Anti cd107a mab

Polyfunctional NK cells were studied with degranulation assays and the cellular production of cytokines was assessed, as previously described (18 (link)). NK cells were pretreated overnight in the presence of interleukin (IL)-12 (10 ng/mL) and IL-18 (100 ng/mL) to measure the intracellular production of IFN-γ and TNF-α by NK cells or incubated with standard HLA class 1-negative K562 target cells (ATCC CCL243) at an effector : target (E : T) cell ratio of 1 : 1 to measure degranulation in the presence of anti-CD107a mAb (#H4A3; Becton Dickinson) (Supplementary Table 1). Cells were incubated for 5 h in the presence of Golgi Stop and Golgi Plug solutions (BD Biosciences; Franklin Lakes, NJ, USA) and then stained with NK cell surface markers. NK cells were thereafter fixed, permeabilized with a Cytofix/Cytoperm kit (Becton Dickinson; Franklin Lakes, NJ, USA), and then intracellularly stained with anti-IFN-γ and anti-TNF-α mAbs (Supplementary Table 1), as described previously (18 (link)). Only samples containing at least 100 CD3⁻CD56⁺ NK cells were acquired on a DXFlex flow cytometer and then analyzed with FlowJo version 10; consequently, the number of samples may vary depending on the experiments.

NK cells were pre-treated overnight in the presence of interleukin (IL)-12 (10 ng/ml) plus IL-18 (100 ng/ml) to measure the intracellular production of IFN-γ and TNF-α by NK cells, or incubated with the standard HLA class-1 negative K562 target cells (ATCC CCL243), at an effector:target (E:T) cell ratio of 1:1, to measure degranulation in the presence of anti-CD107a mAb (#H4A3; Becton Dickinson) (Supplementary Table 1). Cells were thereafter incubated for 5 hours in the presence of Golgi Stop and Golgi Plug solutions (BD Biosciences) and then stained with NK cell-surface markers. Next, NK cells were fixed, permeabilized with a cytofix/cytoperm kit (Becton Dickinson) and then intracellularly stained with anti-IFN-γ and anti-TNF-α mAbs (Supplementary Table 1), as previously described (Carbonnel et al., 2022 (link)). At least 1000 CD3^-CD56⁺ NK cells were acquired on a Gallios flow cytometer (Beckman Coulter) and then analyzed with Flow Jo version 10 (TreeStar, state).