Il 12 il 23 p40

Immune cell surface staining was used with the following antibodies, including anti-CD3ε PE, anti-CD4 PerCP-Cy5.5, anti-CD8α PerCP-Cy5.5, anti-NK1.1 FITC, anti-CD11b FITC, anti-CD11c APC, anti-Gr-1 PerCP-Cy5.5, anti-CD103 PE, anti-80 PE, anti-86 PE, anti-MHC-II PE, and anti-F4/80 PerCP. All of these antibodies were purchased from BioLegend (San Diego, CA, USA). For 293 cells and tumor cells, an anti-GPC3 antibody (APC-conjugated, Sino Biological) was used. The target cells were incubated with antibodies at 4 °C for 30 min. For intracellular cytokine staining, splenocytes were stimulated with 10 μg/mL hGPC3 protein for 72 h. Ionomycin (500 ng/mL, Sigma-Aldrich), PMA (50 ng/mL, Sigma-Aldrich), and Brefeldin A (5 ng/mL BFA, eBioscience, San Diego, CA, USA) were used to stimulate splenocytes at 37 °C and 5% CO₂ for the last 5 h. The cells were firstly stained with antibodies against CD8α (PerCP-Cy5.5) and then conducted using intracellular staining (IFN-γ, APC; TNF-α, FITC; IL-2, PE). For IL-12 staining, 293 cells were performed using intracellular staining with IL-12/IL-23p40 (PE, BioLegend). Flow cytometry analysis was performed using BD FACS CantoII (BD Biosciences, Shanghai, China) or Cytek^® Northern Lights (Cytek Biosciences, Fremont, CA, USA) and analyzed by FlowJo software (Tree Star Inc., San Francisco, CA, USA).

In the supernatant of the matured moDCs, IL-12/IL-23 (p40) was measured according to the manufacturer's protocol (BioLegend, Koblenz, Germany). As such, IL-13, IFNγ, and IL-10 (from BioLegend, Koblenz, Germany) were determined in the supernatant of the DC–T-cell co-cultures. All samples were measured using a Tecan Infinite 200PRO (Tecan, Männedorf, Switzerland).