Anti nkg2d clone 1d11

2 × 10⁵ NK-92 cells were co-incubated with 2 × 10⁵ target cells in a final volume of 200 μl in V-bottomed 96-well plate at 37°C and 5% CO₂ for 4 h. In blocking experiments, target cells were pre-incubated with the corresponding 25 μg/mL blocking antibodies [anti-CD155 (clone SKII.4, Biolegend), anti-DNAM1 (clone DX11, BD Pharmingen) and anti-NKG2D (clone 1D11, Biolegend)] for 15 min on ice prior to co-culture. Fluorochrome-conjugated anti-CD107a-PE (H4A3, BD Biosciences) mAb was added at the initiation of the assay. After 1 h of co-incubation, GolgiStop (BD Biosciences) was added at a 1:300 dilution. The cells were then washed, resuspended in ice-cold PBS and stained with surface anti-CD56 (NCAM 16.2, APC, or BV421, BD Biosciences or Biolegend) to be analyzed by flow cytometry. For flow cytometry, cells were first gated on FSC-A vs. SSC-A, followed by gating on single cells via FSC-A vs. FSC-H, then GFP⁺CD56⁺ cells were selected and lastly CD107a⁺CD56⁺ percentage was recorded for analysis. All flow cytometry analysis was performed with FlowJo software v10.5 (BD Biosciences).

Ex vivo-expanded Vγ9Vδ2 T cells were activated as described above and extensively washed with medium to remove cytokines. RPMI8226 myeloma cells were labeled with 1 μM carboxyfluorescein succinimidyl ester (CFSE) (Dojindo), and 1 × 10⁵ CFSE-labeled tumor cells were incubated with 4 × 10⁵ Vγ9Vδ2 T cells in RPMI-1640 for 2 h in a 24-well plate. MG-63 osteosarcoma cells were cultured overnight at a density of 6 × 10⁴ cells per well in a 48-well plate and then labeled with 1 μM CFSE before incubation with 4.8 × 10⁵ Vγ9Vδ2 T cells in RPMI-1640 for 4 h. Cells were collected and stained with Annexin V-APC (BioLegend) and subjected to FACS analysis. CFSE⁺Annexin V⁺ cells were considered to be dead tumor cells. In some experiments, IL-12/IL-18 activated Vγ9Vδ2 T cells were treated with 100 ng/mL of concanamycin A (Santa Cruz Biotechnology, Dallas, TX), 10 μg/mL of anti-NKG2D (clone 1D11) (BioLegend) or 10 μg/mL of anti-DNAM-1 (clone 102511) (R&D Systems, Minneapolis, MN) for 30 min before the coincubation with MG-63 cells.