Monoclonal antibodies specific

For flow cytometry, NK-92 cells were stained with monoclonal antibodies specific for CD56, CD3, CD45, CD28, CD16, CD11a, CD2, NKG2D, NKp30, NKp46, DNAM-1, CD95 (BD Bioscience) for 30 minutes on ice. CAR expression on the surface of NK-92 cells was detected with an anti-mouse F(ab’)2 antibody (Jackson Immunoresearch). For checkpoint molecules, antibodies specific for PD-1, LAG-3 and TIM-3 (BD Biosciences) were used. Dead cells were excluded using 7-AAD viability dye (BD Biosciences). Samples were acquired using an LSR-Fortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo 10 software (BD Biosciences).

For bronchoalveolar lavages (BAL) sampling, the trachea was cannulated, and 1 ml of PBS plus 1 mM EDTA was instilled six times and recovered by gentle aspiration. BAL cells were washed in PBS containing 2 mM EDTA and 1% fetal bovine serum (FACS-EDTA). For immunophenotyping, 1x10⁶ cells were seeded per tube, and Fc receptors were blocked by incubation with 1 μl of Fc Block (BD) for 20 minutes at 4°C. Then, cells were labeled with specific monoclonal antibodies (BD) (Ly6G-PE, CD11b-APC-Cy7) for 30 minutes at 4°C and finally washed and fixed with 4% formaldehyde and stored at 4°C in dark. Cells were acquired into a FACS Canto II cytometer using BD FACSDiva™software (BD) for acquisition and analysis.