Multiparameter Flow Cytometry Analysis

The following antibodies were used for the analysis of surface and intracellular markers: anti-CD73 (2C5) antibody was discovered at AstraZeneca (18 (link)) and conjugated to fluorochrome in house; anti-CD73 (TY/11.8) was purchased from BioLegend; anti-Ki-67 (SolA15), anti-granzyme B (GB11), and anti-CD8 (OKT8) were purchased from eBioscience; and FcR blocking reagent was purchased from Miltenyi Biotec. Intranuclear staining was performed using Fixation/Permeabilization buffer solution (eBioscience) according to the manufacturer’s instructions. MART-1 dextramer was purchased from Immudex, and the staining was performed according to the manufacturer’s instructions. In some experiments, cells were treated with EGF (50 ng/ml), EGFR inhibitors (osimertinib, 1 μM, AstraZeneca), NF-κB inhibitor (IKK inhibitor VII, 1 μM, MilliporeSigma), ERK inhibitor (LY3214996, 1 μM, Selleckchem), or PKC inhibitor (Go 6983, 10 μM, MilliporeSigma) for 48 hours before analysis. Stained cells were analyzed on a LSRFortessa (BD Biosciences), and data were analyzed with FlowJo software.