Single-cell RNA-seq of the mouse lymph node lymphatic vasculature: SMART-seq2

Lymph nodes (LNs) serve as hubs for the interaction and communication between tissue-derived and blood-derived immune cells. Here we analyzed mouse lymph node (LN) lymphatic endothelial cells (LEC) at single cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not recognized previously as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates known and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells, (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation, and (3) medullary subset specialization for pathogen interactions and LN remodeling. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Overall design: Female 20-week-old Prox1-GFP/C57BL/6J inguinal LNs were processed by the SMART-seq2 workflow for scRNA-seq.

淋巴结（Lymph nodes, LNs）是组织源性与血液源性免疫细胞相互作用及信息交流的核心枢纽。本研究以单细胞分辨率分析了小鼠淋巴结（LN）淋巴管内皮细胞（Lymphatic endothelial cells, LEC）。聚类分析鉴定出五个界限清晰的细胞亚群，其中包含两个此前未被识别为独立类别的髓窦亚群。通过拟时序空间内的最近邻比对，主要亚群被排布为一条序列，该序列既重现了已知的淋巴结淋巴管组织特征，又揭示了全新的组织结构特性，同时构建了淋巴管内皮微环境及其亚群间转化过程的转录组图谱。基因表达差异揭示了三类特化的细胞功能程序：(1) 被膜下穹窿内皮细胞与被膜结缔组织及其他细胞的相互作用；(2) 被膜下窦底调控淋巴源性细胞进入淋巴结实质并参与抗原呈递；(3) 髓质亚群特化以介导病原体相互作用及淋巴结组织重塑。被膜下窦底及髓质的淋巴管内皮细胞分别是细胞进出淋巴结实质的主要位点，二者在恶唑酮（oxazolone）炎症刺激下均产生强烈应答，其富集的信号通路同时参与先天免疫与适应性免疫应答。实验设计：采用SMART-seq2流程对20周龄雌性Prox1-GFP/C57BL/6J小鼠的腹股沟淋巴结进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。