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 (mTEC ^hi ) are critical for the induction of central tolerance. In turn, thymocytes regulate the cellularity of Aire ⁺ mTEC ^hi . However, it remains unknown whether thymocytes control Aire ⁺ mTEC ^hi -precursors that are contained in mTEC ^lo cells or other mTEC ^lo 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 mTEC ^lo 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 mTEC ^lo subset composition, including Aire ⁺ mTEC ^hi -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.