Blockade of chronic type I interferon signaling to control persistent LCMV infection

Abstract

Type I interferons (IFN-I) are critical for antiviral immunity; however, chronic IFN-I signaling is associated with hyperimmune activation and disease progression in persistent infections. We demonstrated in mice that blockade of IFN-I signaling diminished chronic immune activation and immune suppression, restored lymphoid tissue architecture, and increased immune parameters associated with control of virus replication, ultimately facilitating clearance of the persistent infection. The accelerated control of persistent infection induced by blocking IFN-I signaling required CD4 T cells and was associated with enhanced IFN-γ production. Thus, we demonstrated that interfering with chronic IFN-I signaling during persistent infection redirects the immune environment to enable control of infection.

Fig. 1. Prolonged IFN-I signaling during persistent infection

(A) Splenic virus titers ± SD after acute LCMV-Arm (black) and persistent LCMV-Cl13 (red) infection (left). Gene expression kinetics by microarray analysis of the indicated IFNR-inducible, STAT, and IFN-I regulatory factors (IRFs) in whole spleen tissue after LCMV-Arm (black) or LCMV-Cl13 (red) relative to naïve (uninfected) mice. Each value indicates the average of three to four mice per group per time point. Values are shown without error bars for clarity, and the P values for each gene are indicated in table S1. PFU, plaque-forming units. (B) IFNα (left) and IFNβ (right) mRNA expression relative to a control gene, HPRT, in splenic DCs from naïve or LCMV-Arm– or LCMV-Cl13–infected mice (day 9). N.D. indicates that IFNα or IFNβ transcripts were not detected after 40 cycles of amplification. HPRT mRNA expression was measurable in all samples. (C) Mx1, OAS, and IRF3 mRNA expression relative to HPRT in the indicated IL-10+ (GFP+, green) or IL-10− (GFP−, black) splenic DCs (top) or macrophages (bottom) 9 days after LCMV-Cl13 infection of Vert-X IL-10 reporter mice. For (B) and (C), each group is a pool of cells from six to eight mice and is representative of two independent experiments.

Fig. 2. The immunosuppressive program during persistent infection is dependent on IFN-I signaling

(A) Representative histograms and summarized quantification of geometric mean fluorescence intensity (GMFI) show PD-L1 on splenic DCs and macrophages 9 days after LCMV-Cl13 infection in wild-type (WT, black) and Ifnar1^−/− (red) mice. Scatter plots show plasma IL-10 levels on day 9 after LCMV-Cl13 infection. Bar graphs measure IL-10 production by cultured splenocytes (in the absence of exogenous stimulation) isolated 9 days after LCMV-Cl13 infection from WT or Ifnar1^−/− mice. Plasma viral titers in WT or Ifnar1^−/− mice on day 9 after LCMV-Cl13 infection are shown to the far right. Dashed lines indicate the level of detection of the plaque assay (200 PFU). (B to D) WT mice were treated with isotype or IFNR1 blocking antibody beginning 1 day before LCMV-Cl13 infection. (B) Graphs indicate GMFI of PD-L1 on splenic CD45+ cells (left), plasma levels of IL-10 (middle), and plasma viral titers (right) of untreated mice (B6), isotype-treated mice, IFNR1 blocking antibody (αIFNR1)–treated mice, and untreated Ifnar1^−/− mice. Plasma IL-10 levels on day 15 after LCMV-Cl13 infection are shown on the far right. (C) Flow cytometry plots of IL-10 reporter expression (GFP) in splenic DCs from Vert-X mice treated with isotype or IFNR1 blocking antibody. Bar graphs show the frequency of IL-10 expression and the GMFI of PD-L1 expression by splenic DCs. The ratio of IL-10–nonproducing to IL-10–producing DCs is shown on the far right. (D) Hematoxylin and eosin staining of spleens from naïve mice (left) or on day 9 after LCMV-Cl13 infection of mice treated with isotype (middle) or IFNR1 blocking antibody (right). Symbols represent individual mice with bars indicating the mean of the group. In bar graphs, the data represent the average ± SD of three to six mice per group. All data are representative of two or more independent experiments. *P < 0.05.

Fig. 3. IFNR1 blockade enhances control of persistent infection

WT mice were treated with isotype or IFNR1 blocking antibody beginning 1 day before LCMV-Cl13 infection. (A) Plasma virus titers at the indicated time points after infection. (B) Viral titers in plasma, liver, and kidney 30 days after infection. (C) Graphs indicate total numbers of IFN-γ–expressing and of multicytokine-producing (polyfunctional) LCMV-GP_61–80–specific CD4 T cells and LCMV-GP_33–41–specific CD8 T cells. (D) Plasma viral titers at day 30 after LCMV-Cl13 infection in mice that were either undepleted of cells and treated with isotype (iso/iso) or IFNR1 (αIFNR1/iso) blocking antibody or depleted of CD4 T cells (αCD4) or NK cells (αNK) before infection and IFNR1 blocking antibody treatment. (E) Plasma virus titers at the indicated time point after LCMV-Cl13 infection in mice that were either treated with isotype or IFNR1 blocking antibody with or without anti–IFN-γ (αIFN-γ). x axis labels indicate (top) IFNRI or isotype and (bottom) cell or IFN-γ depleting antibody treatments. Each symbol in the scatter plots represents an individual mouse with bars indicating the mean of the group. Dashed lines indicate the level of detection of the plaque assay (200 PFU). Data are representative of two or more independent experiments. *P < 0.05.

Fig. 4. Therapeutic IFNR1 antibody blockade dampens the immunosuppressive program and facilitates clearance of persistent infection

WT mice were treated with isotype or IFNR1 blocking antibody beginning on day 25 after LCMV-Cl13 infection. (A) Plasma viral titers at the indicated time points. The percent of mice at each time point exhibiting detectible virus titers is summarized in the kinetic graph to the right, with the shaded area representing the time of treatment. The data are combined from two experiments. (B) Viral titers in the plasma, liver, and kidney at day 46 after LCMV-Cl13 infection. (C) OAS, Mx1, and IRF7 mRNA expression relative to HPRT in splenocytes from naïve mice (white) or on day 30 after LCMV-Cl13 infection in mice treated with isotype (black) or IFNR1 (red) blocking antibody. (D) Graphs indicate GMFI of PD-L1 on splenic dendritic cells (left) and plasma IL-10 levels (right) on day 30 after LCMV-Cl13 infection in mice treated with isotype or IFNR1 blocking antibody. Each symbol in the scatter plot represents an individual mouse, and bar graphs indicate the average value ± SD. All data are representative of two to five independent experiments. *P < 0.05.