Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: 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 . 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

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Phospho-proteomics, Activation Assay, Staining, Flow Cytometry, Cell Culture, Virus, Standard Deviation, Purification, Blocking Assay, Control, Expressing

Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: Gene expression profiling of NK cells following reovirus treatment. (a) Summary of the approach. PBMC from five healthy donors were treated at an MOI of 1 with reovirus (or left untreated). After 48 h, NK cells were purified using immunomagnetic selection and a small aliquot of cells was removed for flow cytometry to assess CD69 induction (Figure ). The remainder of the NK cells were used in the gene expression profiling. (b) Differentially expressed genes in total human NK cells following reovirus treatment. Genes that were up (green columns) or downregulated (red columns) together with examples and fold change are indicated. Data underlying this graph is shown in Table . (c) Gene set enrichment analysis (GSEA). The left panel shows the top 10 pathways identified by GSEA of differentially expressed genes. Data were generated using Enrichr and the output from Reactome 2016; the adjusted p ‐value of enrichment is shown. Full data is provided in Table . The right panel shows the top 10 transcription factors associated with differentially expressed genes. Data were generated using Enrichr and the output from ENCODE and ChEA Consensus Transcription factors from ChIP‐X; the adjusted p ‐value of enrichment is shown. Full data are provided in Table . (d) Venn diagrams showing the overlap of the differentially expressed genes (DEGs) from NK cells following reovirus treatment (Reo) with the interferon‐stimulated genes listed in Schoggins et al (; S‐Set) or the core mammalian ISGs identified by Shaw et al (; Core m‐ISGs). Overlaps were determined for all reovirus DEGs (left and right panels) or the upregulated genes only (centre panel). (e) Expression of genes from selected pathways. The graph shows the fold change in gene expression in NK cells following reovirus treatment, with genes and pathways indicated. A green circle below the x ‐axis indicates that the gene is induced by IFN‐I in haematopoietic cells, as determined using data from the Interferome database . (f) TRAIL expression by CD56 dim and CD56 bright NK cells following reovirus treatment. The histograms show flow cytometry from a single representative donor, with the different treatments (and isotype control antibody) indicated, along with the NK cell subset analysed via gating. The graphs on the right show the mean and standard deviation of the median fluorescence intensity (MFI), from three separate donors; data were analysed by a repeated‐measures one‐way ANOVA, with Tukey's multiple comparison test; ** p < 0·01

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Gene Expression, Purification, Selection, Flow Cytometry, Generated, Expressing, Control, Standard Deviation, Fluorescence, Comparison

Journal: Immunology

Article Title: Oncolytic virus treatment differentially affects the CD56 dim and CD56 bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity

doi: 10.1111/imm.13453

Figure Lengend Snippet: Reovirus‐induced IFN‐I production inhibits IL‐15‐mediated proliferation of NK cells. (a) Summary of the approach; Conditioned media (CM) was collected from reovirus treated (or untreated) PBMC and filtered to remove virions. This CM was applied to fresh PBMC (labelled with CFSE) together with IL‐15, in the presence or absence of IFN‐I blocking antibodies. PBMC were cultured for 5 days and proliferation of CD56 bright NK cells analysed by CFSE content. (b) Inhibition of CD56 bright NK cell proliferation by reovirus‐induced IFN‐I. CFSE analysis of CD56 brigh t NK cells following treatment described in (a). The percentage of cells that have proliferated after 5 days are indicated. These data are from a single donor, representative of three donors. (c) IFN‐I blocks IL‐15 induced S phase. Purified NK cells were cultured with IL‐15 or IL‐15+IFN‐I (all at 100 ng/ml) for 3 days and S phase assessed by propidium iodide staining, as shown in panel (a). This data is from one donor, representative of three donors tested. (d) Reovirus treatment blocks IL‐15 induced expression of cell cycle mediators. PBMC were cultured for 4 h alone or primed with 1 MOI reovirus (Reo). 10 ng/ml IL‐15 was then added directly to all samples for 3 days. Total NK cells were isolated and MCM4, cyclin B and CDK2 analysed by immunoblotting along with β‐actin as a loading control. The blot image has been cut between the unstimulated control and cytokine treated lanes as shown by the boxing. These data are representative of three donors tested. (e) Modulation of IL‐15 mediated signalling by reovirus treatment. PBMC were cultured for 48 h alone (no virus) or primed with 1 MOI reovirus (+reovirus). After 48 h, 0, 1 or 10 ng/ml IL‐15 was added (as indicated) for 30 min. Phospho‐STAT1, STAT3, STAT5, mTOR and Akt were analysed by intracellular staining and flow cytometry, gating on the NK cell population. Graphs show median fluorescence intensities (MFI) for two or three separate donors, with standard deviation. Data were analysed by two‐way repeated‐measures ANOVA. When the effect of the virus was statistically significant, a post hoc Sidak multiple comparison tests was applied to identify statistically significant differences between “no virus” and “reovirus” MFI values; * p < 0·05

Article Snippet: For IFN‐I ELISA, 96 well plates were coated with a mixture of antibodies against IFN‐α (Mabtech, MT1/3/5) overnight at 4°C, and blocked with PBS supplemented with 10% FCS.

Techniques: Blocking Assay, Cell Culture, Inhibition, Purification, Staining, Expressing, Isolation, Western Blot, Control, Virus, Flow Cytometry, Fluorescence, Standard Deviation, Comparison