Single-cell gene expression reveals a landscape of regulatory T cell phenotypes shaped by the TCR

CD4⁺ T regulatory cells (T_reg) are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells that they regulate. To understand this heterogeneity, we combined single-cell RNA-seq, activation reporter and T cell receptor (TCR) analysis to profile thousands of T_reg or conventional CD4⁺FoxP3^- T cells (T_conv) from mouse lymphoid organs and human blood. T_reg and T_conv pools showed areas of overlap, as resting 'furtive' T_regs with overall similarity to T_convs or as a convergence of activated states. All T_regs expressed a small core of FoxP3-dependent transcripts, onto which additional programs were added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasiconstant, inhibitory cytokines being more sparsely distributed. TCR signal intensity did not affect resting/activated T_reg proportions but molded activated T_reg programs. The main lines of T_reg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations of T_reg heterogeneity.