For in vitro NK-cell co-cultures,
FCS was replaced by 5% normal human AB serum (NHS) (Nabi, Boca Raton, FL, USA). Recombinant human (rh)IL-12p70 and rhIL-18 were purchased from R&D Systems (Minneapolis, MN, USA) and from Bender MedSystems (Burlingame, CA, USA), respectively. Ficoll-Paque™ was obtained from Amersham Biosciences AB (Uppsala, Sweden). Monensin and Brefeldin A were purchased from eBioscience (San Diego, CA, USA) and Sigma (St. Louis, MO, USA), respectively. Autologous LCL was generated in our laboratory as previously described and was used as EBV+ stimulators in functional assays 38. NK-cell phenotype was determined by seven-color flow cytometric analysis as previously described 8. Briefly, 100 μL whole blood or 0.1×106 PBMC aliquots were incubated for 30 min at room temperature or 4°C, respectively, in the dark with different Sirolimus combinations
of fluorochrome-conjugated mAbs, such as anti-CD3, anti-CD19, anti-CD56, anti-CD16, anti-NKG2D and anti-PD-1 (all from e-Bioscience), anti-NKp46 (Miltenyi Biotech GmbH, Auburn CA, USA). Stained aliquots from whole blood were further incubated for 10 min at room temperature with MK-8669 concentration 2 mL/tube of lysing buffer (BD Bioscience) to allow red blood cell lysis. All tubes were then washed twice with FACS buffer (PBS supplemented with 1% FCS, and 0.05% NaN3) and fixed with 2% paraformaldehyde-containing FACS buffer (Sigma). Appropriate isotype negative controls were always used to define background staining. Data acquisition was performed using an LSR II (BD Biosciences) and analyzed using the FlowJo software (Tree Star, Ashland OR, USA). Thawed PBMC (1×106 cells/mL) were plated in 48-well plates (Costar Corning, Corning, NY, USA) in the presence
of (i) hrIL-12p70 Montelukast Sodium (10 ng/mL)+hrIL-18 (20 ng/mL) or (ii) autologous LCL cells (at 5:1 NK:LCL ratio) for 18 h at 37°C, 5% CO2. PBMCs cultured in media alone were used as negative controls. In selected experiments, neutralizing antibodies against PD-1 (R&D Systems) were added at 20 μg/mL at co-culture initiation. NK-cell degranulation upon activation, as a direct measurement of cytotoxicity, was assessed by CD107a staining, as previously described 39. Briefly, anti-CD107a mAb (eBioscience) was added at co-culture initiation. During the last 4 h of co-culture, monensin (2 μM) and brefeldin A (15 μg/mL) were added to each condition according to the manufacturer instructions. Cells were then harvested, washed, surface stained and fixed as described above. NK-cell intracellular cytokine staining was detected simultaneously by further cell permeabilization with 2% saponin (Sigma) and intracellular staining with anti-IFN-γ mAb (eBioscience). Appropriate isotype negative controls were always used to define background staining.