GENERATION OF CYTOLYTIC T CELLS IN MAN

人类溶细胞 T 细胞的生成

• 批准号: 3814612
• 负责人: ANGELA GRANELLI-PIPERNO
• 金额: --
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3814612
• 关键词: AIDS; CD4 molecule; CD8 molecule; antibody; antibody dependent killer cell; autoradiography; cell aggregation; cell differentiation; clone cells; electrofocusing; esterase; gel electrophoresis; genetic library; genetic manipulation; helper T lymphocyte; high performance liquid chromatography; human immunodeficiency virus 1; human subject; hybridomas; interleukin 2; ion exchange chromatography; laboratory mouse; laboratory rabbit; lymphokines; mixed lymphocyte reaction test; molecular cloning; monoclonal antibody; monocyte; nucleic acid probes; pore forming protein; radiotracer; suppressor T lymphocyte; thymus

A combined biochemical and immunologic approach will be applied to the differentiation of human cytolytic T lymphocytes (CTL) in culture. A major aim is to identify a human cytokine termed cytolytic differentiation factor (CDF), which synergizes with IL-2 to induce the formation of CTL in a 2 day, polyclonal, thymocyte bioassay. Preliminary experiments have pinpointed several procedures which will be used to enrich the factor and produce an initial panel of anti-CDF polyclonal and monoclonal Ab. Human cDNA libraries have been prepared from the mRNA of mitogen-stimulated, T cells. The mRNA translates active CDF in Xenopus oocytes. The cDNA library will be screened by differential hybridization with CDF-rich and CDF-depleted mRNA probes and with polyclonal anti-CDF antibodies. Candidate cDNA's will be tested for their capacity to hybridize mRNA that is active in the oocyte assay. Active cDNA's will generate full length probes as well as DNA/amino acid sequence information. The immunologic studies will pursue initial evidence on the important accessory role of dendritic cells (DC) in stimulating alloreactive CTL. In addition to triggering CD4+ helper cells, DC seem to bind alloreactive CD8+ cells directly to generate clusters of CD8+ lymphoblasts. We will verify that an anti CD4 MAb does not block the formation of CD8+ blasts. Then we will determine the lymphokine requirements for differentiating the blasts, and memory cells derived from the blasts, into active CTL. To monitor differentiation in the MLR and in the polyclonal CDF assay (above), we will develop and test antibodies that are specific for CTL granule constituents. This laboratory has purified granule perforin and serine esterase and has shown that perforin is a pore forming protein with immunologic cross reactivity to anti-human C9. The data on the accessory and lymphokine requirements of alloreactive CD8+ cells will be applied to the generation of human immunodeficiency virus (IDV)-specific CTL. We will stimulate the CD8+ subset from patients with AIDS. We will add appropriate accessory cells, cytokines, and different sources of antigen such as infected H-9 cells and IDV particles. CTL will be tested for function in retarding productive infection of CD4+ cells in vitro. IDV-specific CTL may provide an important resistance mechanism which is compromised in AIDS when accessory and/or helper T cell function is altered.

生物化学和免疫学相结合的方法将是 在人溶细胞T淋巴细胞分化中的应用 (CTL)在培养中。一个主要目标是识别一种人类细胞因子 称为细胞溶解分化因子(CDF)，它协同 用IL-2在2天内诱导CTL的形成，多克隆， 胸腺细胞生物测定。初步实验已经准确地确定了 将使用几个程序来丰富因子和 制备抗CDF多克隆和单抗的初步试板 阿布。 从人脑脊髓瘤病毒的mRNA中制备了人的cDNA文库 有丝分裂原刺激的T细胞。该基因翻译活性CDF 在非洲爪哇的卵母细胞中。文库将通过以下方式进行筛选 CDF富含和CDF缺失的差异杂交 MRNA探针和多克隆抗CDF抗体。 候选的cDNAs将接受杂交能力测试 在卵母细胞试验中活跃的mRNA。活性基因的遗嘱 生成全长探针以及DNA/氨基酸序列 信息。 免疫学研究将寻求关于 树突状细胞(DC)在刺激中的重要辅助作用 异体反应性CTL。除了触发CD4+辅助细胞外， DC似乎直接结合同种异体反应性CD8+细胞产生 成簇的CD8+淋巴母细胞。我们将证实一种抗CD4抗体 单抗不能阻断CD8+母细胞的形成。那我们就会 确定区分淋巴因子的要求 母细胞和源自母细胞的记忆细胞变为活跃 CTL. 监测MLR和多克隆CDF的分化 化验(如上)，我们将开发和测试符合以下条件的抗体 专用于CTL颗粒成分。这个实验室有 纯化的穿孔素和丝氨酸酯酶颗粒 穿孔素是一种与免疫交叉的成孔蛋白 对抗人C9的反应性。 关于辅助性和淋巴因子需求的数据 同种异体反应性CD8+细胞将用于生成 人类免疫缺陷病毒(IDV)特异性CTL。我们会 刺激艾滋病患者的CD8+亚群。我们将添加 适当的辅助细胞、细胞因子和不同来源的 抗原，如受感染的H-9细胞和IDV颗粒。CTL将 被测试是否具有延缓CD4+的生产性感染的功能 体外培养的细胞。IDV特异性CTL可能提供一种重要的 当艾滋病患者的耐药机制受到损害时 辅助和/或辅助T细胞功能改变。