Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection

Abstract

Regulatory T (T reg) cells play an essential role in preventing autoimmunity but can also impair clearance of foreign pathogens. Paradoxically, signals known to promote T reg cell function are abundant during infection and could inappropriately enhance T reg cell activity. How T reg cell function is restrained during infection to allow the generation of effective antiviral responses remains largely unclear. We demonstrate that the potent antiviral type I interferons (IFNs) directly inhibit co-stimulation-dependent T reg cell activation and proliferation, both in vitro and in vivo during acute infection with lymphocytic choriomeningitis virus (LCMV). Loss of the type I IFN receptor specifically in T reg cells results in functional impairment of virus-specific CD8(+) and CD4(+) T cells and inefficient viral clearance. Together, these data demonstrate that inhibition of T reg cells by IFNs is necessary for the generation of optimal antiviral T cell responses during acute LCMV infection.

Figure 1.

T reg cells are dynamically regulated during LCMV infection. (a) Proportion of Ki-67⁺ cells among CD4⁺Foxp3⁺ T reg cells (left) and absolute number of CD4⁺Foxp3⁺ T reg cells (right) in spleens of mice left uninfected (U) or at various dpi with LCMV-Armstrong. (b) Proportion of Ki-67⁺ cells (left) and absolute number (right) of CD4⁺Foxp3⁻ T cells (bottom) and CD8⁺ T cells (top) in spleens of mice infected with LCMV-Armstrong. (c) Fold induction of Ifnb mRNA from whole spleens of mice infected with LCMV-Armstrong relative to uninfected controls. Ifnb mRNA expression was normalized to Gapdh expression. (d) Median fluorescence intensity (MFI; top) and representative staining (bottom) of phospho-STAT1 in gated CD4⁺Foxp3⁺ T reg cells directly ex vivo from spleens of mice infected with LCMV-Armstrong. For all panels, n = 3–4 mice per group. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (a–d). *, P < 0.05; **, P < 0.005; ***, P < 0.0001. Data are representative of two independent experiments with 3–4 mice per time point. All data are presented as the mean values ± SEM.

Figure 2.

Type I IFNs directly inhibit T reg cell proliferation and activity in vitro. (a, Left) T reg cell proliferation assay showing CPDye dilution of gated WT (left) or Ifnar1^−/− (right) CD4⁺Foxp3⁺ T reg cells cultured with WT (top) or Ifnar1^−/− APCs (bottom) in the absence (gray) or presence (black) of 50 U/ml IFN-β after 72 h of culture with soluble CD3 and IL-2. (a, Right) Division index (top) and absolute number (bottom) of WT (black) or Ifnar1^−/− (gray) T reg cells after 72 h of culture in the indicated conditions. (b, Left) In vitro suppression assay showing CPDye dilution of Ifnar1^−/− CD4⁺Foxp3⁻ T_conv cells in the presence of WT or Ifnar1^−/− T reg cells at the indicated T reg/T_conv cell ratios in the absence (gray) or presence (black) of 50 U/ml IFN-β after 72 h of culture with soluble CD3 and irradiated Ifnar1^−/− APCs. (b, Right) Absolute number of and percent suppression by WT (black) or Ifnar1^−/− (gray) T reg cells at decreasing T reg/T_conv cell ratios after 72 h culture in the absence (−) or presence (+) of 50 U/ml IFN-β. Data are representative of 3 independent experiments.

Figure 3.

Type I IFNs directly inhibit T reg cell proliferation and activity during LCMV infection. (a, Left) Ratio of Ifnar1^−/−/WT CD4⁺Foxp3⁺ T reg cells in spleens of mixed bone marrow chimeric mice left uninfected or 7 dpi with LCMV Armstrong. (a, Right) Absolute number of WT (black) and Ifnar1^−/− CD4⁺Foxp3⁺ T reg cells in spleens of mixed bone marrow chimeric mice left uninfected or 7 dpi. (b, Left) Summary of Ki-67 expression by WT (black) and Ifnar1^−/− (gray) CD4⁺Foxp3⁺ T reg cells from spleens of mixed bone marrow chimeric mice left uninfected or 7 dpi. (b, Right) Representative flow cytometry analysis of Ki-67 expression by gated CD45.1⁺ WT (black) and CD45.2⁺ Ifnar1^−/− (gray) CD4⁺Foxp3⁺ T reg cells in spleens of mixed bone marrow chimeric mice 7 dpi. (c) Summary of Ki-67 expression by WT (black) and Ifnar1^−/− (gray) CD8⁺ and CD4⁺Foxp3⁻ T cells from spleens of mixed bone marrow chimeric mice left uninfected or 7 dpi. (d) Representative flow cytometry analysis and summaries of the indicated markers expressed by WT (black) and Ifnar1^−/− (gray) CD4⁺Foxp3⁺ T reg cells in spleens of mixed bone marrow chimeric mice left uninfected or 7 dpi with LCMV-Armstrong. (e, Left) Ratio of Ifnar1^−/−/WT CD4⁺Foxp3⁺ T reg cells in small intestinal LP (SI-LP) of mixed bone marrow chimeric mice left uninfected or 7 dpi with LCMV Armstrong. (e, Right) Absolute number of WT (black) and Ifnar1^−/− CD4⁺Foxp3⁺ T reg cells in SI-LP of mixed bone marrow chimeric mice left uninfected or 7 dpi. (f, Left) Summary of Ki-67 expression by WT (black) and Ifnar1^−/− (gray) CD4⁺Foxp3⁺ T reg cells from SI-LP of mixed bone marrow chimeric mice left uninfected or 7 dpi. (f, Right) Representative flow cytometry analysis of Ki-67 expression by gated CD45.1⁺ WT (black) and CD45.2⁺ Ifnar1^−/− (gray) CD4⁺Foxp3⁺ T reg cells in SI-LP of mixed bone marrow chimeric mice 7 dpi. Statistical significance was determined using two-tailed paired t test when comparing WT and Ifnar1^−/− cells within the same chimeric mouse. Two-tailed unpaired Student’s t test was used when comparing cells from different mice. n = 4 per group. Data are representative of 6 (a–d) or 2 (e and f) independent experiments with 3–4 mice per group. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. All data are presented as the mean values ± SEM.

Figure 4.

Neither WT nor Ifnar1^−/− T reg cells are GP_66-77 specific during LCMV infection. Left: representative histograms showing I-A^b/GP_66-77 tetramer staining among gated WT (left) or Ifnar1^−/− (right) CD4⁺Foxp3⁻ T cells (top) and CD4⁺Foxp3⁺ T cells (bottom) from spleens of the same mixed bone marrow chimeric mouse 7 dpi with LCMV-Armstrong. Numbers represent frequency of tetramer-positive cells among the indicated populations. Right: absolute number of the indicated tetramer-positive populations in spleens of chimeric mice 7 dpi with LCMV-Armstrong. Statistical significance was determined using two-tailed paired Student’s t test. n = 4 per group. Data are representative of 2 independent experiments with 3–4 mice per group. *, P < 0.05, P < 0.0001. All data are presented as the mean values ± SEM.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.