ANALYSIS OF T CELL RECEPTOR FUNCTION BY GENE TRANSFER

通过基因转移分析 T 细胞受体功能

• 批准号: 3134558
• 负责人: Anjana Rao
• 金额: $ 16.8万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3134558
• 关键词: Retroviridae; T cell receptor; T lymphocyte; arsenic; chimeric proteins; clone cells; complementary DNA; cytochrome c; electrophoresis; gene expression; gene mutation; genetic manipulation; genetic markers; genetic transduction; haptens; hybridomas; immunochemistry; immunoglobulin genes; laboratory mouse; leukocyte activation /transformation; ligands; major histocompatibility complex; membrane proteins; messenger RNA; molecular cloning; mutagens; mutant; nucleic acid hybridization; nucleic acid probes; oligonucleotides; plasmids; point mutation; protein sequence; radioassay; site directed mutagenesis; surface antigens; transfection; transposon /insertion element

Activation of helper/inducer T lymphocytes initiates the immune response to foreign pathogens, and triggers the processes of transplant rejection and the onset of autoimmune disease. Activation occurs when these cells recognise foreign antigen in association with histocompatibility proteins on the surface of antigen-presenting cells. To be able to clinically manipulate the immune response, it is important to understand the mechanisms of antigen/MHC recognition and activation. The long-term objectives of this research are to analyse the detailed structure of antigen/MHC receptors from selected T cell clones. Arsonate-reactive inducer T cell clones will be used as a model system, since the functions of their receptors can be assayed both with antigen-binding assays and with assays for activation. Receptor genes from these clones will be transferred into other T cells using retroviral vectors, and the recipient cells will be tested for arsonate responsiveness. In future studies site-directed mutagenesis will be used to define receptor residues involved in antigen/MHC recognition and subsequent activation. The specific aims of this proposal are to subclone receptor genes of the arsonate-reactive inducer T cell clone Ar-5 into pZIP retroviral vectors, transfect them into Psi-2 and Psi-AM packaging cell lines, infect recipient T cells with recombinant retroviruses using a cocultivation procedure, and test the infected cells for acquisition of arsonate responsiveness. Two kinds of recipient cells will be used: individual genes will be transferred into antigen-unresponsive variants of clone Ar-5, which are likely to have lost expression or function of one receptor chain; and both receptor chains will be transferred into the unrelated inducer T cell clone BCC 11, which shares neither antigen nor MHC responsiveness with clone Ar-5. Acquisition of a functional clone Ar-5 receptor will be tested by arsonate binding, activation by arsonate plus I-Ad, binding and activation by clone-specific antibodies, and expression of receptor polypeptides with the righy electrophoretic characteristics.

辅助/诱导T淋巴细胞的激活启动免疫应答， 外来病原体，并触发移植排斥反应的过程， 自身免疫性疾病的发病 当这些细胞 识别与组织相容性蛋白相关的外来抗原 在抗原呈递细胞的表面。 能够在临床上 操纵免疫反应，重要的是要了解 抗原/MHC识别和激活的机制。 本研究的长期目标是分析 来自所选T细胞克隆的抗原/MHC受体的结构。 将使用胂酸盐反应性诱导剂T细胞克隆作为模型系统， 因为它们的受体的功能可以用 抗原结合试验和活化试验。 受体基因 这些克隆将通过逆转录病毒转移到其他T细胞中， 载体，和受体细胞将被测试的纵火 响应能力。 在未来的研究中，将使用定点突变 确定参与抗原/MHC识别的受体残基， 后续激活。 该建议的具体目标是亚克隆受体基因的 将胂酸盐反应性诱导剂T细胞克隆Ar-5导入pZIP逆转录病毒载体， 将其分别转染到Psi-2和Psi-AM包装细胞系中， - 使用共培养程序将T细胞与重组逆转录病毒一起培养，和 测试受感染的细胞是否获得对胂酸盐的反应。 两 将使用多种受体细胞：单个基因将被 转移到克隆Ar-5的抗原无应答变体中， 可能失去一条受体链的表达或功能; 受体链将转移到无关的诱导T细胞克隆中 BCC 11与克隆BCC 11既不共享抗原也不共享MHC反应性， Ar-5 功能性克隆Ar-5受体的获得将通过 胂酸盐结合，胂酸盐加I-Ad激活，结合和激活 通过克隆特异性抗体，和受体多肽的表达， 具有良好的电泳特性。