Enhanced proliferation of Ifnar1^−/− T reg cells is ICOSL- and CD28-dependent. (a, Left) Summary of Ki-67 expression and absolute number of transferred WT (black) and Ifnar1^−/− (open squares) CD4⁺Foxp3⁺ T reg cells in WT mice 4 dpi with LCMV-Armstrong and treated with IgG or blocking anti–IL-2 antibody (αIL-2). (a, Right) Representative flow cytometric analysis showing pSTAT5 and CD25 expression by total CD4⁺Foxp3⁺ T reg cells in spleens of mice 4 dpi treated with IgG or αIL-2. Numbers represent frequency of the indicated quadrant among total CD4⁺Foxp3⁺ T reg cells. (b) Summary of Ki-67 expression and absolute number of transferred WT (black) and Ifnar1^−/− (open squares) CD4⁺Foxp3⁺ T reg cells in WT or Il15^−/− recipient mice 4 dpi with LCMV-Armstrong. (c) summary of Ki-67 expression and absolute number of transferred WT (black) and Ifnar1^−/− (open squares) CD4⁺Foxp3⁺ T reg cells in WT mice 4 dpi with LCMV-Armstrong and treated with IgG or blocking anti–B7-1/B7-2 antibodies (αB7-1/B7-2). (d) Summary of Ki-67 expression and absolute number of transferred WT, Ifnar1^−/−, or Ifnar1^−/−Cd28^−/− T reg cells in spleens of WT mice 4 dpi with LCMV-Armstrong. (e) Summary of Ki-67 expression and absolute number of transferred WT (black) and Ifnar1^−/− (open squares) CD4⁺Foxp3⁺ T reg cells in WT mice 4 dpi treated with IgG or blocking anti-ICOSL antibody (αICOSL). a–c, e: statistical significance was determined using paired two-tailed Student’s t test when comparing WT and Ifnar1^−/− T reg cells in the same recipient mice or unpaired two-tailed Student’s t test when comparing T reg cells in different mice. d: statistical significance was determined using one-way ANOVA with Tukey’s post-test. For all panels, n = 3 per group. Results are representative of 2 independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. All data are presented as the mean values ± SEM.