CD161+ regulatory T cells are stable in vitro and possess a predominantly demethylated TSDR. (A-D) Sorted Treg subpopulations were labeled with CFSE, “spiked” back into autologous PBMC at a frequency of 5% to 7%, and cultured for 3 days on plates coated with or without anti-CD3/CD28 and stimulated with either medium alone or cytokines as shown. Schematic of experimental setup for in vitro stability analysis is shown in panel A; representative overlay histograms of CD161 expression gated on CFSE+ cells and summary plots of CD161 MFI at day 0 (n = 5) (B), after 3-day culture in medium alone (n = 3) (C), and after 3-day culture on anti-CD3/CD28–coated plates ± cytokines (n = 3) (D, top); cytokine production by the labeled CD161– or CD161+ Treg was also assessed on day 3 (C; D, bottom). Data in histograms are events gated on CD4+CFSE+ live cells. For both histograms and graphs, clear symbols represent CD161– Treg, and filled symbols CD161+Treg. In bar graph, bars represent means ± SEM with Wilcoxon matched pairs test between cell subsets, and between groups 1-way nonparametric ANOVA (Kruskal-Wallis with Dunn’s test). **P < .01; *P < .05. (E) Both CD161– Treg and CD161+ Treg cells contain a predominantly demethylated FOXP3 TSDR. DNA was extracted from sorted Tconv, CD161– Treg, and CD161+ Treg (purity >80%) and bisulfite treated, and the TSDR was amplified and then cloned. Twenty to 35 individual clones were sequenced, and the percentage of methylated CpG islands determined. Panel E shows a schematic of the FOXP3 locus representing the location of the 12 CpG islands analyzed (positions +3878 to +4082), with mean % methylation of each CpG island displayed (left) and the average for all islands (right; blue, 100%, and yellow, 0%; 1 of 3 representative individuals shown).