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 the thymus. In turn, thymocytes regulate the cellularity of Aire + mTEC hi . However, it remains unknown whether thymocytes control the precursors of Aire + mTEC hi that are contained in mTEC lo cells or other mTEC lo subsets that have recently been delineated 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 key transcriptional regulators in mTEC lo and control the composition of mTEC lo subsets, including Aire + mTEC hi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEC lo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4 + thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4 + thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.