Anti cd3 apc h7 sk7

NK cell degranulation was assessed as previously described [54 (link)]. Aliquots of one million freshly isolated PBMCs were incubated for 2 h at 37 °C in 200 µL complete RPMI-1640 medium under four stimulation conditions: leukocytes alone, +1 µg/mL elotuzumab, + MM.1R target cells (one million cells), or + both MM.1R and elotuzumab. Samples were centrifuged at 150 relative centrifugal force (RCF) for 3 min before incubation. Anti-CD107a-PE (H4A3, BD Biosciences Inc., San Jose, CA, USA), anti-CD45-PerCP-Cy5.5 (2D1, eBioscience, San Diego, CA, USA), anti-CD3-APC-H7 (SK7, BD Biosciences Inc., San Jose, CA, USA), and anti-CD56-APC (NCAM 16.2, Biolegend, San Diego, CA, USA) antibodies were added in the last 30 min of culture. Cells were centrifuged and rinsed twice with ice-cold wash buffer, with PI added in the second wash. NK cell degranulation was measured as a percentage of CD107a⁺ cells among total PI⁻CD45⁺CD3⁻CD56^dim NK cells.

To assess NK cell degranulation against MM target cells (tumor cells), CD107a expression on NK cells was analyzed using flow cytometry. Target cells were plated in 24 wells plate at a concentration of 2 × 10⁶ cells/mL per well and incubated overnight at 37 °C in humidified air containing 5% CO₂ with 21% O₂ (Sanyo MCO-20AIC, Sanyo Electric Co, Japan) or 0.6% O₂ (Invivo_2, 1000 Ruskinn Technology Ltd, Bridgend, UK). Prior to the assay, IL-2 activated NK cells were harvested, washed, and subjected to 1-h incubation with either 50 mM sodium l-lactate (Sigma), 100 ng/mL prostaglandin (Sigma), or medium. Target cells were pre-incubated for 30 min with 1 µg/mL daratumumab (Genmab) or trastuzumab (Roche) or, as a control, with medium at 21% O₂ (ambient air) or 0,6% O₂ (hypoxia). TMEF-exposed NK cells were then, in duplicate wells, co-cultured with the target cells in 1:1 effector:target ratio and 2 µL anti-CD107a-Horizon V450 (H4A3, BD Biosciences) was added per well. After 1 h of co-culture, monensin (BD Biosciences) was added. After another 3 h, the plate was placed on ice to stop the reaction. Cells were then stained on ice with anti-CD3-APC/H7 (SK7, BD Biosciences), anti-CD56-PeCy7 (B159, BD Biosciences), anti-KIR2DL1-APC (143211, R&D), anti-KIR2DL2/3/S2-PE (DX27, Miltenyi Biotec), anti-KIR3DL1-FITC (DX9, Miltenyi Biotec), and anti-NKG2A-PC5.5 (Z199, Beckman Coulter).

NK cells were isolated from blood by negative MACS selection (Miltenyi Biotec) according to manufacture's instructions. NK cells were activated with 1000 U/ml IL-2 (Proleukin) for 6 h at 21 % O₂. Activated NK cells were co-cultured with target cells at 1:1 ratio in 96-well round-bottom plates in duplicate with anti-CD107a-Horizon-V450 (H4A3, BD). After 1 h, co-culture at 21 % O₂ or 0.6 % O₂, monensin (BD GolgiStop, Cat# 554724) was added. After another 8–9 h, co-cultures were stained on ice with anti-CD3-APC/H7 (SK7, BD), anti-CD56-PeCy7 (B159, BD), anti-KIR2DL1-APC (143211, R&D), anti-KIR2DL2/3/S2-PE (DX27, Miltenyi Biotec), anti-KIR3DL1-FITC (DX9, Miltenyi Biotec) and anti-NKG2A-PC5.5 (Z199, Beckman Coulter). For co-cultures with IL-2-activated NK cells, target cells were pre-incubated for 6 h at 21 % O₂ or 0.6 % O₂. For experiments without IL-2 activation, target cells cultured at 21 % O₂ were used. Target cells were subsequently co-cultured with freshly isolated NK cells in a 10- to 12-h degranulation assay. For HLA-E blocking experiments, target cells were pre-incubated for 30 min at 37 °C with 10 µg/ml of anti-HLA-E (3D12; IgG1 isotype eBioscience) or IgG1 isotype control. Gating strategy is described in supplemental figure S2.