Aire controls the recirculation of murine Foxp3⁺ regulatory T-cells back to the thymus

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4⁺ and CD8⁺ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self-antigen availability for negative selection. Interestingly, alterations in both Foxp3⁺ Regulatory T-cells (T-Reg) and conventional SP thymocytes in Aire^-/- mice suggest additional, yet poorly understood, roles for Aire during intrathymic T-cell development. To examine this, we analysed thymocytes from Aire^-/- mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post-selection CD4⁺ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αβT-cells, it plays a key role in controlling the intrathymic T-Reg pool. Surprisingly, a decline in intrathymic T-Reg in Aire^-/- mice maps to a reduction in mature recirculating Rag2GFP^- T-Reg that express CCR6 and re-enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire^-/- mice, and that CCR6 is required for T-Reg recirculation back to the thymus. Collectively, our study re-defines requirements for late stage intrathymic αβT-cell development, and demonstrates that Aire controls a CCR6-CCL20 axis that determines the developmental makeup of the intrathymic T-Reg pool.