Il 18

Functional assays were performed as previously described [19 (link)]. PBMCs were stimulated for a total of 24 h with IL-12 (10 ng/mL, Peprotech, Rocky Hill, NJ, USA) and IL-18 (100 ng/mL Medical & Biological Laboratories, Nagoya, Japan); monensin (eBioscience, San Diego, CA, USA) and Brefeldin A (BD Biosciences) were added during the last 6 h of stimulation. Data was collected the same day on a 14-color BD LSRII SORP.

NK cell degranulation and cytokine production were assessed by co-culture experiments with target cells. PBMCs were pre-stimulated overnight with IL-12 (10 ng/ml, Peprotech) and IL-18 (100 ng/mL, Medical & Biological Laboratories) and then K562 cells were added at a 1:10 ratio. One hour after addition of target cells, Golgi plug (Brefeldin A, BD Biosciences) and Golgi stop (Monesin, BD Biosciences) were added and the assay was continued for an additional 5 h. NK cells were then stained for analysis using flow cytometry.

Fluorochrome-labeled antibodies used for flow cytometric analyses were obtained from the following vendors: anti-CD3 (UCHT1), anti-CD16 (eBioCB16), anti-CD56 (CMSSB), anti-CD107a (eBioH4A3), anti-CD107b (eBioH4B4), anti-CD300c (TX45), anti-TNFα (MAb11), anti-MIP1α (PFFM3) and anti-CD69 (FN50) were from eBiosciences and anti-IFN-γ (B27) was from BD Biosciences. Purified antibodies anti-human IL-2 (AB12-3G4), mouse IgG2a κ isotype (eBM2a), mouse IgG1 κ (MOPC-21) and anti-CD300c (TX45) were obtained from eBiosciences. For the real time PCR experiments, primers for CD300A, CD300C and 18sRNA genes were purchased from SA Biosciences. STAT5 inhibitor N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide was purchased from Calbiochem/EMD Millipore. Recombinant IL-2 was obtained from National Cancer Institute, Frederick, MD. Cytokines IL-12, IL-15 and IL-4 are from R&D Systems, IL-18 is from Medical and Biological Laboratories (MBL) and IL-21 is from Peprotech. Phospholipids 1-palmitoyl-2-oleoyl (PO) phosphatidylserine (POPS) (PS), phosphatidylethanolamine (POPE) (PE) and phosphatidylcholine (POPC) (PC) were purchased from Avanti Polar Lipids.

Paired PBMCs and perfusate MNCs were resuspended in PBS/0.1% BSA to create a 2x cell solution. This was resuspended in Carboxyfluorecin succinimidyl ester (CFSE) staining solution (CellTrace™ CFSE Cell Proliferation Kit) (Life Technologies™, Paisely, UK) to make a final CFSE concentration of 5 μM and incubated for 10 min, 37°C. Staining was quenched with 5 volumes ice‐cold Roswell Park Memorial Institute Medium (RPMI) 1640 + Glutamax (Gibco®, Life Technologies™) supplemented with 10% fetal bovine serum (Hyclone®, Thermoscienticic, Northumberland, UK), penicillin, streptomycin and glutamine (Gibco®, Life Technologies™) (R‐10) and incubated for 5 min, 4°C. Cells were washed three times in R‐10 then recounted. PBMCs and liver MNCs were incubated in R‐10 supplemented with 5% HS (Sigma) in addition to Recombinant Human IL‐2 100 U/ml (PeproTech, London, UK), IL‐12 10 ng/ml (PeproTech), IL‐15 25 ng/ml (R&D Systems, Oxford, UK), IL‐18 100 ng/ml (Medical and Biological Laboratories, Japan), or a cocktail of all four for 6 days. Media and cytokines were changed every 2–3 days. A CFSE FMO was included. On day 0 and 6 PBMCs and liver MNCs underwent staining with Zombie Violet™ Fixable Viability Kit (Biolegend®), CD3‐BV510 (Biolegend®), CD56‐PECy7 (Biolegend®), NKG2C‐APC (Miltenyi Biotec), CXCR6‐PerCP/Cy5.5 (Biolegend®), and CD49a‐PE (BD Biosciences).

IFN-γ and TNF-α expression in NK cells were detected by intracellular cytokine flow cytometry as previous described [20 (link)]. Briefly, freshly-isolated PBMCs (1 × 10⁶/well) were incubated in 1 mL complete media. The media consisted of RPMI 1640, 2 mM _L-glutamine, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. It was supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY). The incubation period was 24 hours in the presence of IL-12 (50 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-18 (50 ng/mL; Medical and Biological Laboratories, Woburn, MA). For intracellular cytokine staining, we added 10 μL of brefeldin A (GolgiPlug; BD Biosciences, San Diego, CA). The final concentration of brefeldin A was 10 μg/mL. After incubation for an additional four hours, cells were stained with APC-Cy7-conjugated anti-CD3, PE-conjugated anti-CD56 and APC-conjugated anti-CD69 mAbs for 20 minutes at 4°C, fixed in 4% paraformaldehyde for 15 minutes at room temperature, and permeabilized with Perm/Wash solution (BD Biosciences) for 10 minutes. Cells were then stained with FITC-conjugated anti-IFN-γ and PE-Cy7-conjugated anti-TNF-α mAbs for 30 minutes at 4°C and analyzed by flow cytometry.