Single-Cell Analysis Reveals Heterogeneity of High Endothelial Venules and Different Regulation of Genes Controlling Lymphocyte Entry to Lymph Nodes

Abstract

Summary: High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-β receptor (LTβR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTβR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease. : High-endothelial venules (HEVs) are specialized blood vessels that allow lymphocyte recirculation through the different lymphoid organs of the body. Using single-cell RNA sequencing, Veerman et al. uncover the cellular and spatial heterogeneity of HEVs in lymph nodes and unveil important differences in the regulation of genes controlling lymphocyte trafficking. Keywords: high-endothelial venule, peripheral lymph node, endothelial cell, lymphocyte trafficking, lymphocyte homing, homeostasis, inflammation, single-cell RNA sequencing, scRNA-seq, lymphotoxin-beta receptor