Tnfα mab11

NK cells were stained with antibodies recognizing CD56 (NCAM16.2), CD3 (UCHT1), CD34 (581), CD4 (RPA-T4), CD19 (HIB19), TNF-α (Mab 11), and IFN-γ (B27) from BD Biosciences (Franklin Lakes, NJ, USA), CD107a (H4A3), CD34 (561), and CXCR4 (12G5) from BioLegend (San Diego, CA, USA), along with propidium iodide (BD Biosciences), annexin V (BioLegend), and/or a Live/Dead fixable aqua dead cell stain kit (Thermo Fisher Scientific). Cells were examined on a BD Biosciences LSRFortessa instrument, and data were analyzed using FlowJo v10.1 (BD Biosciences). Statistical analyses were performed with GraphPad Prism 8.4.1.

For flow cytometry, carboxyfluorescein succinimidyl ester (CFSE) was obtained from Invitrogen (Carlsbad, CA, USA). Phycoerythrin (PE)-conjugated antibodies to human IL-15 were obtained from R&D Systems (catalog number, 34559; Minneapolis, MN, USA). TNF-α (MAb11), TNF-related apoptosis-inducing ligand (TRAIL; RIK-2) and CD107a (H4A3) were from BD Biosciences (Franklin Lakes, NJ, USA). PerCP-Cy5.5-conjugated antibodies to human CD3 were from BioLegend (HIT3a; San Diego, CA, USA). Alexa Fluor 647-conjugated antibodies to human CD56 (B159) and Ki67 (B56) were purchased from BD Biosciences. Alexa Fluor 647-conjugated 161519 antibody was labeled by an Antibody Labeling Kit according to the manufacturer’s instructions (GeneCopoeia, Rockville, MD, USA).
For staining of TRAIL, CD107a, and intracellular cytokines, PBMCs were co-cultured with tumor cells in the presence of 161519 TriKE and monensin (2.5 μg/mL; eBioscience, San Diego, CA, USA) for 4 h. After incubation, tumor cells were stained for surface markers, fixed, and permeabilized with FoxP3 fixation buffer according to the manufacturer’s instructions (eBioscience). The fixed cells were then stained with antibodies to IFN-γ and TNF-α. All samples were acquired on an LSR-II or FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and were analyzed using FlowJo (TreeStar, Ashland, OR, USA).