KILLER CELL SURFACE ANTIGENS--BIOCHEMISTRY AND FUNCTION

杀伤细胞表面抗原——生物化学和功能

• 批准号: 3174703
• 负责人: ROBERT E HALL
• 金额: $ 11.82万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3174703
• 关键词: cell mediated cytotoxicity; genetic translation; immunochemistry; membrane activity; membrane structure; mixed lymphocyte reaction test; monoclonal antibody; monocyte; neoplasm /cancer immunology; surface antigens; tissue /cell culture

Natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and mononuclear phagocytes represent three major systems of cell-mediated cytotoxicity against tumors. In order to dissect cell surface events important in the cytolytic mechanism, monoclonal antibodies (MABs) against killer cell surfaces have been screened for the ability to block cell-mediated cytotoxicity in the absence of complement. One MAB (RH1-38) specifically blocks cytotoxicity mediated by at least three different effector cells, including NK cells, CTLs, and a monocyte-like cell (phorbal myristate acetate-stimulated HL-60). The antibody immunoprecipitates from PMS-stimulated HL-60 a bimolecular complex (195,000 and 125,000 daltons) which is biochemically similar to the LFA-1 antigen, a previously described cell surface antigen found on CTLs. However, RH1-38, unlike previously described anti-LFA-1 antibodies, does not inhibit effector-target binding. In addition, kinetic studies and single cell cytotoxicity assays have demonstrated that RH1-38 blocks a late step in the cytolytic mechanism. Thus, RH1-38 recognizes either a functionally different epitope on the LFA-1 molecule or alternatively a distinct, functionally important cell surface molecule. Using already developed techniques to determine molecular weight, subunit structure, and surface antigen density, the principal investigator proposes to test the hypothesis that differences in molecular structure and/or surface antigen density are related to cytotoxic activity. In addition, using the HL-60 cell line as a source of antigen, the molecule recognized by RH1-38 will be further characterized biochemically. A variety of other, noncytotoxic immunologic functions will be examined with respect to inhibition by this monoclonal antibody, in order to survey its effect on the immune response. As time permits, it is further proposed to: 1) examine molecular structure and antigen density on lymphocytes from select NK-deficient disease states; 2) develop methods to insert the antigen into the surface on noncytotoxic cells using antigen reconstituted into phosphatidyl choline vesicles; and 3) develop methods for exogenous cell-free translation of the mRNA that encodes for the relevant antigen. Characterization of this and other MABs and the functionally important cell surface molecules they recognize is likely to yield complementary information leading to a dissection of cell surface events important in the cytotoxic response. This will lead to a better understanding of host defense against infectious and malignant disease. (CS)

自然杀伤（NK）细胞、细胞毒性T淋巴细胞（CTL）和单核细胞 吞噬细胞代表了细胞介导的细胞毒性的三个主要系统 对抗肿瘤 为了剖析细胞表面的重要事件， 细胞溶解机制，针对杀伤细胞的单克隆抗体（MAB） 已经筛选了表面阻断细胞介导的 在缺乏补体的情况下的细胞毒性。 一种MAB（RH 1 -38）， 阻断由至少三种不同效应细胞介导的细胞毒性， 包括NK细胞、CTL和单核细胞样细胞（肉豆蔻酸佛波酯 乙酸盐刺激的HL-60）。 抗体免疫沉淀来自 PMS刺激的HL-60 a双分子复合物（195，000和125，000道尔顿） 其在生物化学上类似于LFA-1抗原， 细胞表面抗原。 然而，RH 1 -38与以前不同， 描述的抗LFA-1抗体，不抑制效应子-靶标 约束力 此外，动力学研究和单细胞细胞毒性试验 已经证明RH 1 -38阻断了细胞溶解的晚期步骤， 机制 因此，RH 1 -38识别功能上不同的表位 LFA-1分子上，或者可替代地， 细胞表面分子 使用已经开发的技术来确定分子量，亚基 结构和表面抗原密度，首席研究员提出， 为了检验分子结构和/或 表面抗原密度与细胞毒活性有关。 此外，本发明还提供了一种方法， 使用HL-60细胞系作为抗原来源，该分子识别 RH 1 -38将进一步进行生物化学表征。 其他各种各样， 将检查非细胞毒性免疫功能， 抑制这种单克隆抗体，以调查其对 免疫反应。 如果时间允许，还建议：1） 检查淋巴细胞的分子结构和抗原密度， NK缺陷性疾病状态; 2）开发将抗原插入 使用抗原将非细胞毒性细胞的表面重建成 磷脂酰胆碱囊泡; 3）开发外源性 编码相关抗原的mRNA的无细胞翻译。 该MAB和其他MAB以及功能重要的细胞的表征 它们识别的表面分子很可能产生互补的 导致细胞表面事件的解剖的信息， 细胞毒性反应 这将有助于更好地了解主机 防御传染病和恶性疾病。 （中文）