Abstract
Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.
Highlights
Peripheral lymph nodes (LNs) are essential secondary lymphoid organs that mediate interactions between antigen-presenting cells and lymphocytes for the initiation of adaptive immune responses
Unsupervised clustering suggested the existence of at least 4 lymphatic endothelial cell (LEC) subtypes: the largest cluster comprised 364 cells (40.8%), cluster 1 comprised 283 cells (31.7%), cluster 2 comprised 194 cells (21.7%), and the smallest cluster, located between cluster 2 and 3, comprised 52 cells (5.8%) (Fig 1B). All of these cells showed robust expression of the 2 markers used for fluorescence-activated cell sorting (FACS) sorting, CD31 and Pdpn (Fig 1C), and the LEC marker genes prospero homeobox 1 (Prox1) and Fms related receptor tyrosine kinase 4 (Flt4, called Vegfr3) (Fig 1C), confirming their lymphatic endothelial identity
This suggests that the clusters we identified based on gene expression correspond to LECs in different anatomical locations in the LN, with cluster 1 representing floor-lining LEC (fLEC) and cluster 2 ceiling LEC (cLEC), whereas clusters 3 and 4 most likely represent cortical and/or medullary LEC subsets
Summary
Peripheral lymph nodes (LNs) are essential secondary lymphoid organs that mediate interactions between antigen-presenting cells and lymphocytes for the initiation of adaptive immune responses. Apart from lymphocytes, LNs comprise several stromal cell types— including fibroblastic reticular cells (FRCs), blood vascular endothelial cells, and lymphatic endothelial cells (LECs)—that are crucial for LN development and function. LECs provide structure to the LN sinuses that allow lymph percolation through the node and control the access of soluble molecules and subcellular particles (including viruses) to the conduit system that guides them to dendritic cells residing in the LN cortex [2,3]. Under steady-state conditions, LN LECs control lymphocyte egress from LNs via generation of an S1P gradient [4] and regulate
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