Medullary stromal cells define small intestinal lymph node identity in humans and mice

Lymph nodes (LNs) along the murine gastrointestinal tract are immunologically distinct. Here, we investigate whether such an immune dichotomy globally defines gut LN identity and what drives the regional differences. However, we find that, transcriptionally, it is genes associated with stromal cells that define LN location along the intestine, with high conservation between human and mouse. Using LN single-cell RNA sequencing databases and imaging, we pinpoint the underlying reason for our transcriptional signature: a selective enrichment in the small intestine-draining LN medulla for fibroblastic reticular cell subpopulations implicated in extracellular matrix remodeling and stromal cell replenishment, lymphatic endothelial cells, and macrophages. In mice, the unique medullary cellular network is established around weaning age by vitamin A, while the gut microbiota regulates the effect’s amplitude. Our study implicates the LN medulla as contributing to tissue-specific immunity and uncovers privileged access to lipid-soluble vitamins as the pivotal driving force for small intestinal LN architecture. • Gut lymph nodes differ most in their stromal cell composition • This is true in humans and mice • This difference is induced by vitamin A and amplified by the microbiota Fatkhullina et al. discover that in humans and mice, the dominant difference between lymph nodes draining the small intestine versus colon and other sites is their stromal cell and macrophage composition. This difference is induced by dietary vitamin A and amplified by the microbiota.