GSE193449
Single-cell RNA sequencing reveals tissue-associated fibroblastic reticular cell heterogeneity in human secondary lymphoid organs
Fibroblastic reticular cells (FRCs) actively support secondary lymphoid organ (SLO) architecture. They regulate innate and adaptive immunity by compartmentalizing immune cells in specialized niches, and tightly interacting with them. The phenotypic and functional diversity of FRCs in human SLOs remains largely uncharacterized. We performed a comprehensive profiling of FRCs by single-cell RNA sequencing on human tonsils and lymph nodes. We provide the first complete description of the tonsillar CD45-negative compartment. We identified and validated in tonsil, a population of follicular dendritic cells restricted to the mantle zone and discovered two CCL21-negative FRC subsets intermingling with T-cell zone reticular cells in the extrafollicular space. We also identified podoplanin-positive epithelial cells as a potential plasma cell (PC) niche. We found two levels of FRC-associated diversity: i) tissue-specific subsets; ii) shared subsets with tissue-specific gene signatures. These findings shed new light on the role of FRCs in supporting SLO-specific immune responses. Overall design: Palatine tonsils were obtained following routine tonsillectomy for recurrent sleep apnea or airway obstruction at Robert Debré and Bichat Hospitals, in Paris. None of the patients had tonsillitis at the time of surgery. Patients with immunodeficiency or serious infections were excluded. LNs were obtained from cadaveric donors and patients at Bichat Hospital, Paris. Tumor draining LNs were obtained from consenting patients undergoing resection surgery. Resting lymph nodes were obtained from cadaveric donors. After tissue dissociation and CD45-negative single-cell suspension generation, CD45-CD31- cell were stained and sorted by FACS. Sorted CD45-CD31- cells were analyzed with the 10X chromium system (10X Genomics). Single-cell libraries were generated according to the manufacturer's instructions (chromium 10X Next GEM Single-cell 3' Kit (V.3.3)). Next-generation sequencing was performed on an Illumina NovaSeq 6000. ScRNAseq data processing and FASTQ file analysis were performed at the NGS platform of the Curie Institute, Paris (Tonsil 1) and the sequencing platform of the Paris Brain Institute (Tonsil 2-4, LNs) with the Cell Ranger pipeline (10X Genomics, version 3). Reads were aligned with the human reference genome hg38.