Highlights d Single-cell RNA sequencing of human lymph nodes unveils six types of LECs d LECs lining the floor and ceiling of the SCS, MS, and valve are the main types d LECs of the SCS floor and MS highly express neutrophil chemoattractants d Human MS LECs support neutrophil adhesion in the LN medulla via CD209

Xiang et al. scRNA-seq of Lymph Node Lymphatic Vasculature HIGHLIGHTS Computational alignments ("trajectories") predict LN LEC organization in situ, revealing a continuum of phenotypes punctuated by specialized clusters. Multiple intermediate phenotypes suggest LEC malleability. Gene profiles define niche-specific functional specialization. Medullary sinus LECs are comprised of Ptx3-LECs and Marco-LECs.-Distinct mechanisms for pathogen interactions and matrix modeling.-Ptx3-LECs: paracortical and central medullary sinuses near hilus; enriched for genes driving lymphangiogenic responses and lymphocyte egress.-Marco-LECs: peri-follicular medullary sinuses; macrophageassociated genes, complement and coagulation cascade. Niche-specific responses to inflammation.-IFN gene responses in SCS floor and medullary sinus LECs.-Suppression of LEC identity genes in responding subsets. Conserved and unique LEC subsets and gene programs across species.-Core subsets common to mouse and human.-Greater diversity of subsets and intermediates in human LN LECs.

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis.

Single-cell transcriptomics promises to revolutionize our understanding of the vasculature. Emerging computational methods applied to high dimensional single cell data allow integration of results between samples and species, and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in analysis of 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. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN.