Distinct mutations in the autoimmune regulator gene differentially affect transcriptional and functional properties...

Autoimmune Polyendocrine Syndrome Type 1 (APS-1) is a rare monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene. Although AIRE is essential for central immune tolerance, how distinct APS-1-associated mutations differentially affect medullary thymic epithelial cell (mTEC) biology remains incompletely understood. Here, we investigated the molecular and functional consequences of three Aire/AIRE variants using complementary murine mTEC models. To define transcriptional effects, we performed single-cell RNA sequencing (scRNA-seq) on mTECs carrying a heterozygous genomic Aire c.735delG mutation generated by CRISPR-Cas9. This analysis revealed reduced transcriptional heterogeneity, decreased expression of tissue-restricted antigens (TRAs) mRNAs (including Col4a3, Col7a1, and Neto2), and downregulation of key mTEC lineage markers (Epcam, Cldn4, Krt14). Mutant cells also displayed altered expression of mRNAs involved in chemokine-mediated migration (Ccl25, Cxcl16), extracellular matrix and cell adhesion (Fn1, Lama5, Col4a1, Nectin1, Cdh1), and actin cytoskeleton organization (Gsn, Rac1, Wasl, Actn1), indicating broad disruption of pathways governing mTEC identity and cell-cell interactions. Guided by these findings, we assessed mutation-specific functional outcomes using a CRISPR-derived Aire functional knockout and lentiviral expression of the human AIRE missense variants p.G229W and p.C313Y in wild-type mTECs. Functional assays revealed mutation-dependent alterations in mTEC morphology, thymocyte migration, and adhesion, with the p.C313Y variant exerting the strongest effects. Together, these data demonstrate that heterozygous and missense AIRE mutations exert distinct yet convergent effects on mTEC transcriptional programs and cellular behavior, providing mechanistic insight into AIRE-dependent immune tolerance failure in APS-1.