Figure 2.

IFN-γ effect on PD-L1 and NK cell ligand expression and susceptibility to Avelumab-mediated ADCC of TNBC cell lines. TNBC cells lines were treated with 10 U/ml IFN-γ for 24 h or left untreated before being used in the assays. In all of the figures, cells with higher basal expression of PD-L1 are shown in orange and the ones with lower basal expression, in gray. Lysis of IFN-γ treated and untreated cancer cells coated with IgG1 or Avelumab was evaluated using PBMC as effector cells at 5:1 or 10:1 NK:BC ratios (n = 6–7): (A) Changes in lysis (Δ % Lysis) of treated compared to untreated cancer cells were quantified for each donor and results are shown as box plots; (B) Percentage of lysis against MDA-MB-231 cells at 5:1 NK:BC ratio; (C) Percentage of lysis against BT-549 cells at 5:1 NK:BC ratio; (D) Percentage of lysis against MDA-MB-468 cells at 10:1 NK:BC ratio. Bars with different letter are statistically different, p < 0.05 (ANOVA). (E) PD-L1 expression in IFN-γ treated and untreated TNBC cell lines represented as normalized MFI (MFI of cells stained with specific mAb/MFI of cells stained with isotype control). ^*p < 0.05; ^**p < 0.01 (paired t-test). (F) Heat-map representing basal expression of NK cell ligands in TNBC cell lines, showed as log₂ normalized MFI (left) and heat-map representing fold change in NK cell ligand expression after IFN-γ treatment of TNBC cell lines, showed as log₂ fold change in normalized MFI (right).