Table_5_Translatome analyses by bio-orthogonal non-canonical amino acid labeling reveal that MR1-activated MAIT cells induce an M1 phenotype and antiviral programming in antigen-presenting monocytes.xlsx

MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related protein 1 (MR1). However, our understanding of MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR1 ligand stimulations. Translation in both cell types was found to be increased by 5-OP-RU, which correlated with their conjugation frequency and CD3 polarization at MAIT cell immunological synapses in the presence of 5-OP-RU. In contrast, Ac-6-FP only regulated a few protein translations, including GSK3B, indicating an anergic phenotype. In addition to known effector responses, 5-OP-RU-induced protein translations uncovered type I and type II Interferon-driven protein expression profiles in both MAIT and THP-1 cells. Interestingly, the translatome of THP-1 cells suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1β, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

黏膜相关恒定T细胞（Mucosal-associated invariant T cells，MAIT）是一类兼具多种功能的天然样效应细胞，可识别由非多态性MHC I类相关蛋白1（MR1）呈递的细菌源性维生素B代谢产物。然而，目前学界对于MAIT细胞与其他免疫细胞相互作用时，MR1介导的MAIT细胞应答的认知仍存在不足。本研究首次在双细胞共培养体系中，开展了原代人MAIT细胞与THP-1单核细胞相互作用的翻译组学研究。本研究分别在激活型MR1配体5-OP-RU或抑制型MR1配体Ac-6-FP存在的条件下，分析了MAIT细胞与THP-1细胞的相互作用过程。借助生物正交非经典氨基酸标记（bio-orthogonal non-canonical amino acid tagging, BONCAT）技术，我们可选择性富集MR1依赖的细胞相互作用过程中新生合成的蛋白质。随后，通过超灵敏蛋白质组学技术对两种细胞的新生合成蛋白质进行细胞类型特异性定量检测，以解析两种细胞中共存的免疫应答特征。该策略在MR1配体刺激后，分别鉴定出2000余种MAIT细胞相关活性翻译蛋白，以及3000余种THP-1细胞相关活性翻译蛋白。研究发现，5-OP-RU可上调两种细胞的蛋白质翻译水平，且该效应与5-OP-RU存在时MAIT细胞免疫突触处的细胞结合频率及CD3极化水平呈正相关。与之相反，Ac-6-FP仅调控少量蛋白质的翻译过程，其中包括GSK3B，提示MAIT细胞呈现免疫无反应性表型。除已报道的效应应答外，5-OP-RU诱导的蛋白质翻译特征还在MAIT细胞与THP-1细胞中均揭示了I型与II型干扰素驱动的蛋白质表达谱。值得关注的是，THP-1细胞的翻译组学数据显示，活化的MAIT细胞可调控该细胞的M1/M2极化状态。实验证实，CXCL10、IL-1β、CD80及CD206的基因表达与表面蛋白表达结果表明，在5-OP-RU活化的MAIT细胞存在时，可诱导巨噬细胞呈现M1样表型。此外，本研究验证了干扰素驱动的翻译组学特征伴随THP-1细胞抗病毒表型的诱导；研究发现，THP-1细胞在与MR1活化的MAIT细胞结合后，可抑制病毒复制。综上，BONCAT翻译组学技术拓展了我们在蛋白质层面对MAIT细胞免疫应答的认知，并证实MR1活化的MAIT细胞足以诱导巨噬细胞发生M1极化及启动抗病毒程序。