Lamp1 apc

For flow cytometry, cells were washed with PBA [PBS supplemented with 0.5% bovine serum albumin (BSA), 1% FBS and 0.1% sodium azide] and incubated with antibodies against surface markers [CD3-CF (Immunostep); CD3-FITC, CD56-PC5, CD16-PE-Cy7, CD4-PC5.5, CD8-PC7 and LAMP1-APC (Biolegend)] at 4°C for 30 min in the dark. For intracellular staining, after surface labelling, cells were fixed with 1% p-formaldehyde for 10 min at RT, permeabilized with 0.2% saponin for 10 min at RT and stained with Perforin-PE, Granzyme B-PE (Biolegend) for 30 min. Cells were washed in PBA and analyzed using a Gallios Flow Cytometer (Beckman Coulter). Analysis of the experiments was performed using Kaluza software.

10⁴ lung cancer cells (H1322 and H2188) cells were plated in 96-well flat-bottom plates in triplicates. The next day, cells were labeled with calcein-AM (Molecular Probes, C3100MP) and pre-treated with HP1F7 antibody to block MHC-I. PBMCs were co-cultured with target cells for 3 h at an E:T ratio of 5:1 (the percentage of NK cells was previously determined for each donor by flow cytometry). Calcein-AM release was determined by measuring absorbance using BioNova® F5 System. Specific lysis was expressed as a percentage, calculated as the ratio [(value  −  spontaneous release)/(maximum − spontaneous release)] × 100. Spontaneous release corresponds to target cells alone. Maximum release was determined by lysing the target cells in 0.5% Triton X-100 (ThermoScientific). NK cells were identified with the following antibodies: CD3-PacificBlue, CD16-PE-Cy7, and CD56-PE, from Biolegend. For degranulation experiments, cells were additionally stained with LAMP1-APC (Biolegend) for 30 min. After staining, cells were washed in PBS and analyzed in a CytoFLEX (Beckman Coulter). Analysis was performed using Kaluza software and representative gating strategies for CD107a expression on NK cells are shown in Supplementary Fig. 14.