A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3⁺ T_reg cell development

The neonatal thymus generates Foxp3⁺ regulatory T (tT_reg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tT_reg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tT_reg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IA^b-Padi4 with moderate dwell times within a conventional docking orientation are exported as tT_reg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4⁺ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tT_reg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tT_reg selection window.