AIRE Deficiency Impairs Thymic Epithelial Progenitor Differentiation from Mouse Embryonic Stem Cells and Accelerates Type 1 Diabetes in NOD/Ltj Mice

The autoimmune regulator (AIRE) is essential for central immune tolerance, but its role in thymic epithelial cell (TEC) differentiation and development remains incompletely understood. To address this, we knocked down AIRE in mouse embryonic stem cells (mESCs) and analyzed thymic epithelial progenitor (TEP) differentiation using RNA-sequencing and also performed thymic AIRE knockdown in NOD/Ltj mice to evaluate type 1 diabetes (T1D) pathogenesis. AIRE knockdown significantly reduced the proportion of K5+/K8+ TEPs among EpCAM1+ cells and altered the ECM–receptor interaction pathway, with downregulation of SPP1 and upregulation of Fn1 and CD44. Importantly, re-expression of AIRE using an shRNA-resistant rescue vector partially reversed these molecular changes and partially restored TEP differentiation. In vivo, thymic AIRE knockdown accelerated blood glucose elevation, aggravated insulitis, and reduced expression of T1D-associated TSAs (INS2, GAD67) and maturation markers (CD80, MHC-II). Importantly, the changes in SPP1, Fn1, and CD44 were consistent between in vitro and in vivo models. Furthermore, AIRE deficiency increased CD4+ single-positive thymocytes, reduced splenic Treg frequency, and enhanced antigen-specific T cell proliferation and activation. Collectively, these findings demonstrate that AIRE deficiency impairs TEP differentiation and alters the ECM-receptor pathway, while thymic AIRE deficiency compromises immune tolerance and accelerates T1D progression in NOD/Ltj mice.