A molecular map of lymph node blood vascular endothelium at single cell resolution

Abstract

48 49 Blood vascular endothelial cells (BECs) control the immune response by regulating immune 50 cell recruitment, metabolite exchange and blood flow in lymphoid tissues. However, the 51 diversity of BEC and their origins during immune angiogenesis remain poorly understood. 52Here we profile transcriptomes of BEC from mouse peripheral lymph nodes and map key 53 phenotypes to the vasculature. Our analysis identifies multiple novel subsets including a 54 venous population whose gene signature predicts an unexpectedly selective role in myeloid 55 cell (vs lymphocyte) recruitment to the medulla, confirmed by 2 photon videomicroscopy. 56We define five phenotypes of capillary lining BEC including a capillary resident regenerative 57 population (CRP) that displays stem cell and migratory gene signatures and contributes to58 homeostatic BEC turnover and to vascular neogenesis after immunization. Trajectory 59 analyses reveal retention of developmental programs along a progression of cellular 60 phenotypes from CRP to mature venous and arterial BEC subsets. Overall, our single cell 61 atlas provides a molecular blueprint of the lymph node blood vasculature and defines subset 62 specialization for immune cell recruitment and vascular homeostasis. 63 64 Introduction 65The vascular endothelium lining blood vessels regulates exchange of oxygen and 66 metabolites between the blood vascular compartment and tissues. In lymph nodes (LN), 67 additionally, the endothelium plays essential roles in controlling immune cell access. The 68 organization of the vasculature in LN is well characterized: Arteries entering at the hilus lead to 69 capillary arcades in the LN cortex; capillaries link to 'high endothelial venules' (HEV), the post 70 capillary venules that recruit lymphocytes from the blood 1 ; and the vasculature exits the lymph 71 node at the hilus. Upon immune challenge, lymph nodes increase in volume up to 10-fold or more 72 within days, and the vascular endothelium expands roughly proportionally from local precursors 1,2 . 73Vessel expansion involves extensive proliferation of both capillary and high endothelial cells 74 (HEC) 3 , without contribution from blood borne progenitors 4 ; and an increase in vessel numbers 75 through intussusceptive (splitting) angiogenesis 5 . However, the nature and extent of endothelial 76 cell diversity within LN remains incompletely understood. 77Single cell RNA profiling is a transformative technology for the identification of cell 78 diversity and elucidation of developmental and physical relationships. Here we provide a survey 79 of blood vessel endothelial cells (BEC) from mouse peripheral lymph nodes (PLN). We confirm 80 known features of high endothelium 6,7 , identify novel endothelial subsets, uncover unexpected 81 diversity among capillary cells and demonstrate a distinctive role of medullary veins in selective 82 myeloid cell recruitment. We define gene signatures and transcription regulatory factors for these 83 subsets, and map key subsets to the vasculature. We also identify a p...

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

“…Particularly exciting is the power of trajectory inference to recapitulate or predict the organization of endothelial cells within the complex vascular networks. While trajectory analysis has been shown to model sequences of cell phenotypes in development ("pseudotime") (30), our results both in lymphatics (here) and blood vascular EC studies (31) show that computed alignments of cells in trajectory space reflect the tissue architecture (spatial organization) and physical relationships between endothelial cells in situ with surprising faithfulness. This correspondence implies, as an approximation, that endothelial cell phenotypes progress in a gradual and orderly fashion within the linear arrangements (as in vascular tubes) or sheets (as in the SCS and sinuslining LECs in the LN) that make up the endothelium.…”

A single-cell transcriptional roadmap of the mouse and human lymph node lymphatic vasculature

“…Particularly exciting is the power of trajectory inference to recapitulate or predict the organization of endothelial cells within the complex vascular networks. While trajectory analysis has been shown to model sequences of cell phenotypes in development (“pseudotime”) [29], our results both in lymphatics (here) and blood vascular EC studies [30] show that computed alignments of cells in trajectory space reflect the tissue architecture (spatial organization) and physical relationships between endothelial cells in situ with surprising faithfulness. This correspondence implies, as an approximation, that endothelial cell phenotypes progress in a gradual and orderly fashion within the linear arrangements (as in vascular tubes) or sheets (as in the SCS and sinus-lining LECs in the LN) that make up the endothelium.…”

“…In developing cell systems trajectory inference methods can model developmental sequence (“pseudotime”) [29]. In the resting adult vascular endothelium, they facilitate computational modeling of vessel architecture [30]. Subsets and alignments were shared in mice with different genetic backgrounds (C57BL/6 and BALB/c) (Figure S1A).…”

A Single‐Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

“…In developing cell systems, trajectory inference methods can model developmental sequence ("pseudotime") (30). In the resting adult vascular endothelium, they facilitate computational modeling of vessel architecture (27,31). Subsets and alignments were shared in mice with different genetic backgrounds (C57BL/6 and BALB/c) (Figure S1A).…”