Mechanism of Lymphocyte-Mediated Cytotoxicity

淋巴细胞介导的细胞毒性的颗粒胞外分泌模型假设抗原触发预先形成的强效裂解剂穿孔素（以及其他介质）快速分泌到细胞毒性淋巴细胞与其结合靶标之间的突触样空间。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对细胞毒性T细胞和NK细胞提供这种自我保护。支持这一模型的两个主要证据：1)当在膜外组织蛋白酶b特异性抑制剂的存在下触发脱颗粒时，细胞毒性淋巴细胞经历快速的穿孔依赖，fasl独立死亡；2)组织蛋白酶B的一种蛋白水解活性形式在细胞毒性T细胞脱颗粒后的表面表达。由于先前已有研究表明组织蛋白酶B在肿瘤细胞表面表达，因此我们在体外测试了它是否可能负责抵抗CTL致死性损伤。筛选了一系列体外肿瘤系，以确定用膜外组织蛋白酶b特异性抑制剂预处理它们是否使它们更容易受到重定向ctl诱导的死亡。在同等的CTL脱粒后，大多数非淋巴性肿瘤的溶解性似乎比淋巴性肿瘤（如Jurkat）低10倍。在某些情况下，用CA074进行预处理可显著提高溶出度。我们已经在一些但不是全部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系。

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.