Skin and Skin Draining lymph node dendritic cells from neonatal mouse colonized with a commensal or control. Skin and Skin Draining lymph node dendritic cells from neonatal mouse colonized with a commensal or control

Early life establishment of tolerance to commensal bacteria at barrier surfaces carries enduring implications for immune health but remains poorly understood. Here we show that this process is controlled by microbial interaction with a specialized subset of antigen presenting cells. More particularly, we identify CD301b+ type 2 conventional dendritic cells (DC) as a subset in neonatal skin specifically capable of uptake, presentation and generation of regulatory T cells (Tregs) to commensal antigens. In early life, CD301b+ DC2 are enriched for programs of phagocytosis and maturation, while also expressing tolerogenic markers. In both human and murine skin, these signatures were reinforced by microbial uptake. In contrast to their adult counterparts or other early life DC subsets, neonatal CD301b+ DC2 highly expressed the retinoic acid-producing enzyme, RALDH2, deletion of which limited commensal-specific Tregs. Thus, synergistic interactions between bacteria and a specialized DC subset critically support early life tolerance at the cutaneous interface. Overall design: We performed scRNAseq on neonatal mice colonized with S.epidermidis or S.aureus, and look at skin dendritic cells and skin draining lymph nodes migratory dendritic cells to understand specificities of neonatal DCs and uptake/carriage of bacterial antigens. CT-subpop corresponds to cells from uncolonized mice. SEneg-subpop corresponds to cells without bacteria from a mouse colonized with S.epidermis, SEpos-subpop corresponds to cells with bacteria from a mouse colonized with S.epidermis, SAneg-subpop corresponds to cells without bacteria from a mouse colonized with S.aureus, SApos-subpop corresponds to cells with bacteria from a mouse colonized with S.aureus. Cells were stained for ADT: CD103_ADT, CD301B_ADT, CD11B_ADT, EpCAM_ADT, PDL-1_ADT, CD86_ADT

在机体屏障表面建立针对共生菌的免疫耐受，这一过程对免疫健康具有持久影响，但其具体机制目前仍未被充分阐明。本研究证实，该过程由微生物与一类特殊的抗原呈递细胞亚群的相互作用所调控。具体而言，我们鉴定出CD301b+ 2型常规树突状细胞（type 2 conventional dendritic cells, DC）是新生小鼠皮肤中的特异性亚群，能够摄取、呈递共生菌抗原，并诱导调节性T细胞（regulatory T cells, Tregs）的生成。在早期生命阶段，CD301b+ DC2高表达吞噬作用与细胞成熟相关的基因程序，同时还表达免疫耐受相关标志物。在人类和小鼠的皮肤组织中，微生物的摄取进一步强化了这些基因表达特征。与成年小鼠的对应细胞群或其他早期生命阶段的DC亚群相比，新生小鼠的CD301b+ DC2高表达视黄酸合成酶RALDH2，敲除该酶会削弱共生菌特异性Tregs的生成。综上，细菌与特殊DC亚群之间的协同互作，是皮肤屏障表面早期免疫耐受建立的关键支撑机制。

实验设计：我们对定殖了表皮葡萄球菌（S.epidermidis）或金黄色葡萄球菌（S.aureus）的新生小鼠进行单细胞RNA测序（single-cell RNA sequencing, scRNAseq），并分析皮肤树突状细胞及皮肤引流淋巴结中的迁移性树突状细胞，以解析新生DC的特异性特征以及细菌抗原的摄取/携带情况。
CT亚群（CT-subpop）对应未定殖小鼠的细胞；SE阴性亚群（SEneg-subpop）对应定殖表皮葡萄球菌的小鼠中未携带细菌的细胞；SE阳性亚群（SEpos-subpop）对应定殖表皮葡萄球菌的小鼠中携带细菌的细胞；SA阴性亚群（SAneg-subpop）对应定殖金黄色葡萄球菌的小鼠中未携带细菌的细胞；SA阳性亚群（SApos-subpop）对应定殖金黄色葡萄球菌的小鼠中携带细菌的细胞。所有细胞均通过抗体偶联寡核苷酸标签（Antibody-Derived Tag, ADT）进行染色，标记物包括CD103_ADT、CD301B_ADT、CD11B_ADT、EpCAM_ADT、PD-L1_ADT及CD86_ADT。