Runx3 instructs Aire⁺ mTEC development, TSA gene expression, and central tolerance

The transcription factor Runx3 modulates key gene expression programs important for the development of multiple tissues. Here, we uncover new functions for Runx3 in thymic epithelial lineage development and show Runx3 expression in medullary thymic epithelial cells (mTECs) is required for both Autoimmune Regulator + (Aire⁺) mTEC development and tissue-specific antigen (TSA) gene expression. Consequently, TEC-specific deletion of Runx3 in mice results in a profound decrease in Aire ⁺ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, loss of Runx3 in TECs results in an expansion of immature CCL21⁺ mTECs and a loss of Aire-dependent mimetic cells. Single-cell analysis reveals Runx3 modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Runx3 in mTEC development and thymic central tolerance.