Inhibition of T reg cells by type I IFNs is necessary for optimal antiviral immune responses. (a) Representative flow plots showing gated CD4⁺ T cells from spleens of Foxp3^DTR mice left untreated (left) or treated daily for 7 d with 5 µg/kg DT. DT-treated mice were reconstituted with either no T reg cells (center left), WT T reg cells (center right), or Ifnar1^−/− T reg cells (right) at the start of DT treatment. Numbers represent frequency of Foxp3⁺ T reg cells among gated CD4⁺ T cells. (b) Representative flow cytometry analysis showing IFNαR1 expression on gated CD4⁺ T cells (left) and summary of IFNαR1 expression on CD4⁺Foxp3⁺ T reg cells (right) from peripheral blood of Foxp3^DTR mice replaced with WT or Ifnar1^−/− T reg cells one day before infection with LCMV. Numbers represent frequency of the indicated quadrants among total CD4⁺ T cells. n = 5 per group. Data are representative of three independent experiments. (c) Representative flow plots (left) and summary (right) showing frequency of Foxp3⁺ T reg cells among gated CD4⁺ T cells in WT- and Ifnar1^−/−-replaced Foxp3^DTR mice 7 dpi with LCMV-Armstrong. Numbers represent frequency of CD4⁺Foxp3⁺ T reg cells among total CD4⁺ T cells. (d, Left) Representative flow cytometric analysis of IFN-γ production by gated CD8⁺ T cells (top) and gated CD4⁺Foxp3⁻ T cells (bottom) by intracellular cytokine staining 7 dpi in WT- and Ifnar1^−/−-replaced Foxp3^DTR mice after 5-h stimulation of whole splenocytes with GP_33-41 (top) or GP_61-80 (bottom) peptide. Numbers represent frequency of IFN-γ⁺ cells among CD8⁺ (top) and CD4⁺Foxp3⁻ (bottom) T cells. (d, Right) Absolute number of LCMV peptide-specific IFN-γ⁺CD8⁺ (top) and IFN-γ⁺CD4⁺Foxp3⁻ T cells (bottom). (e) Absolute number of CD8⁺ T cells staining positively for D^b/GP_33-41 tetramer as assessed by flow cytometry. (f) Representative flow cytometric histograms from in vivo cytotoxicity assay showing frequency of CPDye^hi-labeled GP_33-41 peptide-pulsed splenocytes relative to CPDye^lo-labeled unpulsed splenocytes in WT- and Ifnar1^−/−-replaced Foxp3^DTR mice left uninfected or 7 dpi, 1 h after transfer of splenocytes. (g) Summary of percent killing of peptide-pulsed splenocytes in WT- and Ifnar1^−/−-replaced Foxp3^DTR mice 7 dpi. (h) LCMV GP RNA expression measured by qPCR in infected spleens and normalized to a standard curve generated using an LCMV-GP plasmid. c–e: n = 10 per group; data are summarized from 3 independent experiments. For all panels, statistical significance was determined using a two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. All data are presented as the mean values ± SEM.