A novel method to identify Post-Aire stages of medullary thymic epithelial cell differentiation

Autoimmune regulator⁺ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire⁺ mTECs differentiate further into Post-Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate-mapping mouse models. Herein, we resolve this limitation by segmenting the mTEC^hi (MHCII^hi CD80^hi ) compartment into mTEC^A/hi (CD24^- Sca1^- ), mTEC^B/hi (CD24⁺ Sca1^- ), and mTEC^C/hi (CD24⁺ Sca1⁺ ). While mTEC^A/hi included mostly Aire-expressing cells, mTEC^B/hi contained Aire⁺ and Aire^- cells and mTEC^C/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor-product relationship between these subsets. Strikingly, transcriptomic analysis of mTEC^A/hi , mTEC^B/hi , and mTEC^C/hi sequentially mirrored the specific genetic program of Early-, Late- and Post-Aire mTECs. Corroborating their Post-Aire nature, mTEC^C/hi downregulated the expression of tissue-restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire-deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.