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.