Tnfα mab11

Fluorophore-conjugated murine antibodies were purchased from eBioscience, including antibodies specific for CD8a (53–6.7; catalog #48-0081-80), Thy1.1 (HIS 51; 25–0900-82), Tim3 (RMT3-23; 14–5870-81), Lag3 (ebioC9B7W; 12–2231-81), CD137 (17B5; 17-1370-80), KLRG1 (2F1; 175893–81), and CD25 (PC 61.5; 17–0251-81). Other antibodies to FasL (K10; 106805), vβ9 (MR10-2; 553201), CD69 (H1.2F3; 104502), CD127 (A7R34; 135013), CD80 (16-10A1; 553768), CD86 (GL-1; 105011), IL-2 (JES6-5H4; 503807), IFNγ (XMG1.2; 505809), TNFα (Mab11; 502913), Bax (6A7; 633801), and Bcl-2 (10C4; 633507) were purchased from Biolegend. 7AAD (A1310) and other antibodies were purchased from BD Biosciences, including Fas (Jo2; 563647), Thy1.2 (53–2.1; 561616), CD103 (M290; 557495), purified rat anti-mouse CD16/CD32 (2.4G2; 12–4875-80), PD-1 (J43; 11–9985-81), and Vβ8.1/8.2 (553185). Antibodies against pAKT (S473; D9E; 5315S) were purchased from Cell Signaling Technology. Anti-Eomes (DAN11 mag; 12–4875-80) and the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (L34957) were purchased from ThermoFisher. For FasL detection, Panc1 cells were treated for 24 h with 3 µg/ml Batimastat (BB-94; ab146619) purchased from Abcam. Flow cytometry data were acquired on a BD FACS Canto II instrument or LSR II and analyzed with Flowjo v9 software.

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.