Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control Aire+ mTEChi-precursors that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated or identified by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen-presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce in mTEClo the expression of tissue-restricted self-antigens, cytokines, chemokines and adhesion molecules important for T-cell development. This gene activation program is combined with a global increase of the active H3K4me3 histone mark. Finally, we show that these interactions induce key mTEC transcriptional regulators and govern mTEClo subset composition, including Aire+ mTEChi-precursors, post-Aire and tuft-like mTECs. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.