A conserved transcriptional program for MAIT cells across mammalian evolution [Rnorvegicus_thymus_MAIT]

Mucosal-Associated Invariant T (MAIT) cells recognize the riboflavin pathway-derived metabolite 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) presented by the MHC-Ib molecule MR1. Both MR1 and the T cell receptor genes used by MAIT cells are under strong evolutionary pressure in mammals, suggesting an important role of 5-OP-RU-specific T cells across species. In humans and mice, MAIT cells acquire distinctive effector functions linked to the expression of the master transcription factor ZBTB16 (PLZF) during thymic development. Conservation of a unique differentiation program in 5-OP-RU-specific T cells from other species is unclear. Here, we used single-cell RNA sequencing to characterize the development of 5-OP-RU-specific T cells in 6 species spanning 110 million years of mammalian evolution. Cross-species comparative analyses revealed a conserved sequence of transcriptional events underlying the maturation of 5-OP-RU-specific thymocytes, marked by the early expression of ZBTB16 in all species. MAIT cells also co-expressed the transcription factors TBX21 (Tbet) and RORC (RORgt) in human, sheep, cattle and opossum. By contrast, Tbet and RORgt were expressed by distinct subsets of MAIT cells in the thymus of rodents, including pet mice and >30 genetically diverse mouse strains, dismissing a laboratory mouse artifact. In mice, RORgt+ MAIT cells further matured in the mesenteric lymph nodes and intestines to acquire a transcriptional program remarkably conserved in MAIT cells from non-rodent species and characterized by co-expression of type 1 and type 17 effector genes, but also genes associated with cytotoxicity and tissue repair. Thus, we define a deeply conserved transcriptional program for 5-OP-RU-specific T cells, which may help understand their functions.黏膜相关恒定T细胞（Mucosal-Associated Invariant T, MAIT）可识别由MHC-Ib类分子MR1呈递的、源自核黄素代谢通路的代谢物5-(2-氧代亚丙基氨基)-6-D-核糖胺基尿嘧啶（5-OP-RU）。在哺乳动物中，MR1以及MAIT细胞所使用的T细胞受体基因均处于强烈的进化选择压力之下，这提示5-OP-RU特异性T细胞在各物种中均发挥着重要功能。在人类和小鼠体内，MAIT细胞在胸腺发育过程中会获得独特的效应功能，这一过程与主转录因子ZBTB16（PLZF）的表达密切相关。但其他物种的5-OP-RU特异性T细胞是否也存在保守的独特分化程序，目前尚不清楚。本研究借助单细胞RNA测序技术，对涵盖1.1亿年哺乳动物进化历程的6个物种体内的5-OP-RU特异性T细胞发育过程进行了表征。跨物种比较分析结果显示，5-OP-RU特异性胸腺细胞的成熟过程背后存在一套保守的转录事件序列，其特征为所有物种中ZBTB16的早期表达。在人类、绵羊、牛以及负鼠体内，MAIT细胞可共表达转录因子TBX21（T-bet）与RORC（RORγt）。与之相反，在啮齿类动物（包括宠物小鼠及30余种遗传背景各异的小鼠品系）的胸腺中，T-bet与RORγt由不同的MAIT细胞亚群分别表达，这排除了实验小鼠特有的实验假象的可能性。在小鼠体内，RORγt阳性MAIT细胞会在肠系膜淋巴结与肠道中进一步成熟，获得一套在非啮齿类物种的MAIT细胞中高度保守的转录程序，该程序以1型与17型效应基因的共表达为特征，同时还包含与细胞毒性及组织修复相关的基因。综上，本研究明确了一套高度保守的5-OP-RU特异性T细胞转录程序，该程序或有助于解析这类细胞的功能。