Apoptotic Death in T Lymphocytes

我们继续使用一系列细胞渗透性荧光肽底物研究T细胞凋亡。先前对胸腺细胞的研究表明，Fas交联可触发caspase级联反应，其中用流式细胞术检测到的活性最早为IETD （caspase 8选择性）底物。鉴于有大量证据表明caspase 8激活是Fas死亡途径的启动事件，这一结果是意料之中的。然而，当我们用这种方法检测抗fas在细胞毒性T淋巴细胞中诱导的细胞凋亡时，ietase的激活出乎意料地伴随着lehase、yvhase和VEIDase活性的增加（选择性caspase 9、1和6）。通过共聚焦显微镜检查，抗fas处理的CTL显示早期出现含有ietase的质膜囊泡，它们从CTL表面释放出来，随后细胞内部其他caspase活性被激活。由于这些囊泡不包括在流式细胞术分析中，早期的ietase活性被低估了。与抗fas相反，IL-2戒断诱导这些CTL凋亡导致细胞质中早期的ietase活性，而没有质膜泡化。因此，抗fas诱导的在质膜上启动caspase活性可能在某些细胞中导致亚膜蛋白的局部蛋白水解，从而产生高度富集活性caspase 8的膜囊泡。

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.