示。本提案将提供 CD2细胞内的详细结构功能分析细胞外节段。首先，CD2信号传导与CD3zeta的耦合将进行调查，CD3zeta 与确定CD2细胞质区域。 CD2 的毫克量细胞质结构域将由大肠杆菌表达产生，用作亲和配体检测 CD2 相关细胞质蛋白并使用 4-D NMR 解析 CD2 胞质结构域的结构。 CD2 与细胞骨架成分的潜在关联，特别是 LFA-3 连接 CD2 胞外片段后将调查了。其次，LFA-3结合区域的详细映射 CD2细胞外片段将通过定点进行与人类 CD2 的溶液结构相结合的诱变。这暗示可能存在额外的 CD2 细胞外配体利用重组CD2胞外片段可以直接解决该问题。将测试此类配体直接结合 CD2 ex 的能力体内以及利用标记的 CD2 进行免疫组织化学分析细胞外段。如果配体被识别，它们将被克隆以及它们在 CD2 细胞外片段上的结合位点定义为相对至 LFA-3。最后，利用同源重组技术制作 CD2-/- 小鼠或表达改变的 CD2 形式的小鼠。 CD2的作用在胸腺细胞发育中，重点是曲目的形成，包括将确定正选择和负选择。