Postnatal expansion of the lymph node stromal cell pool towards reticular and CD34+ stromal cell subsets [WGBS]. Postnatal expansion of the lymph node stromal cell pool towards reticular and CD34+ stromal cell subsets [WGBS]

Gut-draining mesenteric lymph nodes (mLNs) provide the framework to shape intestinal adaptive immune responses. We previously delineated transcriptional signatures in LN stromal cells (SC), pointing to tissue-specific variability in SC composition and immunomodulatory function. Here, using scRNA-seq we dissected the developmental trajectory of SCs within mLNs derived from postnatal to aged mice, and identified two putative progenitors, namely CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors, which both feed the rapid LN expansion postnatally. We further unraveled the tissue-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs, and identified a microbiota-independent core epigenomic signature, showcasing differences between SCs from mLN and skin-draining peripheral LN. Irf3 was inferred from the epigenomic landscape of SCs, being dynamically expressed along the differentiation trajectories of FRCs. Lentiviral overexpression of Irf3 in a mesenchymal stem cell line established a Cxcl9+ FRC molecular phenotype. The relevance of Irf3 for SC biology was further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs from Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs. Overall design: Whole Genome Bisulfite Sequencing on FACS-isolated CD45-Ter119-CD31-Pdpn+ cells from skin- or gut-draining lymph nodes of SPF or GF housed mice

肠道引流肠系膜淋巴结（gut-draining mesenteric lymph nodes，mLNs）为塑造肠道适应性免疫应答提供了结构框架。本研究团队此前已明确淋巴结基质细胞（lymph node stromal cells，SC）的转录特征，揭示了基质细胞组成与免疫调节功能存在组织特异性差异。本研究借助单细胞RNA测序（single-cell RNA sequencing，scRNA-seq），解析了新生至衰老阶段小鼠肠系膜淋巴结内基质细胞的发育轨迹，并鉴定出两种潜在祖细胞：CD34+基质细胞与成纤维网状基质细胞（fibroblastic reticular stromal cell，FRC）祖细胞，二者均可促进出生后淋巴结的快速扩张。本研究进一步解析了非内皮基质细胞的组织特异性染色质可及性与DNA甲基化图谱，并鉴定出一类不依赖微生物群的核心表观基因组特征，揭示了肠道引流肠系膜淋巴结与皮肤引流外周淋巴结的基质细胞之间的差异。研究人员通过基质细胞的表观基因组图谱推断出干扰素调节因子3（interferon regulatory factor 3，Irf3），其在成纤维网状基质细胞的分化轨迹中呈动态表达模式。在间充质干细胞系中通过慢病毒介导的Irf3过表达，可诱导出表达Cxcl9的成纤维网状基质细胞分子表型。Irf3基因敲除（Irf3-/-）小鼠的淋巴结中，表达Ccl19与Cxcl9的成纤维网状基质细胞比例显著降低，进一步证实了Irf3在基质细胞生物学功能中的重要性。综上，本研究数据构建了生命早期淋巴结基质细胞发育的完整转录组与表观基因组图谱，并解析了非内皮基质细胞的位置特异性、不依赖微生物群的生物学特性。整体实验设计：对来自无特定病原体（specific pathogen free，SPF）或无菌（germ free，GF）环境饲养小鼠的皮肤引流或肠道引流淋巴结中，经荧光激活细胞分选（fluorescence-activated cell sorting，FACS）分离得到的CD45-Ter119-CD31-Pdpn+细胞进行全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing，WGBS）。