Figure 1.

PM21-NK cells effectively lyse lung cancer cells infected with PIV5 P/V oncolytic virus. (A–C) A549 cells in 2D culture were mock infected or infected at an MOI of 5 for the indicated times with either P/V mutant or WT PIV5 virus and cells were assayed by flow cytometry for GFP expression (panel A) or annexin V staining (panel B). Alternatively, cells incubated for 45 min with PM21-NK cells at an E:T ratio of 1:1 before determining the cytotoxicity using a flow cytometric assay (panel C) as described in Methods section. Values are the mean of three replicates and per cent cytotoxicity of mock is compared with virus-infected samples at 16 hpi. Data is representative of four independent experiments conducted with four different NK cell donors with triplicates. (D) A549 cells were infected at the indicated MOIs with the P/V mutant virus for 16 hours, and then incubated for 4 hours with PM21-NK cells at the indicated E:T ratios. Cytotoxicity was assayed using CytoTox-Glo assay. (E) A549 cells were mock infected or infected at an MOI of 5 with P/V virus. At 16 hpi, cells were pretreated with anti-HN monoclonal antibodies 1b or 4b or isotype control antibody and were analyzed for HN surface expression by flow cytometry (panel E) prior to incubation with PM21-NK cells at an E:T of 10:1 (panel F). Per cent cytotoxicity was determined by CytoTox-Glo assay. In all panels, values are the mean of three samples with error bars representing SD. Graphs were analyzed using two-way ANOVA and one-way ANOVA. ****p<0.0001. ANOVA, analysis of variance; E:T, effector:target; GFP, green fluorescence protein; HN, hemagglutinin-neuraminidase; MOI, multiplicity of infection; WT, wild type.