Postnatal expansion of the lymph node stromal cell pool towards reticular and CD34+ stromal cell subsets [BRB-seq]

Gut-draining mesenteric lymph nodes (mLNs) provide the framework to shape intestinal adaptive immune responses. We previously delineated transcriptional signatures in LN stromal cells (SC), pointing to tissue-specific variability in SC composition and immunomodulatory function. Here, using scRNA-seq we dissected the developmental trajectory of SCs within mLNs derived from postnatal to aged mice, and identified two putative progenitors, namely CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors, which both feed the rapid LN expansion postnatally. We further unraveled the tissue-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs, and identified a microbiota-independent core epigenomic signature, showcasing differences between SCs from mLN and skin-draining peripheral LN. Irf3 was inferred from the epigenomic landscape of SCs, being dynamically expressed along the differentiation trajectories of FRCs. Lentiviral overexpression of Irf3 in a mesenchymal stem cell line established a Cxcl9+ FRC molecular phenotype. The relevance of Irf3 for SC biology was further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs from Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs. Overall design: 3'RNA-seq (methods used BRB-seq) on C3H10T1/2 lentivirally transduced with indicated transcription factor.