Recombinant il 18

MLKL^−/− mice (seven mice per group) were injected daily i.p. with PBS alone or with recombinant IL-18 (MBL International) at a dose of 1.0 µg per mouse in 100 µl PBS on days 0, 1, and 2. WT mice were injected daily i.p. with PBS alone.

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.

PBMC (4x10⁵ cells/well) and purified CD56^bright NK cells (1x10⁵ cells/well) were cultured in VLE RPMI 1640 medium supplemented with 10% autologous serum, 100 U/ml Penicillin, and 100 μg/ml Streptomycin in flat-bottom 96-well plates (BD Falcon, Heidelberg, Germany). All cultures were set up in triplicates in a total volume of 200 μl/well. PBMC were stimulated with 0.05% (v/v) inactivated S. aureus bacteria (Pansorbin, Merck, Darmstadt, Germany). Unstimulated PBMC served as negative control. Where indicated, recombinant human IL-12 (0.2–0.4 ng/ml as indicated; Biolegend, San Diego, USA) or antibodies (Abs) against human IL-12 (10 μg/ml; clone 24910, R&D, Wiesbaden, Germany) were added to the cultures 1 h prior to stimulation with S. aureus. Previous experiments have verified that the isotype control Abs against IL-12 (mouse IgG2a isotype control, clone 20102, R&D) did not influence the responsiveness of PBMC to S. aureus (data not shown). After 19 h, supernatants were harvested and stored at -20°C until use. The cells were used for intracellular staining (see below). Purified CD56^bright NK cells were cultured in the presence or absence of 1 ng/ml recombinant IL-12 and 10 ng/ml recombinant IL-18 (MBL International, Woburn, MA). After 19 h, supernatants were harvested and stored at -20°C until use. Cell culture was performed at 37°C in 5% CO₂ humidified atmosphere.