Cell-intrinsic role of caspase-1 in regulating antigen-specific CD8+ T cell responses

摘要 半胱天冬酶-1是炎性小体复合物的重要组成部分，负责切割前体蛋白， 炎性细胞因子IL-1β和IL-18，并通过Gasdermin D的裂解引发焦亡。使用 西尼罗河病毒（WNV）感染的小鼠模型，我们和其他人以前已经证明了 炎性体信号转导促进IL-1β介导的保护性免疫。我们现在已经扩展了这些 观察并发现了caspase-1在调节抗原特异性CD 8 + T细胞中的一种新的细胞内在作用 感染WNV时的反应。CD 8 + T细胞的缺乏导致不受控制的WNV复制， 脑内严重的神经元损伤为了了解大脑中的环境线索如何影响T细胞 在WNV感染期间的应答中，我们对分离的抗原特异性CD 8 + T细胞进行了转录谱分析。 在西尼罗河病毒感染后的早期和晚期，通过分析，我们发现， 半胱天冬酶-1，以及其他炎性体信号成分，在脑驻留抗原中富集， 与从脾分离的抗原特异性CD 8 + T细胞相比，特异性CD 8 + T细胞。我们确认 与脾CD 8 + T细胞相比，活性半胱天冬酶-1在脑驻留的CD 8 + T细胞中表达更丰富。 T细胞。使用共过继转移模型，我们观察到抗原特异性Casp 1-/-CD 8 + T细胞增加， 与感染期间脾和脑内的WT CD 8 + T细胞相比。值得注意的是，CD 8 + T细胞没有被感染， 这表明caspase-1的激活不依赖于病毒感染。接下来，我们描述 使用体外T细胞受体（TCR）刺激测定法体外研究胱天蛋白酶-1的功能，并发现胱天蛋白酶-1是 在CD 3/CD 28刺激后自蛋白水解裂解。此外，我们发现ASC，而不是NLRP 3或IL-10， 1β在初始和刺激的CD 8 + T细胞中表达。最后，最近的研究将caspase-1与 线粒体功能与这些观察结果一致，我们发现α CD 3/α CD 28启动了Casp 1-/-CD 8 + 与WT CD 8 + T细胞相比，T细胞显示出增强的氧化磷酸化，表明 caspase-1可能通过重编程线粒体动力学和代谢来调节CD 8 + T细胞功能。基于 基于这些发现，我们假设TCR刺激CD 8 + T细胞触发caspase-1活化， 其功能是以组织特异性方式限制T细胞活化、线粒体生物能量学和细胞扩增。在 反过来，我们认为，活性caspase-1是控制CNS内病毒复制所必需的， 通过防止病毒感染期间过度旺盛的T细胞反应， 感染为了验证这一假设，我们有两个具体的目的：1）caspase-1是如何介导CD 8 + T细胞的 感染WNV时的反应？（2）目标2。caspase-1的分子机制是什么 活化的CD 8 + T细胞这些目标的完成将使我们对 caspase-1在病毒感染期间调节CD 8 + T细胞应答的潜在机制。

ABSTRACT Caspase-1 is an essential component of the inflammasome complex and is responsible for cleaving pro- inflammatory cytokines IL-1β and IL-18 and initiating pyroptosis through cleavage of Gasdermin D. Using a murine model of West Nile virus (WNV) infection, we and others have previously demonstrated the importance of inflammasome signaling for promoting IL-1β-mediated protective immunity. We have now extended these observations and discovered a novel, cell-intrinsic role for caspase-1 in regulating antigen-specific CD8+ T cells responses during WNV infection. The absence of CD8+ T cells leads to uncontrolled WNV replication and significant neuronal injury within the brain. To understand how environmental cues within the brain impact T cell responses during WNV infection, we performed transcriptional profiling on antigen-specific CD8+ T cells isolated from the spleen and brain at early and late times post-WNV infection. Through this analysis, we found that caspase-1, as well as other inflammasome signaling components, were enriched within brain-resident antigen- specific CD8+ T cells as compared to antigen-specific CD8+ T cells isolated from the spleen. We confirmed that active caspase-1 is more abundantly expressed within brain-resident CD8+ T cells as compared to splenic CD8+ T cells. Using a co-adoptive transfer model, we observed increased antigen-specific Casp1-/- CD8+ T cells as compared to WT CD8+ T cells within the spleen and brain during infection. Of note, CD8+ T cells are not infected by WNV, suggesting that the activation of caspase-1 is independent of virus infection. Next, we characterized caspase-1 function in vitro using an in vitro T cell receptor (TCR) stimulation assay and found that caspase-1 is autoproteolytically cleaved following CD3/CD28 stimulation. Moreover, we found that ASC, but not NLRP3 or IL- 1β, is expressed in naïve and stimulated CD8+ T cells. Lastly, recent studies have linked caspase-1 with mitochondrial function. Consistent with these observations, we found that αCD3/αCD28 primed Casp1-/- CD8+ T cells displayed enhanced oxidative phosphorylation as compared to WT CD8+ T cells, suggesting that caspase-1 may regulate CD8+ T cell function by reprogramming mitochondrial dynamics and metabolism. Based on these findings, we hypothesize that TCR stimulation of CD8+ T cells triggers caspase-1 activation, which functions to limit T cell activation, mitochondrial bioenergetics, and cell expansion in a tissue-specific manner. In turn, we believe that active caspase-1 is critically required for controlling virus replication within the CNS and minimizing consequential neuronal damage by preventing an over-exuberant T cell response during viral infection. To address this hypothesis, we have two specific aims: 1) How does caspase-1 mediate CD8+ T cell responses during WNV infection? and 2) Aim 2. What are the molecular mechanisms of caspase-1 function in activated CD8+ T cells? The completion of these aims will provide us with a broader understanding of the underlying mechanisms by which caspase-1 regulates CD8+ T cells responses during virus infection.