Microbiota are dispensable for early stages of de novo regulatory T cell induction within mesenteric lymph nodes

Intestinal Foxp3+ regulatory T cell (Treg) subsets are crucial players for tolerance towards microbiota-derived and food-borne antigens, and compelling evidence suggests that intestinal microbiota modulate their differentiation and maintenance. Selected bacterial species and microbiota-derived metabolites such as short-chain fatty acids (SCFAs) have been reported to foster Treg homeostasis in the intestinal lamina propria. Furthermore, gut-draining mesenteric lymph nodes (mLNs) are particularly efficient sites of de novo Treg induction, and we could previously show that mLN stromal cells contribute to this process. Yet, it is not fully elucidated which direct role microbiota and their metabolites play for the early stages of de novo Treg induction and in shaping the Treg transcriptome already during the initial priming within mLNs. Here, we show that neither dysbiotic microbiota nor dietary SCFA supplementation impact de novo induction of Foxp3+ Tregs within mLNs. Even mice housed under germ-free (GF) conditions displayed equivalent frequencies of de novo induced Foxp3+ Tregs within mLNs. Further dissection of the accessible chromatin and transcriptome revealed that microbiota indeed have a limited impact on fostering the establishment of peripherally induced Tregs and do not contribute to the initialization of the epigenetic landscape for an extensive Treg signature. Viewed as a whole, our data suggest that microbiota are dispensable for the early stages of de novo Treg induction within mLNs, while being required to foster further Treg differentiation and homeostasis at later stages within the intestinal lamina propria. ATAC-seq with 2-4 replicates per condition for regulatory or conventional T cells ex vivo isolated.

肠道Foxp3+调节性T细胞（Treg）亚群是介导针对菌群衍生及食源性抗原免疫耐受的关键效应群体，且已有确凿证据表明肠道菌群可调控其分化与维持。已有研究报道，特定细菌物种及菌群衍生代谢物（如短链脂肪酸SCFAs）可促进肠道固有层内Treg的稳态维持。此外，肠道引流肠系膜淋巴结（mLNs）是高效介导从头Treg诱导的关键位点，我们前期研究也曾证实肠系膜淋巴结基质细胞参与这一过程。然而，菌群及其代谢物在从头Treg诱导早期阶段，以及在肠系膜淋巴结初始致敏阶段塑造Treg转录组谱中的直接作用，尚未完全阐明。本研究显示，失调菌群与膳食SCFA补充均未对肠系膜淋巴结内Foxp3+ Treg的从头诱导产生影响。即便是饲养于无菌（GF）环境中的小鼠，其肠系膜淋巴结内从头诱导的Foxp3+ Treg细胞频率也无显著差异。进一步剖析开放染色质与转录组谱发现，菌群仅对外周诱导Treg的建立存在有限调控作用，且未参与初始化与广泛Treg特征相关的表观遗传图谱。综上，本研究数据表明，菌群对于肠系膜淋巴结内从头Treg诱导的早期阶段并非必需，但在肠道固有层内的后续阶段，其对于Treg的进一步分化与稳态维持是不可或缺的。本研究对离体分离的调节性T细胞与常规T细胞开展ATAC-seq测序，每组设置2-4次生物学重复。