A new blueprint of medullary thymic epithelial cells of the Aire lineage

Medullary thymic epithelial cells (mTECs) provide critical microenvironments for tolerance induction in the thymus. Recent reports demonstrated that mTEC define an heterogenous functional compartment. While mTEChi include mature Aire+ cells, mTEClo contain mTEC-restricted precursors and CCL21+, post-Aire and Tuft-like cells. Still, the prospective isolation of these populations remains challenging due to the lack of suitable markers. Combining the simultaneous analysis of CD24 and Sca1, we identified three novel mTEC subsets in the postnatal thymus termed mTECA (CD24+Sca1-), mTECB (CD24+Sca1+) and mTECC (CD24-Sca1-). Notably, mTECB/lo and mTECC/hi defined CCL21+ and Aire+ subsets. We showed that mTECB/lo contained cells with the potential to generate Aire+ cells, suggesting their relationship to the Aire lineage. Strikingly, mTECB/lo, mTECC/hi and mTECB/hi presented molecular signatures that mapped them to Pre-, Early- and Late/Post-Aire stages. Supportive of their link to Aire-mediate differentiation program, Aire-deficient thymus lacked mTECB/lo and mTECB/hi and accumulated mTECC/hi. Our novel phenotypic profiling resolves distinct stages of the CCL21 and Aire mTEC lineages.