TARGET CELL DEATH BY CYTOTOXIC LYMPHOCYTES

细胞毒性淋巴细胞导致靶细胞死亡

• 批准号: 6289235
• 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/6289235
• 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 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. - Apoptosis, caspase, CTL, Cytotoxicity, Death, granzyme, lymphocyte, NK, protease,

淋巴细胞介导的细胞毒性的颗粒胞吐作用模型假定抗原触发将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中。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素聚集并形成孔之前使其失活。我们通过询问是否 1) 在细胞毒性淋巴细胞表面可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止自我保护并导致细胞毒性淋巴细胞脱颗粒诱导死亡来测试该模型。我们通过用生物素化组织蛋白酶亲和试剂和蛋白质抑制剂胱抑素 B 标记细胞毒性 T 淋巴细胞表面，检测到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，并且它们在 TcR 触发时增加。它们可能参与自我保护的证据来自于研究，在这些研究中，当这些组织蛋白酶抑制剂在涂有与 T 细胞受体交联的抗体但不与对照 Mab 交联的孔中孵育时，4 小时内会诱导 CTL 细胞凋亡。这些组织蛋白酶抑制剂杀死静息 CTL 的能力可以忽略不计，而具有相同反应基团但不适当肽的对照试剂在 TcR 接合后未能诱导死亡。 CTL 的这种死亡是钙依赖性的，可被颗粒毒素刀那霉素 B 阻断，并且在穿孔缺陷的 CTL 中未见。当在涂有脱颗粒抗 CD16 Mab 的孔中孵育时，人 NK 细胞在存在组织蛋白酶抑制剂的情况下表现出类似的死亡，但对照 Mab 则不然。这些结果支持细胞毒性淋巴细胞利用表面组织蛋白酶的蛋白水解作用来保护自身免受分泌性穿孔素造成的膜损伤的假设。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 是一种新型组织蛋白酶，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。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前组织蛋白酶 W，并制备了一系列针对它的单克隆抗体。在蛋白质印迹中，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 预期的大小。目前正在进行实验，看看组织蛋白酶 W 是否定位在颗粒中、细胞表面或其他地方，以及它是否参与自我保护。 - 细胞凋亡、半胱天冬酶、CTL、细胞毒性、死亡、颗粒酶、淋巴细胞、NK、蛋白酶、