Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520.
Centre de Recherche, Institut Curie, 75005 Paris, France.
Proc Natl Acad Sci U S A. 2017 Jul 18;114(29):E5910-E5919. doi: 10.1073/pnas.1705242114. Epub 2017 Jul 5.
Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8 T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
虽然抗原交叉呈现在树突状细胞（DC）中对于大多数细胞毒性免疫反应的启动至关重要，但涉及的细胞内机制和运输途径仍不清楚。在这个过程中最关键的步骤之一，即将内化的抗原输出到细胞质中，据推测是由 Sec61 介导的。Sec61 是一种将带有信号肽的新生多肽易位到内质网（ER）中的通道，它也被提议在 ER 相关降解（称为 ERAD）过程中介导蛋白质逆行转运。在这里，我们使用新鉴定的 Sec61 阻断剂，mycolactone，来分析 Sec61 对抗原交叉呈递、ERAD 和内化抗原进入细胞质的运输的贡献。如先前在其他细胞类型中所示，mycolactone 阻止了蛋白质向 DC 的 ER 内的导入。mycolactone 介导的 Sec61 阻断还强烈抑制了抗原交叉呈递和合成肽对 CD8 T 细胞的直接呈递。相比之下，它不影响 ER 腔或内体中的蛋白质从 ER 腔或内体向细胞质的输出，表明抑制交叉呈递与这两种运输途径都无关。暴露于 mycolactone 的 DC 的蛋白质组学分析表明，抗原呈递的介质表达，包括 MHC 类 I 和β2 微球蛋白，高度易受 mycolactone 处理的影响，这表明 Sec61 阻断间接影响抗原交叉呈递。总之，我们的数据表明 Sec61 底物的转运和随后的降解缺陷是 Sec61 阻断的 DC 中改变抗原交叉呈递的原因。
