Target Cell Death by Cytotoxic Lymphocytes

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。我们最近进行了一系列实验，旨在测试一种新的自我保护模型，该模型提出效应细胞膜上的半胱氨酸蛋白酶在插入的穿孔蛋白聚集和形成孔之前使其失活。我们测试了这个模型，询问是否1)在细胞毒性淋巴细胞表面检测到活性半胱氨酸蛋白酶，以及2)半胱氨酸蛋白酶抑制剂阻断自我保护并导致细胞毒性淋巴细胞脱颗粒诱导的死亡。我们用生物素化组织蛋白酶亲和试剂和蛋白抑制剂半胱抑素B标记了细胞毒性T淋巴细胞表面的活性半胱氨酸蛋白酶，随后的流式细胞术分析表明，通过这种方法可以在细胞毒性T淋巴细胞上检测到半胱氨酸蛋白酶，并且在TcR触发时它们会增加。它们可能参与自我保护的证据来自于这些组织蛋白酶抑制剂在4小时内诱导CTL凋亡的研究，这些组织蛋白酶抑制剂在涂有T细胞受体交联抗体而不是对照单克隆抗体的孔中孵育。这些组织蛋白酶抑制剂对静止CTL的杀伤能力可以忽略不计，而具有相同反应基团但不合适的肽的对照试剂在TcR参与后不能诱导死亡。CTL的死亡是钙依赖性的，被颗粒毒素concanamycin B阻断，在穿孔素缺陷CTL中未见。人NK细胞在组织蛋白酶抑制剂存在下表现出类似的死亡，当在包被抗cd16单抗的孔中孵育时，而不是对照单抗。这些结果支持了细胞毒性淋巴细胞利用表面组织蛋白酶的蛋白水解来保护自己免受分泌穿孔素对膜的损伤的假设。导致上述结果的组织蛋白酶的性质正在研究中，一些证据表明溶酶体组织蛋白酶B和L都在T淋巴细胞表面表达。组织蛋白酶W是一种新的组织蛋白酶，最初被描述为一个表达序列标签，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。由于它是上述活性的明显候选者，我们在大肠杆菌中表达了人蛋白酶前原W，并制备了一系列针对它的单克隆抗体。在Western blots中，其中一些与小鼠CTL中的单个30kd条带发生反应，这是活性组织蛋白酶W的大小，目前正在进行实验，以确定组织蛋白酶W是否定位于颗粒，细胞表面或其他地方，以及它是否参与自我保护。

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 carried out a series of experiments designed to test a new model for self-protection which proposes that cysteine proteases on the effector cell membrane inactivate inserted perforin before it can aggregate and form pores. We have tested this model by asking if 1) active cysteine proteases are detectable on the surface of cytotoxic lymphocytes, and 2) cysteine protease inhibitors block self-protection and result in degranulation-induced death in cytotoxic lymphocytes. We have detected active cysteine proteases on the surface of cytotoxic T lymphocytes by labeling them with a biotinylated cathepsin affinity reagent and with the protein inhibitor cystatin B Subsequent analysis by flow cytometry shows that cysteine proteases are detectable on cytotoxic T lymphocytes by this approach, and that they increase upon TcR triggering. Evidence that they may be involved in self-protection comes from studies in which these cathepsin inhibitors induce apoptotic death in CTL within 4 hours when incubated on wells coated with antibodies cross-linking the T cell receptor but not control Mabs. These cathepsin inhibitors show negligible ability to kill resting CTL, and control reagents with the same reactive group but an inappropriate peptide fail to induce death after TcR engagement. This death of CTL is calcium-dependent, blocked by the granule toxin concanamycin B, and is not seen in perforin-deficient CTL. Human NK cells show a similar death in the presence of cathepsin inhibitors when incubated on wells coated with degranulating anti-CD16 Mab, but not control Mab. These results support the hypothesis that cytotoxic lymphocytes use proteolysis by surface cathepsins to protect themselves against membrane damage by secreted perforin.The nature of the cathepsin responsible for the above results is under investigation, and some evidence suggests both the lysosomal cathepsins B and L are expressed on the T lymphocyte surface. Cathepsin W is a novel cathepsin originally described as an espressed sequence tag and subsequently found in mRNA expression studies to be expressed exclusively in cytotoxic lymphocytes (both T and NK cells). Since it is an obvious candidate for the above activity, we have expressed human pre-procathepsin W in E. coli and made a series of monoclonal antibodies against it. In Western blots, some of these react with a single 30kd band in mouse CTL, a size expected for active cathepsin W. Experiments are currently under way to see if cathepsin W is localized in granules, on the cell surface, or elsewhere, and if it is involved in self-protection.