Rat thymic epithelium-free areas are specialized medullary niches enriched for mature thymocytes and distinct stromal subsets

In contrast, rat thymic medulla contains medullary epithelium-free areas (mEFAs), but the cellular composition and functional significance of these regions remain unclear. Here, we combined spatial transcriptomics, scRNA-seq, and robust cell-type decomposition (RCTD) to characterize mEFAs in Lewis rat thymus.

These analyses revealed that mature single-positive thymocytes, including CD4SP, CD8SP, and regulatory T-cell-lineage cells, were preferentially localized in mEFAs, whereas immature SP subsets were enriched in medullary epithelium-containing areas. Rat thymic mesenchymal cell-3 and -4 (TMC3 and TMC4) subsets were also enriched in mEFAs. These subsets were broadly similar to mouse medullary fibroblasts but displayed distinct predicted interactions with SP thymocytes, including costimulatory molecule–receptor, chemokine–receptor, and ECM–integrin axes.