Small intestinal  lymph node identity is defined by medullary stromal cells in humans and mice [CD169_RPL22HA_lns_RNAseq]
Lymph nodes (LNs) along the murine gastrointestinal tract are immunologically distinct. Here, we investigated what defines gut LN identity at large in humans and mice and drives their regional differences. Using transcriptional profiling and imaging, we found that stromal cells in the LN medulla and sinus are susceptible to location along the intestine, with high conservation between human and mouse. Specifically, the medulla of small intestine-draining LNs was enriched for fibroblastic reticular cell subpopulations implicated in extracellular matrix remodeling and stromal cell replenishment, lymphatic endothelial cells, and macrophages compared to LNs draining colon and other sites like lung. In mice, this unique medullary cellular network was established around weaning age by Vitamin A, while the gut microbiota regulated the effect's amplitude. Our study posits the LN medulla as a location for tissue-specific immune responses and uncovers privileged access to lipid-soluble vitamins as a pivotal driving force for small intestinal LN properties. Overall design: RiboTag mice were crossed to a CD169-Cre recombinase-expressing mouse to activate the Rpl22HA allele in all tissues. Homozygous CD169RPL22HA mice lymph nodes were collected and used for the RiboTag immunoprecipitation. RNA was extracted using the Arcturus PicoPure RNA isolation kit (Applied Biosystems) according to the manufacturer's instructions.