CD161+FoxP3+ regulatory T cells share phenotypic features with memory effector T cells. (A) Production of cytokines ex vivo in Treg subpopulations was assessed by intracellular flow cytometric staining after stimulation with PMA and ionomycin. Dot plots show intracellular cytokine staining for IL-17A (top left), IFN-γ (top right), and IL-2 (bottom left) gated on FoxP3+CD161– and FoxP3+CD161+ peripheral blood Treg, respectively, in 1 representative healthy adult, with summary plots (bottom right) of staining on healthy adult (n = 13-19) and child (n = 4-6) peripheral blood samples. (B) Representative staining showing double positive cytokine production (left, IL-17A vs IFN-γ; right, IFN-γ vs IL-2) comparing CD161–FoxP3+ to CD161+FoxP3+ cells. (C) Expression of effector cell markers on CD161+FoxP3+ cells. Representative plots showing expression of CCR6 (top), IL-23R (middle), and CD45RO (bottom) on FoxP3+CD161– and FoxP3+CD161+ Treg, respectively, with summary plots (right) for healthy adult (n = 16-28) and child (n = 6-8) peripheral blood samples. (D) Transcription factor expression in CD161+FoxP3+ cells. Fold change in mRNA expression of FOXP3, RORCv2, and TBX21 (Tbet) in sorted CD161+ Treg relative to sorted CD161– Treg assayed by RT-PCR (n = 3-6; top). RORCv2 and Tbet protein expression was assessed by flow cytometry ex vivo. Representative FACS plots of expression of RORCv2 (middle left) and Tbet (middle right) in CD161–FoxP3+ (open histogram) compared with CD161+FoxP3+ cells (filled histogram) and isotype (gray histogram); summary graphs of FACS data are shown below (n = 7). Data in dot plots and histograms are derived from gated CD4+FoxP3+ lymphocytes. In scatter plots, horizontal lines represent median; in bar graph, bars represent mean ± SEM with Wilcoxon matched pairs test between cell subsets, and between groups 1-way nonparametric ANOVA (Kruskal-Wallis with Dunn’s test). ***P < .001; **P < .01; *P < .05.