A single-cell transcriptional roadmap of the mouse and human lymph node lymphatic vasculature
Authors: Xiang Menglan , Grosso Rubén Adrián , Takeda Akira , Pan Junliang , Bekkhus Tove , Brulois Kevin , Dermadi Denis , Nordling Sofia , Vanlandewijck Michael , Jalkanen Sirpa , Ulvmar Maria H. , Butcher Eugene C. 
Published: January 2020

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.

Highlights

Computational alignments (“trajectories”) predict LN LEC organizationin 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; macrophage-associated 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