Treg-specific physiological regulation of TCR signaling molecules differentially affects Treg and Tconv TCR signaling. (A) ZAP-70 and Nur77 levels in Foxp3⁻CD4⁺ Tconv and Foxp3⁺ Treg cells from ZAC or BALB/c WT mice upon in vitro αCD3/αCD28 antibody stimulation for 24 h. Numbers indicate median fluorescence intensity (MFI; n = 5). (B and C) Foxp3 binding (peaks) to Zap70 and genes encoding TCR signaling molecules (B), and gene expression levels of TCR signaling molecules by CD4⁺ Tconv and Treg cells before and after stimulation (C). Expression levels as indicated by tag counts per million (tpm) were analyzed from DeepCAGE database (Morikawa et al. 2014); *Foxp3-bound genes commonly detected in two ChIP-seq databases. The arrows indicate transcription start site and boxes indicate exons in B. (D) Proliferation of Foxp3⁺ Treg and Foxp3⁻CD4⁺ T cells from WT or rtTA⁺hZAP-70⁺ mice cultured with αCD3 antibody and 1 μg/ml Dox (n = 3).