Fig 1: OrfV-stimulated neutrophils exhibited enhanced tumor necrosis factor (TNF)-ɑ-mediated cytotoxicity against tumor cells: (a) naïve seven- to nine-week-old female C57BL/6 mice (n = 8 per group) were injected intraperitoneally with phosphate-buffered saline (PBS) or 5 × 107 PFU of OrfV. Two hours following treatment, bone marrow was flushed from femurs to isolate viable neutrophils (defined by a CD45+ Ly6Ghi CD11bhi phenotype) by flow cytometric cell sorting. Neutrophils were co-cultured in vitro with B16F10 melanoma cells at increasing tumor-to-neutrophil ratios for 16 h. The viability of tumor cells was quantified via flow cytometry; (b) twenty-four hours after intravenous treatment with OrfV or PBS, B16F10 metastasis-bearing lungs were collected from the mice, and the cytokine response of immature and mature neutrophils (defined by CD101 expression) was quantified with flow cytometry after intracellular cytokine staining. TNF-α, tumor necrosis factor-alpha; IL-10, interleukin 10; IL-17A, interleukin 17A, IFNγ, interferon-gamma; PD-1, programmed cell death protein 1; IL-12p40, interleukin 12p40; IL-6, interleukin 6; (c) two hours after treating mice with OrfV, bone marrow-derived neutrophils (CD45+ Ly6Ghi CD11bhi) were flow-sorted. The isolated neutrophils were incubated with TNF-ɑ-neutralizing antibodies and co-cultured in vitro with B16F10 melanoma cells at increasing tumor-to-neutrophil ratios for 16 h. The cell viability of tumor cells was quantified with flow cytometry. Statistical analysis was performed using two-way analysis of variance. Statistical significance was designated as * p ≤ 0.05, *** p ≤ 0.001, and **** p ≤ 0.0001.