(A) Frequency of CD3⁺CD4⁺Foxp3⁺ Tregs in the indicated tissues of adult mice. (B) Percentage (left) and number (right) of CD3⁺CD4⁺Foxp3⁺ Tregs in the peripheral lymph node of adult and old mice. (C) Representative flow cytometry from the peripheral blood of adult mice. (D) Representative flow cytometry from the peripheral lymph node showing staining for the indicated markers, gated on WT conventional T cells (T_conv) (CD3⁺CD4⁺EGFP^–), or Tregs (CD3⁺CD4⁺EGFP⁺) from WT and G421R mice. Data are representative of at least 9 mice and 4 independent experiments. (E) Suppressive capacity of WT and G421R Tregs as measured by Tag-it Violet dilution on responder T cells. Graph depicting the mean ± SEM of the percent suppression, at each Treg/CD4 ratio (left). Representative histograms are shown along with the average proliferation of CD4⁺ T cells for selected Treg to CD4 ratios (right). Percent suppression = (percent proliferation of the CD4⁺ T cells at the 0:1 Treg/Teff ratio — percent proliferation of CD4⁺ T cells for each Treg to CD4 ratio)/percent proliferation of the CD4⁺ T cells at the 0:1 Treg/Teff ratio. Data were from 3 experiments consisting of 8–9 individual assays. (F) In vitro–derived iTregs were generated from WT or G421R CD4⁺EGFP^–CD45RB^hi naive T cells with anti-CD3, anti-CD28, TGF-β1, and IL-2. Representative flow cytometry (left) and scatter plot showing the frequency of CD4⁺Foxp3⁺ Tregs (right). Each point represents an individual mouse and at least 3 independent experiments. An unpaired t test was used for comparisons with 2 groups, a Welch’s t test was used for unequal variance, and — for 3 or more groups — 1-way ANOVA was used, except for in E, which was analyzed with 2-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.