Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse
Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse
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DOI：
10.4049/jimmunol.1301205
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发表时间：
2013-09-01
影响因子：
4.4
通讯作者：
Voskoboinik, Ilia
中科院分区：
文献类型：
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作者：
Lopez, Jamie A.;Jenkins, Misty R.;Voskoboinik, Ilia
细胞毒性淋巴细胞（CLs）与其靶细胞的有效结合对于从体内清除病毒感染细胞和恶性细胞至关重要。由于缺乏生物学报告，定义CL参与和杀伤靶细胞的时空特性在很大程度上仍未表征。我们使用了一种新颖的活细胞显微镜技术来观察人类和小鼠原发CL与其靶标的接触以及随后的致命打击。对单个效应靶细胞偶联物的大量实时定量分析表明，单个效应钙通量事件足以使人细胞脱颗粒，导致穿孔蛋白在65-100秒内破坏靶细胞膜。相比之下，小鼠CLs表现出不同的钙信号传导谱，导致脱颗粒：小鼠nk细胞需要一次钙通量事件，而CD8(+) T细胞通常在穿孔素递送之前需要多次钙通量事件。不管它们的信号结构如何，每一个被穿孔素损伤的靶细胞都死于细胞凋亡。据我们所知，我们首次证明了穿孔递送是单向的，只发生在靶细胞膜上，而不影响杀伤细胞。尽管如此，CTL膜本身并不具有穿孔素抗性，因为完整的CTL作为效应CTL的靶标能够被穿孔素依赖机制杀死。我们的研究结果强调了CLs射孔输送的显著效率和特异性。
The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.