Figure 1.
Type I IFNs are required for NK cell activation in response to flu infection.
Following i.v. infection with flu, splenic NK cells (NK1.1+CD3−CD19−) from B6 WT, IFNAR−/−, IL-12R−/− and IL-18R−/− mice were analyzed at 9h post-infection. (A) IFN-γ and granzyme B expression are shown. Mouse genotypes are indicated above the dot plots or histograms. Inset values represent the percentages of IFN-γ+ (upper panels) or granzyme B+ (lower panels) NK cells. (B) CD69 expression levels on NK cells from uninfected and infected mice are shown. Percentages of NK cells located within the CD69+gate are indicated. (C) CD107a expression and IFN-γ production were analyzed in NK cells after incubation with YAC-1 cells. Percentages of NK cells within each quadrant are indicated. Data are representative of at least three mice per group.
Figure 2.
Direct action of type I IFNs is critical for activation of NK cells following flu infection.
Splenocytes from IFNAR+/− or IFNAR−/− (CD45.2+) were transferred into CD45.1+ B6 WT recipients by i.v. injection prior to infection with flu. Splenic NK cells from indicated mice were analyzed post-infection. (A) Expression levels of IFN-γ (upper panels) and granzyme B (lower panels) were analyzed in NK cells after transfer and infection. Donor and recipient genotypes are indicated, and upper and lower quadrants for each dot plot represent the donor and host NK cells, respectively. Inset values indicate the percentages of IFN-γ+ or granzyme B+ NK cells. (B) CD69 expression of NK cells from infected (open histograms) and uninfected (shaded histograms) mice. Percentages of NK cells located within the CD69+gate are indicated. (C) IFN-γ and CD107a expression were analyzed after in vitro stimulation of splenocytes with YAC-1 cells. Numbers represent the relative percentages of CD107a+ and IFN-γ+ NK cells for donor and host cells. Data are representative of four separate experiments with 2–4 mice per group.
Figure 3.
Highly purified NK cells exhibit enhanced cytotoxicity toward target cells in response to treatment with IFN-α.
Sorted NK cells (>99% pure) were cultured in vitro for 6 hours with or without indicated cytokines, then combined with 51Cr-labeled YAC-1 targets (E:T = 10∶1) to assess cytotoxic activity. Bars represent the average specific lysis of target cells for each condition. Error bars denote the SEM. Data are representative of two independent experiments.
Figure 4.
Activation of STAT1 in NK cells requires the direct action of type I IFNs during influenza infection.
NK cells from infected (open histograms) and uninfected (shaded histograms) mice were analyzed for intracellular pSTAT1 following adoptive transfer (A) or co-culture (B). Adoptive transfer was as described in Figure 2. For in vitro infection, CD45.2+ splenocytes from IFNAR+/− or IFNAR−/− mice were combined with CD45.1+ B6 splenocytes at 1∶1 ratio, then infected with flu. NK cells (NK1.1+CD3−) from infected (open histograms) and uninfected (shaded histograms) samples were analyzed for intracellular pSTAT1. Values represent the percentages of pSTAT1+ NK cells. Data are representative of three independent experiments with 2–4 (A) or 1–3 (B) mice per group.
Figure 5.
Activation of STAT4 is partially dependent on the direct action of type I IFNs during influenza infection.
NK cells from infected (open histograms) and uninfected (shaded histograms) mice were analyzed for intracellular pSTAT4 following adoptive transfer (A) or co-culture (B). Adoptive transfer was as described in Figure 2. For in vitro infection, CD45.2+ splenocytes from IFNAR+/− or IFNAR−/− mice were combined with CD45.1+ B6 splenocytes at 1∶1 ratio, then infected with flu. NK cells (NK1.1+CD3−) from infected (open histograms) and uninfected (shaded histograms) samples were analyzed for intracellular pSTAT4. Values represent the percentages of pSTAT4+ NK cells. Data are representative of three independent experiments with 2–4 (A) or 1–3 (B) mice per group.
Figure 6.
STAT1, but not STAT4, is required for granzyme B induction by NK cells in response to flu infection.
CD45.2+ splenocytes from 129/Sv WT and STAT1−/− (A) or from BALB/c WT and STAT4−/− (B) mice were combined with CD45.1+ B6 splenocytes in vitro, then infected with flu. NK cells (DX5+CD3−CD19−) were analyzed for expression levels of IFN-γ (left panels) and granzyme B (right panels). Bar graphs represent the mean differences in percentage of IFN-γ+ or granzyme B+ CD45.2+ NK cells from infected samples over uninfected controls. Error bars represent the SEM of triplicate samples. Data are representative of four independent experiments. **, P<0.001; ***, P<0.0001; ns, not significant.