a, Expression of CD25 on CD4+Foxp3–YFP+ cells isolated from the spleen and lymph nodes of three-week-old RISP KO and RISP wild-type mice. b, Surface expression of CTLA-4 and GITR on CD4+Foxp3–YFP+CD25+ cells from three-week-old RISP KO and RISP wild-type mice. c, Cellular expression of EOS and Helios from CD4+Foxp3+CD25+ cells isolated from the spleen and lymph nodes. d, Surface expression of activation makers CD44, CD69, CD103, ICOS and OX40 on CD4+Foxp3–YFP+CD25+ cells isolated from the spleen and lymph nodes. e, Ki-67 expression in CD4+Foxp3+CD25+ cells from the spleen and lymph nodes. f, Percentage of central T reg (cT reg ) and effector T reg (eT reg ) cells among T reg cells isolated from the spleen and lymph nodes of three-week-old RISP KO (n = 4 for both tissues) and RISP wild-type (spleen, n = 6, lymph nodes, n = 8) mice with representative contour plot. g, Representative histograms of CD69, CD73 and NRP1 expression on cT reg and eT reg cells isolated from lymph nodes of three-week-old RISP KO and RISP wild-type mice. h, Cell trace violet (CTV) dilution in CD8+ effector T cells stimulated to proliferate with varying ratios of RISP wild-type and RISP KO CD4+Foxp3–YFP+CD25+ cells isolated from the lymph nodes of three-week-old mice. Data are representative of five mice, performed on at least three separate days. Numbers in top left corner represent the division index of each histogram. i, Representative images of the colons from RAG1-deficient mice, one month following adoptive transfer. Images are representative of at least three mice collected on independent days. Contour plots are representative at least three independent experiments with at least four mice. Numbers in dot plot quadrants indicate percentage of cells. Histograms are representative of at least three independent experiments with at least five mice in total. In graphs: blue, RISP wild type; red, RISP KO. Numbers on histograms represent mean fluorescence intensity (MFI) of depicted samples; y axis of all histograms represents percentage of maximum. Data in f analysed with multiple two-tailed t-tests using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 0.01; Q values in Source Data. Each cell type was analysed individually, without assuming a consistent s.d. All data points on graphs represent individual mice isolated and analysed on at least two separate days. Source Data