Il 12

NK cell degranulation and cytokine production was evaluated by mixing ADAPT-NK products at different effector to target (E:T) ratios with tumor targets in 6 hour co-cultures. To measure ADCC, anti-CD20 (MabThera, 1 µg/mL) was added to co-cultures with 721.221 cells. Phorbol-12-myristate-13-acetate (50 ng/mL) + ionomycin (1 µg/mL) (Sigma) was used as positive control. In cytotoxicity assays, target cells were pre-stained with CellTrace Violet (Invitrogen), and FITC-DEVD-FMK (Abcam) to detect active Caspase-3, was added at the start of the incubation. Dead/dying cells were defined as CellTrace⁺ Caspase-3⁺ and/or dead cell marker⁺ and specific cytotoxicity was calculated as: (% dead ^experimental- % dead ^{target only}) ÷ (100% % dead ^{target only}) x 100%. For competitive cytotoxicity assays, K562 expressing an HLA-C1-or HLA-C2-dimer were stained with two different concentrations of CellTrace Violet prior to being mixed at a 1:1 ratio and subsequently seeded at different E:T ratios with ADAPT-NK cells. Cytokine stimulations with IL-12 (10 ng/mL) and IL-18 (10 ng/mL) (both Biotechne) were performed for 25 hours. Further experimental details are provided in online supplemental methods.

TGFβ1, IL-12 and IL-10 ELISAs (all Bio-Techne) were performed as per the manufacturer’s instructions.

PBMC were isolated using the standard protocol for Ficoll density gradient centrifugation followed by erythrocyte lysis as described before [13 (link)]. Briefly, PBMC were cultured in “Very low endotoxin” VLE Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 100 U/mL Penicillin, 100 µg/mL Streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 10% (v/v) serum. Cultures of PBMC were set up at least in triplicates (4 × 10⁵ cells/well) in 96-well flat bottom plates (BD Biosciences, Heidelberg, Germany) as described [13 (link)] and rested for 30 min. Where indicated, recombinant human IL-2 (1 ng/mL; Biotechne), IL-12 (1 ng/mL; Biotechne), or both was added before stimulation of the cells with heat-inactivated S. aureus (Pansorbin Cells Standardized, 0.05% (v/v), Calbiochem, Merck, Darmstadt, Germany) 30 min later. Unstimulated PBMC served as negative control. After 16 h, PBMC were harvested for further analyses.
In some experiments, PBMC from healthy donors were cultured in the presence of 4% (v/v) serum from patients. Therefore, the sera from all patients were pooled for each time point.