How autoreactive thymocytes differentiate into regulatory versus effector CD4⁺ T cells after avoiding clonal deletion

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (T_reg) or effector T (T_eff) CD4⁺ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that T_reg and T_eff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4⁺CD25⁺ precursors. Disruption of agonist signaling induces CD4⁺CD25⁺ precursors to initiate Foxp3 expression and become T_reg cells, whereas persistent agonist signaling induces CD4⁺CD25⁺ precursors to become IL-2⁺ T_eff cells. Notably, we discovered that transforming growth factor-β induces Foxp3 expression and promotes T_reg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.