Recombinant human il 12

Purified CD14⁺ monocytes (1 x 10⁶ cells/mL) (N=4) were pre-treated with LEAF purified mouse IgG1, k isotype control, Ultra-LEAF purified anti-human IL-12p40 or Ultra-LEAF purified anti-human IL-18 neutralizing antibodies (each 10 µg/mL, Biolegend) for 2h at 37°C humidified incubator (19 (link)), followed by stimulation with TL8-506 or ssRNA40/Lyovec for 1.5h. For recombinant protein stimulation assay, isolated autologous naïve CD4⁺ T cells were treated with recombinant human IL-12 or recombinant human IL-18 (each 20 ng/mL, Biolegend) followed by co-culture experiments as described above.

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.

Human PBMC samples were obtained from iQ Biosciences, CA. 1.0 × 10⁶/mL PBMCs were seeded in complete RPMI at a sterilized U-shaped 96-well plate with a volume of 200 μL/well. PBMCs were treated by serially-diluted test compounds, and incubated with 40 ng/mL recombinant human IL12 (Biolegend, #573002) for overnight in a humidified, 5% CO₂ cell culture incubator at 37°C. The supernatant was then collected for detection of IFNγ secretion. IFNγ level was measured by Human IFNγ AlphaLISA Kit (PerkinElmer, AL217C/F) according to the manufacturer’s instructions. Inhibition data were calculated by comparison to vehicle control wells for 0% inhibition and non-stimulated control wells for 100% inhibition. Dose response curves were then generated to determine the concentration required to suppress 50% of cellular response (IC₅₀) as derived by non-linear regression analysis using GraphPad Prism.