Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒性的颗粒胞吐模型假设抗原触发的预先形成的有效溶解剂穿孔素（以及其他介质）快速分泌到细胞毒性淋巴细胞和其结合靶之间的突触样空间。然而，为什么效应细胞本身不会因穿孔素损伤而死亡的问题经常被提出，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为细胞毒性T细胞和NK细胞提供了这种自我保护。两个主要证据支持该模型：1）当在膜不渗透性组织蛋白酶B特异性抑制剂存在下触发脱颗粒时，细胞毒性淋巴细胞经历快速穿孔素依赖性、FasL非依赖性死亡; 2）脱颗粒后，细胞毒性T细胞表面表达组织蛋白酶B的蛋白水解活性形式。由于组织蛋白酶B先前被其他人证明在肿瘤细胞表面表达，我们已经测试了它是否可能负责抵抗体外CTL致死性损伤。筛选了一系列体外肿瘤细胞系，以确定用膜不透性组织蛋白酶B特异性抑制剂预处理是否使它们更容易受到重定向CTL诱导的死亡的影响。大多数非淋巴样肿瘤在等效的CTL脱粒后似乎比淋巴样肿瘤如Jurkat的可裂解性低> 10倍。在某些情况下，用CA 074预处理引起裂解性的显著增加。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细胞系。 我们以前表明，细胞质蛋白Rab 27 a所需的颗粒胞吐在细胞毒性淋巴细胞的后期步骤，但其功能的分子伴侣仍然不确定。我们目前正在探索免疫沉淀为基础的方法，以确定一个假定的亲黑素同系物在CTL中运作。 组织蛋白酶W是一种新的组织蛋白酶，最初被描述为表达序列标签，随后在mRNA表达研究中发现仅在细胞毒性淋巴细胞（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前组织蛋白原W。在CTL的Western印迹中，其中一些与25- 30 kd的单一条带反应，这是活性组织蛋白酶W的预期大小。当这些单克隆抗体用于定位组织蛋白酶W时，通过荧光显微镜观察到颗粒图案。 我们还研究了由CCR 7和CD 45 RO表达定义的人类血液T淋巴细胞表型幼稚和记忆亚群中颗粒蛋白的表达。这些研究表明，CD 4+和CD 8+的记忆亚群表达颗粒标志物，其在初始T细胞中表达最低。

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.