αcd3 clone 17a2

Mouse CD8 + T cells were purified from spleens of C57BL/6 mice using EasySep mouse CD8 + T cell isolation kit (Stem Cell). Purified CD8 + T cells (10 6 cells/mL) were activated in six-well plates precoated with 2 μg/mL α-CD3 (clone 17A2, Bioxcell) and supplemented with soluble 5 μg/mL α-CD28 (clone 37.51, BioLegend) and 30 ng/mL mouse IL-2 (Peprotech) for 3 days. Culture medium was IMDM (Gibco) containing 10% heat-inactivated FBS, 1% Penicillin/Streptomycin and 50 μM 2-mercaptoethanol (Sigma Aldrich). After 3 days of culture, activated CD8 + T cells were rested for 6 hrs in fresh culture medium and were transferred into 96-well plates (50,000 cells/well). Indicated amounts of IL-12 or CBD-IL-12 were applied to CD8 + T cells for 20 min at 37 °C to induce STAT4 phosphorylation. Cells were fixed immediately using BD Phosflow Lyse/Fix buffer for 10 min at 37 °C and then permeabilized with BD Phosflow Perm Buffer III for 30 min on ice. Cells were stained with Alexa Fluor (AF) 647-conjugated antibody against pSTAT4 (clone 38, BD) recognizing phosphorylation of Tyr693. Staining was performed for 1 hr at room temperature (RT) in the dark. Cells were acquired on BD LSR and data were analysed using FlowJo (Treestar). Mean Fluorescence Intensity (MFI) of pSTAT4 + population was plotted against cytokine concentration. Dose-response curve was fitted using Prism (v8, GraphPad).