Recirculating Foxp3⁺ regulatory T cells are restimulated in the thymus under Aire control

Thymically-derived Foxp3⁺ regulatory T cells (T_reg) critically control immunological tolerance. These cells are generated in the medulla through high affinity interactions with medullary thymic epithelial cells (mTEC) expressing the Autoimmune regulator (Aire). Recent advances have revealed that thymic T_reg contain not only developing but also recirculating cells from the periphery. Although Aire is implicated in the generation of Foxp3⁺ T_reg, its role in the biology of recirculating T_reg remains elusive. Here, we show that Aire regulates the suppressive signature of recirculating T_reg independently of the remodeling of the medullary 3D organization throughout life where T_reg reside. Accordingly, the adoptive transfer of peripheral Foxp3⁺ T_reg in Aire^KO recipients led to an impaired suppressive signature upon their entry into the thymus. Furthermore, recirculating T_reg from Aire^KO mice failed to attenuate the severity of multiorgan autoimmunity, demonstrating that their suppressive function is altered. Using bone marrow chimeras, we reveal that mTEC-specific expression of Aire controls the suppressive signature of recirculating T_reg. Finally, mature mTEC lacking Aire were inefficient in stimulating peripheral T_reg both in polyclonal and antigen-specific co-culture assays. Overall, this study demonstrates that Aire confers to mTEC the ability to restimulate recirculating T_reg, unravelling a novel function for this master regulator in T_reg biology.