Thymic regulatory T cells arise via two distinct developmental programs

The developmental programs that generate a broad repertoire of regulatory T cells (T _reg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature T _reg cells were generated through two distinct developmental programs involving CD25 ⁺ T _reg cell progenitors (CD25 ⁺ T _reg P cells) and Foxp3 ^lo T _reg cell progenitors (Foxp3 ^lo T _reg P cells). CD25 ⁺ T _reg P cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3 ^lo T _reg P cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3 ^lo T _reg P cells. The development of both CD25 ⁺ T _reg P cells and Foxp3 ^lo T _reg P cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25 ⁺ T _reg P cells and Foxp3 ^lo T _reg P cells arose by coopting negative-selection programs and positive-selection programs, respectively. T _reg cells derived from CD25 ⁺ T _reg P cells, but not those derived from Foxp3 ^lo T _reg P cells, prevented experimental autoimmune encephalitis. Our findings indicate that T _reg cells arise through two distinct developmental programs that are both required for a comprehensive T _reg cell repertoire capable of establishing immunotolerance.