Figure 2.

Innate Activation Is Dependent on Type I IFNs and CD14⁺ Monocytes

(A) Healthy donor PBMCs or isolated NK cells were treated with HSV^GM-CSF for 48 h and NK cell (CD56⁺/CD3⁻) (i) CD69 expression and (ii) CD107 degranulation (following co-culture with melanoma targets) were determined by flow cytometry (n = 4). (B) Healthy donor PBMCs were treated with HSV^GM-CSF for 48 h, and production of IFNγ, IFNα, IFNβ, and IL-29 was determined by ELISA. The graph shows the mean of at least four independent experiments + SEM. (C) Healthy donor PBMCs were treated overnight with HSV^GM-CSF either alone or in the presence of IFNα/β blocking antibodies or isotype controls before (i) CD69 upregulation on CD56⁺/CD3⁻ NK cells was determined by flow cytometry. The graph shows the average percentage of NK cells expressing CD69 + SEM (n = 3). (ii) PBMCs (with or without IFN blockade and with or without HSV^GM-CSF) were co-cultured with MEL888 cells, and NK cell CD107 degranulation was determined by flow cytometry. The graph shows the mean percentage of NK cells expressing CD107a/b + SEM (n = 3). (iii) PBMCs (with or without IFN blockade and with or without HSV^GM-CSF) were co-cultured with MEL888 cells at the indicated E:T ratios, and the percentage of tumor cell lysis was determined by ⁵¹Cr release. The graph shows the mean percentage lysis ± SEM (n = 3). (D) IFNα/β production from whole PBMCs or CD14⁺ monocyte-depleted PBMCs was determined by ELISA. The graph shows the mean + SEM (n = 5). Statistical significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001.