Dnam 1 dx11

Target cells were labeled with 2 µM PKH26 fluorescent dye (Sigma) according to the manufacturer’s instruction and cocultured with EV-DNTs and CAR4-DNTs in a 96-well plate at various effector-to-target ratios for 2 hours or 24 hours at 37°C. Subsequently, cells were stained with annexin V, and apoptosis of the target cells was analyzed by flow cytometry. Per cent specific killing was calculated using the formula: ${\displaystyle \frac{\mathrm{\%}AnnexinV+withDNT-\mathrm{\%}AnnexinV+withoutDNT}{100-\mathrm{\%}AnnexinV+withoutDNT}\times 100}$ as previously described. For transwell assays, an HTS Transwell 96-well permeable support (Sigma) with 0.4 µm pore size was used. For blocking assays, anti-CD18 (TS1/18, BioLegend), NKG2D (1D11, BioLegend), DNAM-1 (DX11, BD Bioscience), TNF-α (MAb11, BioLegend), IFN-γ (MD-1, BioLegend), TRAIL (RIK-2, BioLegend), and FasL (NOK-1, BioLegend) antibodies or immunoglobulin 1 (IgG1) isotype control (QA16A12, BioLegend) was added at 10 µg/mL. For blocking assays with concanamycin A (CMA), EV-DNTs or CAR4-DNTs were treated with CMA (100 nM; Sigma) or dimethyl sulfoxide (DMSO) for 30 min prior to use for in vitro cytotoxicity assay.

To explore receptor-ligand interactions by blocking antibodies, the MaestroZ (Axion Biosystems) impedance-based system was used. To correct for background resistance, wells were first measured in 50 μl medium as a baseline. Target cells were seeded at appropriate cell concentrations in a total volume of 100 μl and were allowed to adhere for 5 hours at 37°C. NK cells were pre-incubated with 10 μg/ml blocking antibody for 15 minutes at 37°C. The following blocking monoclonal antibodies (mAbs) were used: NKG2D (1D11), TRAIL (RIK-2), FASL (NOK-1), NKp30 (P30-15), NKp44 (P44-8), NKp46 (9E2) all from Biolegend and DNAM-1 (DX11) from BD Biosciences. Mouse IgG1, kappa (MOPC-21) from Biolegend was used as isotype control antibody at a maximum concentration of 40 μg/ml (corresponding to the concentration of combining the blocking Abs). After incubation, NK cells were added at a 1:1 E:T ratio. The co-culture was followed for 24 hours, and data was analyzed using the Axis Z software (Axion Biosystems).