Anti ifn gamma

For additional characterization of intracellular markers PBMCs were suspended in culture medium consisting of RPMI 1640 (Biochrom), 5% human AB serum (CC pro), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Biochrom). PBMCs were stimulated with 10 ng/ml phorbol myristate acetate (PMA, Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) in the presence of 0.2 µM Monensin (Biomol) for 6 hours prior to analysis. First, cell surface markers were stained as described above. Before the characterization of intracellular markers, cells were fixed with freshly prepared fixation concentrate, and permeabilized with wash-permeabilization concentrate (Fixation/Permeabilisation Buffer Set, eBioscience). Subsequently, cells were stained using fluorescence-labeled anti-IFN-gamma, anti-IL-17A (BioLegend), anti-IL-4 (BD Bioscience), anti-FoxP3 antibody (Miltenyi Biotec) or isotype-matched irrelevant antibody (BD Biosciences) (Suppl. Fig. 6). After the staining procedure, cells were evaluated by flow cytometry. Cells were measured on a LSR-Fortessa (BD Bioscience) and evaluated by FACS-Diva Software (BD Bioscience).

TLR activation of NK cells was analysed concerning induction IFN-gamma and TNF-alpha as well as CD107a degranulation in response to K562 target cells. Purified NK cells were pre-activated overnight with IL-2 and then cultured with and without Pam3CSK4, LPS-B5 and CpG-ODN-2216, respectively. After 16 h, NK cells were divided into either co-culture with K562 effector cells (effector to target ratio of 1:2) or kept in culture with medium alone. Next, FITC-conjugated CD107a (BD Biosciences, Heidelberg, Germany) was added. After 1 h, Golgi Stop (BD Biosciences) and BFA (Enzo Life Sciences GmbH) were added for additional 3 h. Then, cells were stained with Zombie AquaTM (BioLegend, London, UK) followed by staining with anti-CD3 (APC-Cy7-labelled), anti-CD56 (Brilliant Violet 421-labelled) and anti-CD16 (PerCP-labelled). After fixation and permeabilization, cells were stained intracellularly with anti-IFN-gamma (PE-labelled) and anti-TNF-alpha (PE-Cy7-labelled) (all BioLegend). Percent IFN-gamma-, TNF-alpha- and CD107a-positive CD56^dimCD16^pos, CD56^dimCD16^neg and CD56^highCD16^neg NK cells were measured before and after TLR stimulation as well as after co-culture of NK cells with K562 target cells in the presence and absence of TLR stimulation (Supplementary Figure 2).