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-cony) from mouse lymphoid organs and human blood. T-reg and T-cony 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-reg expressed a small core of FoxP3-dependent transcripts, onto which additional programs were added less uniformly. Among suppressive functions, ll2ra 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.