FIGURE 1.

STAT phosphorylation and IFN‐I dependent NK cell activation following reovirus treatment. (a) STAT phosphorylation in CD56^bright and CD56^dim NK cells (detected by intracellular staining and flow cytometry) in PBMC cultured without virus (untreated; black line) or with 1 MOI reovirus (purple line), for 8, 24 and 48 h. Graphs show mean MFI and standard deviation from three donors. Data were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. *p < 0·05 **p < 0·01. (b) NK cell activation by reovirus is IFN‐I dependent. The flow chart shows the approach taken; PBMC (from one donor) were left untreated or treated with reovirus for 24 h and the conditioned media (CM) filtered to remove viruses. CM was added to purified NK cells in the presence of an IFN‐I blocking antibody cocktail (IFN block), a control blocking cocktail (control block) or no added antibody (no block). CM from untreated PBMC was used a control. After 48 h, the NK cell surface expression of CD69 and tetherin was measured by flow cytometry. Data is from control CM or CM from reovirus‐treated PBMC from a single donor, applied to three NK cell donors. The y‐axes show the percentage of CD69 expressing cells (top panel) or the fold change in MFI of tetherin relative to control CM and no added antibody treatment (bottom panel), due to constitutive low‐level expression of this molecule on unstimulated NK cells [25]. Differences between mean percentage positive values for CD69, or mean fold change MFI for tetherin, were analysed by two‐way repeated‐measures ANOVA, followed by Sidak multiple comparisons test. *p < 0·05 **p < 0·01