Cross-species chromatin accessibility pinpoints HIVEP3 as a driver of thymic epithelial maturation and self-antigen expression, preventing chronic inflammation

Abstract

Promiscuous expression of tissue-specific antigens (TSAs) by medullary thymic epithelial cells (mTECs) underpins central T-cell tolerance, relying on NF-{kappa}B-driven mTEC maturation and induction of the autoimmune regulator AIRE. However, a substantial fraction of TSAs is AIRE-independent, implying additional regulators that remain only partially identified. Using cross-species single-cell chromatin accessibility profiling of human and mouse TECs, we find that HIVEP motifs are among the most accessible regulatory elements across the mTEC lineage. Among HIVEP paralogs, HIVEP3 is robustly expressed in mature mTECs across human, mouse and rat; given that rat immunity more closely mirrors human physiology than the mouse, we generated a CRISPR Hivep3-knockout rat line. Hivep3 deletion impairs mTEC maturation, reduces Aire expression and reshapes the TSA repertoire that underlies negative selection and Treg induction, as Hivep3 constrains canonical NF-{kappa}B1 and sustains non-canonical NF-{kappa}B2, the principal mediator of mTEC maturation. Beyond mTECs, Hivep3 deficiency downregulates Foxn1 target genes in cortical TECs and impairs positive selection. In the periphery, Hivep3-KO rats show reduced splenic CD4 T-cells, a shift from naive towards effector-and central-memory phenotypes, increased regulatory T-cells, and a sustained Th1-biased serum profile (elevated IFN-{gamma}, reduced IL-17A) with concurrent systemic metabolic perturbations across all ages. With age, this culminates in chronic inflammation, with multi-organ CD3 T-cell infiltration accompanied by inflammatory lesions. Together, these findings establish HIVEP3 as a previously unrecognised regulator of thymic epithelial function and central tolerance, whose deficiency leads to systemic T-cell-mediated chronic inflammation.