Target Cell Death by Cytotoxic Lymphocytes

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素聚集和形成毛孔之前将其失活。我们通过询问1)细胞毒性淋巴细胞表面是否可以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是否能阻止自身保护并导致细胞毒性淋巴细胞脱颗粒诱导死亡来测试这一模型。我们用生物素标记的组织蛋白酶亲和试剂和蛋白抑制剂cystatin B标记了细胞毒性T淋巴细胞表面的半胱氨酸蛋白酶，随后用流式细胞仪分析表明，用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触发后半胱氨酸蛋白酶的数量增加。它们可能参与自我保护的证据来自研究，在这些研究中，当这些组织蛋白酶抑制剂孵育在涂有T细胞受体交联型抗体的孔洞中时，4小时内诱导CTL细胞凋亡，但不能控制单抗。这些组织蛋白酶抑制剂对静息CTL的杀伤能力可以忽略不计，并且具有相同反应基团但不适当的多肽在TCR参与后不能诱导死亡的对照试剂。CTL的这种死亡是钙依赖的，被颗粒毒素刀豆素B阻断，在穿孔素缺乏的CTL中看不到。人NK细胞在有组织蛋白酶抑制剂存在的情况下显示出类似的死亡，当孵育在涂有脱颗粒抗CD16单抗的孔洞中时，但不是对照单抗。这些结果支持细胞毒性淋巴细胞通过表面组织蛋白酶的蛋白分解来保护自己免受穿孔蛋白分泌的膜损伤的假说。导致上述结果的组织蛋白酶的性质正在调查中，一些证据表明溶酶体组织蛋白酶B和L都表达在T淋巴细胞表面。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。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Pre-proathepsin W，并制备了一系列针对它的单抗。在Western blotts中，其中一些与小鼠CTL中的一条30kd条带反应，这是活性组织蛋白酶W的预期大小。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.