Age-dependent Zap70 expression in thymocytes regulates selection of the neonatal regulatory T cell repertoire

The Foxp3⁺ regulatory T (T_reg) cell repertoire carries age-dependent biases, with neonatal subsets enriched for highly self-reactive clones. However, the thymocyte features distinguishing neonatal from adult T_reg selection remain unclear. Here, we show that neonatal double-positive mouse thymocytes, unlike their adult counterparts, fail to upregulate Zap70 during thymic selection, creating a calcium signaling bottleneck. This attenuated Zap70-dependent signaling limits negative selection, allowing highly self-reactive clones to evade deletion. Modulating Zap70 expression alters this balance; reducing Zap70 in adults rescues development of these clones, whereas increasing Zap70 in neonates enforces their deletion. Similarly, enhancing neonatal calcium signaling via increased LAT Y136-mediated PLCγ1 activation promotes clonal deletion. Analysis of pediatric human thymi reveals that ZAP70 expression remains low during the first year of life, aligning with the peak window for thymic T_reg cell development. These findings suggest that age-dependent Zap70 expression regulates negative selection and thymic T_reg cell development.