Clone nib42

After cell transfection and/or treatment, supernatants were replaced by mouse T-cell culture medium or AIM V medium (Gibco) for human cells containing polarization cytokines as follows: anti-mouse IFN-γ (50 μg/mL, clone XMG 1.2; BioXCell) and anti-mouse IL-4 (50 μg/mL, clone 11B11; BioXCell) for mouse T_H0 cells; IL-12 (10 ng/mL, Miltenyi Biotec) and anti-mouse IL-4 for mouse T_H1 cells; TGF-β (2 ng/mL, Miltenyi Biotec), IL-4 (20 ng/mL, Miltenyi Biotec), and anti-mouse IFN-γ for mouse T_H9 cells; IL-6 (20 ng/mL, Miltenyi Biotec), TGF-β, anti-mouse IFN-γ, and anti-mouse IL-4 for mouse T_H17, IL-12 (10 ng/mL, R&D System) and anti-human IL-4 (3.5 μg/mL, clone MP4-25D2; BioXCell) for human T_H1 cells, TGF-β (5 ng/mL, Miltenyi Biotec), IL-4 (10 ng/mL, R&D System), and anti-human IFN-γ (3.5 μg/mL, clone NIB42; BioLegend) for human T_H9 cells. Unless specified otherwise, cells were cultured for 3 days at 37°C under 5% CO₂.

5x10⁵ PBMC or 1x10⁵ NK cells isolated from HD were cultured in the absence (medium) or in the presence of 3x10⁵ tumor cells for 48 h, stained for CD3, CD56 and PD-L1, and analyzed by flow cytometry following the gating strategy depicted in Supplementary Figure 2. For blocking experiments, anti-IL-12 p70 (10 μg/ml, clone QS-12p70, Abcam), anti-IL-15 (10 μg/ml, clone ct2nu, eBioscience), anti-IL-18 (10 μg/ml, clone 125-2H, MBL International), anti-IFN-γ (10 μg/ml, clone NIB42, BioLegend), anti-IFN-γR (10 μg/ml, clone GIR-208, BioLegend) or anti-NKG2D (20 μg/ml, clone 1D11, BioLegend) mAb, were added at the beginning of the cocultures. In other experiments, recombinant IFN-γ (200 ng/ml, Peprotech) and recombinant IL-18 (10 ng/ml, MBL International) were used to stimulate cells. In some experiments, 1x10⁵ autologous monocytes were added to NK cell cultures.