Medullary epithelium-free areas in the rat thymus are specialized niches enriched for mature thymocytes and distinct stromal subsets

The thymic medulla provides the microenvironment for negative selection, late thymocyte maturation, and thymocyte egress, and is generally characterized by widespread distribution of medullary thymic epithelial cells (mTECs). 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 and scRNA-seq, using robust cell-type decomposition (RCTD) to characterize mEFAs in Lewis-strain rat thymus. These analyses revealed that more mature-phenotypes of CD4SP, CD8SP, and regulatory T-cell-lineage thymocytes were preferentially localized in mEFAs, whereas immature SP subsets were enriched in medullary epithelium-containing areas. Newly found 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. In addition, the venous endothelial cells (vECs) expressing portal endothelial cell markers were accumulated in mEFAs. The S1P transporter gene Spns2 was preferentially expressed in both TMC4 and vEC subsets, suggesting increased local concentration in mEFAs. These findings indicate that rat mEFAs are specialized medullary niches linking stromal organization, thymocyte maturation, and thymic egress.