Type I IFNs preferentially inhibit CD62L^loCD44^hi effector/memory T reg cells. (a, Left) Representative gating of CD44^lo and CD44^hi T reg cells. (a, Right) Absolute number of CD44^lo and CD44^hi CD4⁺Foxp3⁺ T reg cells in spleens of mice infected with LCMV-Armstrong. Data are representative of two independent experiments. n = 4 mice per group. Statistical significance was determined using one-way ANOVA with Tukey’s post-test. (b) Absolute number of CD44^lo (left) and CD44^hi (right) CD4⁺Foxp3⁺ T reg cells in mixed bone marrow chimeric mice left uninfected or 7 dpi. Data are representative of 6 independent experiments with 3–4 mice per group. Statistical significance was determined by two-tailed paired Student’s t test. Two-tailed unpaired Student’s t test was used when comparing cells from different mice. (c) Representative histograms (right) and MFI summary (left) of phospho-STAT1 in CD44^lo (white) and CD44^hi (black) T reg cells in spleens of mice infected with LCMV-Armstrong. Data are representative of two independent experiments. n = 4 mice per group. Statistical significance was determined using two-tailed paired Student’s t test. (d) Representative histograms (right) and MFI summary (left) of phospho-STAT1 in Ifnar1^−/−, WT CD44^lo and WT CD44^hi T reg cells, CD8⁺, and CD4⁺Foxp3⁻ T cells stimulated for 30 min with IFN-β. Data are representative of 3 independent experiments. (e) Representative histograms (right) and MFI summary (left) of IFNαR1, IFNαR2, and STAT1 in CD44^lo and CD44^hi CD4⁺Foxp3⁺ T reg cells determined by flow cytometry. n = 3 mice per group. Data are representative of three independent experiments. Statistical significance was determined using two-tailed paired Student’s t test. (f) miR-155 microRNA and Socs1 mRNA expression in sorted CD44^lo and CD44^hi CD4⁺Foxp3⁺ T reg cells_, expressed in arbitrary expression units (AEUs) normalized to U6 and Gapdh expression, respectively. Data are representative of two independent experiments with three mice each. Statistical significance was determined using two-tailed paired Student’s t test. (g Left) Summary of Ki67 expression by WT and Ifnar1^−/− CD4⁺Foxp3⁺CD62L^loCD44^hi (CD44^hi) T reg cells from mixed bone marrow chimeric mice left uninfected (black) or infected with LCMV for 7 d (gray). (g, Right) Representative histogram showing Ki-67 expression by WT (black) and Ifnar1^−/− (gray) CD4⁺Foxp3⁺CD62L^loCD44^hi T reg cells in mixed bone marrow chimeric mice 7 dpi with LCMV-Armstrong. n = 4 per group. Data are representative of 6 independent experiments. Statistical significance was determined by two-tailed paired Student’s t test. Two-tailed unpaired Student’s t test was used when comparing cells from different mice. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. All data are presented as the mean values ± SEM.