Effect of WF10 on the cytotoxic activity of NK cells. (a, b) Freshly isolated resting human NK cells or (c, d) IL-2 stimulated NK cells were used in a standard 4 h ⁵¹Cr-release assays against LCL721.221 target cells with or without the addition of 200 μM WF10 at the indicated effector to target (E/T) ratios. Panels (a) and (c) show the results of representative experiments. In panels (b) and (d) the activity of the WF10-treated cells is shown relative to the cytotoxic activity of control cells (set as 100%) as an average value from (b) 24 or (d) 3 independent experiments. The ratio of each outcome and its control was tested against a value of 1 (equality) by two-sided single-sample t-tests (*P < .05).

WF10 specifically affects NK cells. (a) Freshly isolated resting human NK cells were preincubated for 5 h with or without 200 μM WF10 in culture medium, washed and then used in a standard 4 h ⁵¹Cr-release assays against LCL721.221 target cells at the indicated effector to target (E/T) ratios. (b) LCL721.221 target cells were preincubated with or without 200 μM WF10 in culture medium, washed and then used as targets in a standard 4 h ⁵¹Cr-release assays using freshly isolated human NK cells as effectors. The activity of the WF10-treated cells is shown relative to the cytotoxic activity of control cells (set as 100%) as an average value from (a) 4 or (b) 3 independent experiments.

Time-dependent modulation of NK cell responses by WF10. (a) Freshly isolated resting human NK cells or were preincubated for the indicated times with 200 μM WF10 and then used in a standard 4 h ⁵¹Cr-release assays against LCL721.221 target cells. Cytotoxic activity was normalized to the untreated control (set as 100%). Shown are the average values of two independent experiments using NK cells from different donors. (b, c) Freshly isolated resting human NK cells were incubated with or without 200 μM WF10 in culture medium for 18 h. Cells were then resuspended in fresh medium and tested as described above. Panel (b) shows the results of a representative experiment. In panel (c) the activity of the WF10-treated cells is shown relative to the cytotoxic activity of control cells (set as 100%) as an average value from 8 independent experiments. The ratio of each outcome and its control was tested against a value of 1 (equality) by two-sided single-sample t-tests (*P < .05).

The effect of WF10 on the transcription of NK cell genes. Freshly isolated NK cells were incubated with WF10 (at a chlorite concentration of 200 μM) for 0, 3, or 18 h and mRNA was isolated. The indicated transcripts were quantified by quantitative RT-PCR. The number of transcripts of individual genes in each sample was normalised using the number of transcripts of the house-keeping genes β-actin and cyclophilin b. The number of transcripts in the WF10-treated sample was compared with the number of transcripts in the control sample using the formula log  2 (transcripts WF10 sample/transcripts control sample). Average values and standard deviation from 5 independent experiments with different blood donors are shown.

Enhancement of receptor-mediated cytotoxicity of IL-2-activated NK cells by WF10. IL-2-activated NK cells were tested in a redirected-lysis assay against murine P815 cells. NK cells were stimulated using a (a) control antibody, or by antibodies directed against the activating receptors (b) CD16, (c) NKG2D, (d) NKp30, or (e) 2B4. Where indicated, WF10 was added to the assay medium at a final chlorite concentration of 200 μM. Average values of triplicates are shown with the standard deviation. One representative of three independent experiments with different donors is shown.

WF10 enhances NK cell adhesion. (a) NK cells and 721.221 cells were labeled with a different fluorescent dye, washed, and then incubated together with or without WF10 (200 or 600 μM). At the indicated time points the mixed suspension was fixed with ice-cold 4% PFA and analyzed by FACS. The amount of NK: 721.221 conjugates formed by the cells without WF10 was used as a reference (100%). Average values and the standard deviation from 3 independent experiments with different blood donors are shown. (b, c) Freshly isolated NK cells were incubated with PE-conjugated ICAM-1 complex (scICAM-1) with or without WF10 for 3 h. Samples were either left unstimulated (b, left panel), or treated with IL-15 (100 U/ml) (b, right panel). The amount of bound ICAM-1 complexes was measured by FACS. A representative experiment is shown in (b). The ICAM-1 binding to control cells (MFI 44.2 ± 20.4) and IL-15 stimulated cells (MFI 126 ± 60.3) was used as a reference (100%). (c) Average values and standard deviations from 5 independent experiments with different blood donors are shown. The ratio of each outcome and its control was tested against a value of 1 (equality) by two-sided single-sample t-tests (*P < .05).