Identification and mapping of human lymph node stromal cell subsets by combining single-cell RNA sequencing with spatial transcriptomics.

Lymph node stromal cells (LNSCs) have a crucial immunomodulatory function, but their heterogeneity in human is incompletely understood. Here, we report the single cell RNA sequencing (scRNA-seq) of 12’000 LNSCs isolated from a human lymph node (LN). This study comprehensively defines the gene signatures of 10 fibroblast subtypes: CCL21+SC, CCL19+SC, CD34+CXCL14+SC, pericytes, DES+SC, LAMP5+SC, NR4A1+BCAM+ SC, HLA-DR+SC, SEPT4+SC and GLDN+SC. To explore the heterogeneous stromal compartment within the complex LN tissue architecture, we integrated the scRNA-seq profiles of the identified LNSC subsets with a publicly available human spatial transcriptomic LN dataset and predicted their location within the complex LN tissue architecture. Each LNSC subtype was spatially restricted to specific LN regions, indicating different LNSC-lymphocyte interactions which was further investigated using NicheNet. The positioning of distinct LNSC subtypes in different LN regions sets the stage for future research on the relationship between LNSC-specific niches and immunomodulatory function during health and disease. LNs were collected from kidney transplantation recipients during living donor kidney transplantation. We have noticed that HLA-DR+ stromal cells represent only a small population within the stromal compartment of the lymph node. To ensure that sufficient HLA-DR+ stromal cells were included in the sorted cells used for sequencing, we included HLA-DR in the gating strategy of the sorting. We first gated on the HLA-DR+ (plus) and HLA-DR- (minus) stromal cells, subsequently we sorted DN, FRC, BEC and LEC based on PDPN and CD31 expression, resulting in two sorted cell suspensions. To assess the presence of a possible batch effect between these two samples, we added spike-in B cells in each sample.