Figure 1.
Antiretroviral activity of different IFN-α subtypes in vivo.
(B10.A×A.BY) F1 mice were treated daily with 8000 units of IFN-α2, α5 or α11 from −1 through +9 dpi with 7000 SFFU of FV. Ten dpi, disease progression and viral loads were analyzed. Virus-induced splenomegaly was documented by spleen weights (A) and viral loads were measured in the spleen (B) by using an infectious center assay. To analyze the long-term protection of IFN-α11 treatment, we treated mice with IFN-α11 during the acute phase of FV infection (−1 through +9 dpi). At 6 weeks post infection, viral loads were analyzed by an infectious center assay (C). At least 5 mice per group were analyzed and the mean values for each group are indicated by a bar. At least 2 independent experiments were performed. Differences between the control group (FV) and the groups of treated mice (FV+IFN-α2, α5, or α11) were analyzed.
Figure 2.
Analysis of NK cells from IFN-α11-treated mice.
(B10.A×A.BY) F1 mice were treated daily with 8000 units of IFN-α2, α5 or α11 from −1 through +9 dpi with 7000 SFFU of FV. At 10 dpi, splenocytes were analyzed by flow cytometry. The early activation marker CD69 was used to analyze the frequencies of activated NK cells (NK1.1+ CD3− CD49b+) (A). C57BL/6 mice were treated daily with 8000 units of IFN-α11 from −1 through +9 dpi with 20,000 SFFU of FV. NK cells were depleted by 5 injections of supernatant of clone PK136, starting at the day of infection. IFN-α11-treated non-depleted mice and NK cell depleted, non-treated mice were used as control groups. At 10 dpi viral loads in the spleen were determined (B). A minimum of 3 mice per group were analyzed and the mean values are shown by bars. At least 2 independent experiments were performed. Statistically significant differences between the groups are indicated by ** for p<0.005, *** for p<0.0005.
Figure 3.
Analysis of NK cell effector molecules in IFN-α11-treated mice.
(B10.A×A.BY) F1 mice were treated daily with 8000 units of IFN-α2, α5 or α11 from −1 through +9 dpi with 7000 SFFU of FV. At 10 dpi, splenocytes were analyzed by flow cytometry. The frequencies of intracellular expression of granzyme B (GzmB) in NK cells (NK1.1+ CD3− CD49b+) are shown (A). Surface TRAIL expression was measured in the same NK cell population of the IFN-α11-treated mice (shaded histogram = isotype control; dashed line = FV; solid line = FV+IFN-α11) (B). The mean fluorescence intensity of TRAIL expression was evaluated on NK cells (C). A minimum of 3 mice per group were analyzed and the mean values are shown by bars. To analyze whether IFN-α11 acts directly on NK cells, C57BL/6 mice were irradiated and reconstituted with mixed bone marrow cells from CD45.1+ wild type and CD45.2+ IFNAR−/− mice at a ratio of 1∶1. Thirty days later, mice were treated daily with 8000 units of IFN-α11 from −1 through +9 dpi with 20,000 SFFU of FV. As controls, mixed bone marrow chimeras were infected with FV or stayed naive. At 10 dpi, splenocytes were analyzed by flow cytometry. The frequencies of intracellular expression of granzyme B in CD45.1+ wild type (white bars) and CD45.2+ IFNAR−/− (black bars) NK cells are shown (D). Eight mice in each group of infected mice were analyzed and the mean value for each group is indicated by a bar. At least 2 independent experiments were performed. Statistically significant differences between the control group (FV) and the IFN-α11-treated mice are indicated by ** for p<0.005 or *** for p<0.0005.
Figure 4.
Target cell killing by NK cells from IFN-α11-treated mice.
C57BL/6 mice were treated daily with 8000 units of IFN-α11 from −1 through +9 dpi with 20,000 SFFU of FV. Ten dpi, NK cells were isolated from 10 individual mice per group using magnetic bead separation. NK cells were incubated for 4 hours with 2 different target cell lines (YAC-1 and FBL-3) in different effector to target cell (E∶T) ratios (A). In some assays, CMA, Z-VAD-fmk, neutralizing anti-TRAIL, anti-Fas ligand (FasL) mAbs, or isotype control antibodies were added to NK and FBL-3 cells (E∶T = 25∶1) (B). Killing of FBL-3 cells by NK cells (E∶T = 25∶1) isolated from mice deficient in granzyme B (GzmB−/−; grey bars) and wild type mice (C57BL/6; black bars) was investigated (C). Mice were treated daily with 8000 units of IFN-α11 from −1 through +9 dpi with 20,000 SFFU of FV. Ten dpi, NK cells were isolated from six individual mice per group using magnetic bead separation. Specific lysis of target cells was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Mice deficient in granzyme B (GzmB−/−; grey bars) and wild type mice (C57BL/6; black bars) treated with IFN-α11 or left untreated were analyzed at day 10 post FV infection for viral loads in the spleen by using an infectious center assay (D). Adoptive transfer experiments of NK cells were performed as follows. C57BL/6 mice were treated daily with 8000 units of IFN-α11 from −1 through +9 dpi with 20,000 SFFU of FV or left untreated. Nine dpi, NK cells were isolated from 10 individual mice per group. 2×106 NK cells were adoptively transferred into recipient mice, which were infected with FV 6 days before transfer. Four days post transfer, spleen samples were assayed for viral loads by using an infectious center assay (E). Five mice in each group of infected mice were analyzed and the mean value for each group is indicated by a bar. At least 2 independent experiments were performed. Statistically significant differences between the groups are indicated by * for p<0.05, ** for p<0.005 or *** for p<0.0005.
Figure 5.
Treatment with IFN-α11 during acute MCMV infection.
C57BL/6 mice were treated daily with 8000 units of IFN α11 from −1 through +6 dpi with 5×104 PFU of MCMV. Seven dpi, viral loads in the spleen, liver and salivary glands were analyzed by using a plaque assay (A). NK cells in the liver and spleen were analyzed by flow cytometry. The early activation marker CD69 was used to analyze the frequencies of activated NK cells (NK1.1+ CD3− CD49b+) (B). C57BL/6 mice were treated daily with 8000 units of IFN-α11 from −1 through +6 dpi with 5×104 PFU of MCMV. NK cells were depleted by 4 injections of supernatant of clone PK136 starting at the day of MCMV infection. IFN-α11-treated non-depleted mice and NK cell depleted, non-treated mice were used as control groups. At 7 dpi, viral loads in the salivary glands were determined by using a plaque assay (C). A minimum of 4 mice per group were analyzed and the mean values are shown by bars. At least 2 independent experiments were performed. Statistically significant differences between the groups are indicated by * for p<0.05, *** for p<0.0005.