GENERATION OF CYTOLYTIC T CELLS IN MAN

人类溶细胞 T 细胞的生成

• 批准号: 3810048
• 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/3810048
• 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; histocompatibility; 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 应用于人溶细胞性T淋巴细胞的分化 (CTL)在文化中。 一个主要目的是鉴定一种人类细胞因子 称为溶细胞分化因子（CDF），它协同 用IL-2诱导CTL的形成，多克隆， 胸腺细胞生物测定 初步实验已经确定 将用于富集因子的几个程序， 产生抗CDF多克隆和单克隆抗体的初始组 AB. 人cDNA文库已经从以下基因的mRNA制备： 有丝分裂原刺激的T细胞。 mRNA翻译活性CDF 在非洲爪蟾卵母细胞中。 将通过以下方法筛选cDNA文库： 富含和缺失CDF的差异杂交 mRNA探针和多克隆抗CDF抗体。 将测试候选cDNA的杂交能力 在卵母细胞测定中有活性的mRNA。 主动cDNA的意志 产生全长探针以及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 同种异体反应性CTL 除了触发CD 4+辅助细胞外， DC似乎直接结合同种异体反应性CD 8+细胞， CD 8+淋巴母细胞簇。 我们将验证抗CD 4抗体 MAb不阻断CD 8+母细胞的形成。 那么我们将 确定区分淋巴细胞的淋巴因子要求， 原始细胞和来源于原始细胞的记忆细胞， CTL. 监测MLR和多克隆CDF中的分化 检测（上述），我们将开发和测试抗体， 对CTL颗粒成分具有特异性。 本实验室曾 纯化的颗粒穿孔素和丝氨酸酯酶，并且已经表明， 穿孔素是一种具有免疫交叉性的成孔蛋白 对抗人C9的反应性。 关于辅助和淋巴因子需求的数据 同种异体反应性CD 8+细胞将被应用于产生 人免疫缺陷病毒（IDV）特异性CTL。 我们将 刺激来自艾滋病患者的CD 8+亚群。 我们将添加 适当的辅助细胞、细胞因子和不同来源的 抗原如感染的H-9细胞和IDV颗粒。 CTL将 测试其在延缓CD 4+的生产性感染中的功能 体外细胞 IDV特异性CTL可提供重要的免疫应答。 艾滋病的抵抗机制， 辅助和/或辅助T细胞功能改变。