Isotype control

Six days after co-culture, CD8⁺ T cells were harvested and stained to assess CD107a expression following stimulation with PMA (25 ng/ml, Sigma-Aldrich) and ionomycin (1 µM, Sigma-Aldrich) for 3 hr. Cells were stained with anti-CD8-PerCP-Vio700, anti-CD45-RA-FITC, or isotype controls (Miltenyi Biotech, Paris, France). For membrane expression analysis, cells were then stained with anti-CD107a-PE or isotype control (BD Biosciences, San Jose, CA). For total expression analysis, cells were fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter), and stained with anti-CD107a-PE or isotype control. FACS data were acquired using a Canto II 4-Blue 2-Violet 2-Red laser configuration (BD Biosciences).

To evaluate CD16A-dependent functional responses, NK92 hCD16V (see Section 2.4.1) effector cells were incubated with target cells. NK92 hCD16V (5 × 10⁴ cells) were incubated for 4 h with Daudi or SK-BR-3 cells (E:T 1:2) in the presence of 10 µg/mL of mAbs or BsmAbs at 37 °C, anti-CD107a mAb and 0.1 µg/mL BD GolgiPlug containing Brefeldin A (BD Biosciences, San Jose, CA, USA) at 37 °C in 5% CO2 humidified air. Cells were then stained with anti-CD16 and anti-CD56 Abs for 30 min at 4 °C, then fixed and permeabilized by using the BD Cytofix/cytoperm Plus Kit (BD Biosciences, San Jose, CA, USA) and stained for intracellular interferon γ (IFN-γ) for 30 min at 4 °C. The following Abs were used: APC Alexa Fluor 750-conjugated anti-CD16 (clone 3G8), ECD-conjugated anti-CD56 (clone N901), PE-conjugated anti-IFN-γ (clone 45.15) and isotype control (Beckman Coulter, CA, USA), PC5-conjugated anti-CD107a (clone H4A3) and isotype control (BD Biosciences). Functional responses and MFI of each fluorochrome of cell subsets were analyzed by FCM.

EDTA whole blood samples from 5 healthy subjects, 10 patients with mild and 10 patients with severe disease were stained with anti-HLA-DR PE and ECD, anti-CD3 ECD, anti-CD4 PE, anti-CD8 FITC, anti-CD19 PC7, anti-CD14 FITC, anti-CD16 PC5, anti-CD15 PE, anti-CD57 FITC, anti-CD56 PE, anti-CD11c PCP, anti-CD123 PE, anti-CD83 PE, anti-CD38 PE, anti-CD23 ECD and isotype controls (all from Beckman Coulter) for 20 minutes in the dark at +4°C. Samples were analyzed on the flow cytometer Cytomics FC500 (Beckman Coulter). Data were processed by FlowJo V.10.