Apoptotic Death in T Lymphocytes

我们继续使用一系列细胞可穿透的荧光肽底物研究T细胞的凋亡。以往对胸腺细胞的研究表明，Fas的交联会触发caspase级联反应，其中IETD(caspase 8选择性)底物是最早被流式细胞仪检测到的活性。这一结果是预期的，因为有大量证据表明caspase 8激活是Fas死亡途径的启动事件。然而，当我们用这种方法检测抗Fas诱导的细胞毒性T淋巴细胞的凋亡时，IETDase的激活出人意料地伴随着LEHDase、YVHDase和VEIDase活性的增加(对caspase 9、1和6具有选择性)。共聚焦显微镜观察发现，经抗Fas处理的CTL早期出现含有IETDase的质膜囊泡，并从CTL表面释放出来，随后细胞内部的其他caspase活性被激活。由于这些囊泡没有包括在流式细胞仪分析中，早期的IETDase活性被低估了。与抗Fas相反，IL-2撤除诱导这些CTL的凋亡导致IETDase活性早期出现在细胞质中，没有质膜囊泡。因此，在某些细胞中，抗Fas诱导的质膜上caspase活性的启动可能导致亚膜蛋白的局部蛋白降解，从而产生高度富含caspase 8活性的膜小泡。

We have continued studying T cell apoptosis using a series of cell-permeable fluorogenic peptide substrates. Previous studies of thymocytes showed that Fas crosslinking triggered a caspase cascade in which the IETD (caspase 8 selective) substrate was the earliest activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when we examined apoptosis induced by anti-Fas in cytotoxic T lymphocytes by this approach, IETDase activation unexpectedly followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9,1, and 6). When examined by confocal microscopy, anti-Fas treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of sub-membrane proteins, leading to generation of membrane vesicles which are highly enriched in active caspase 8.