Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒性机制

• 批准号: 6762133
• 负责人: Pierre A Henkart
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6762133
• 关键词: CD95 molecule; apoptosis; cell mediated cytotoxicity; cell nucleus; cytotoxic T lymphocyte; endopeptidases; enzyme activity; enzyme mechanism; exocytosis; granule; protease inhibitor; serine proteinases; tissue /cell culture; transfection; zymogens

The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of the preformed potent lytic agent perforin (as well as other mediators) into the synapse-like space between the cytotoxic lymphocyte and its bound target. However, the question of why the effector cell does not itself die of perforin damage has often been raised without a clear answer. We have recently proposed that transiently expressed surface cathepsin B is provides such self protection to cytotoxic T cells and NK cells. Two major lines of evidence support this model: 1) When triggered to degranulate in the presence of membrane impermeant cathepsin B-specific inhibitors, cytotoxic lymphocytes undergo a rapid perforin-dependent, FasL-independent death; 2) A proteolytically active form of cathepsin B is expressed on the surface of cytotoxic T cells after degranulation. Since cathepsin B was previously shown by others to be expressed on the surface of tumor cells, we have tested whether it may be responsible for resisting CTL lethal damage in vitro. A series of in vitro tumor lines was screened to determine if pretreating them with membrane impermeant cathepsin B-specific inhibitors renders them more susceptible to redirected CTL-induced death. Most non-lymphoid tumors seem to be >10x less lysable than lymphoid tumors such as Jurkat after equivalent CTL degranulation. In some cases pre-treatment with CA074 causes a marked increase in lysability. We have seen this behavior with some but not all melanoma and colon tumor cells, and are currently screening a variety of available tumor lines. We previously showed that the cytoplasmic protein Rab27a was required for a late step in granule exocytosis in cytotoxic lymphocytes, but its functional molecular partners remain undefined. We are currently exploring immunoprecipitation-based approaches to identify a putative melanophilin homolog operating in CTL. Cathepsin W is a novel cathepsin originally described as an expressed sequence tag and subsequently found in mRNA expression studies to be expressed exclusively in cytotoxic lymphocytes (both T and NK cells). In order to study the protein and probe its function, we expressed human pre-procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots of CTL, some of these react with a single 25-30kd band, a size expected for active cathepsin W. When these mAb were used to localize cathepsin W, a granular pattern was seen by fluorescence microscopy. We are also examining the expression of granule proteins in naive and memory subpopulations of human blood T lymphocytes phenotypically defined by CCR7 and CD45RO expression. These studies show that memory subsets of both CD4+ and CD8+ express granule markers, which are minimally expressed in naive T cells.

淋巴细胞介导的细胞毒性的颗粒吐出模型假定抗原触发的预形成的有效裂解剂穿孔素(以及其他介质)快速分泌到细胞毒性淋巴细胞与其结合靶细胞之间的突触样间隙中。然而，为什么效应细胞本身不会死于穿孔素损伤的问题经常被提出，但没有明确的答案。我们最近提出，瞬时表达的表面组织蛋白酶B对细胞毒性T细胞和NK细胞具有这样的自我保护作用。支持这一模型的主要证据有两条：1)当在膜上不必要的组织蛋白酶B特异性抑制剂存在的情况下被触发脱颗粒时，细胞毒性淋巴细胞经历快速的穿孔素依赖的、非FasL非依赖的死亡；2)脱颗粒后的细胞毒性T细胞表面表达一种具有蛋白水解性的组织蛋白酶B。由于组织蛋白酶B以前被发现表达在肿瘤细胞表面，我们已经测试了它是否可能在体外抵抗CTL致命性损伤。对一系列体外肿瘤株进行筛选，以确定是否用膜无意义的组织蛋白酶B特异性抑制剂处理它们会使它们更容易受到CTL诱导的重定向死亡的影响。在同等的CTL脱颗粒后，大多数非淋巴样肿瘤的溶解能力似乎比淋巴样肿瘤(如Jurkat)少10倍。在某些情况下，用CA074进行前处理会导致溶解性显着增加。我们已经在一些但不是所有的黑色素瘤和结肠肿瘤细胞中看到了这种行为，目前正在筛选各种可用的肿瘤株。 我们之前的研究表明，细胞毒性淋巴细胞颗粒胞吐的晚期需要胞质蛋白Rab27a，但其功能分子伴侣仍未确定。我们目前正在探索基于免疫沉淀的方法来鉴定CTL中可能存在的亲黑素同源物。 组织蛋白酶W是一种新的组织蛋白酶，最初被描述为一种表达序列标签，后来在mRNA表达研究中发现，它仅在细胞毒淋巴细胞(T细胞和NK细胞)中表达。为了研究该蛋白并探讨其功能，我们在大肠杆菌中表达了人Pre-Proathepsin W，并制备了一系列抗Pre-Prothepsin W的单抗。在CTL的Western blotts中，其中一些细胞与一条25-30kd的条带反应，这是活性组织蛋白W的预期大小。当用这些单抗定位组织蛋白W时，荧光显微镜下可见颗粒状图案。 我们还检测了由CCR7和CD45RO表达所定义的人类血液T淋巴细胞的幼稚和记忆亚群中颗粒蛋白的表达。这些研究表明，CD4+和CD8+的记忆亚群都表达颗粒标记，而颗粒标记在幼稚T细胞中表达最少。