Fitc conjugated anti mouse ifn γ

The cells were treated with or without 1 μg/ml Jagged-1 (R&D Systems, Minneapolis, MN, USA) for 72 h in the presence of anti-CD3/CD28 beads at a bead-to-cell ratio of 1:1 (Invitrogen, Carlsbad, CA, USA), 20 ng/ml IL-6, 10 ng/ml TGF-β, 20 ng/ml IL-23 (PeproTech, Rocky Hill, NJ, USA), 10 μg/ml anti-IFN-γ and 10 μg/ml anti-IL-4 (eBioscience, San Diego, CA, USA). The additional cells were treated with the equal volume of phosphate buffered saline (PBS) as a control. Six hours before the end of the treatment, the cells were stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) plus 1 μg/ml of ionomycin (Alexis, Lausen, Switzerland) for the below experiments. Meanwhile, 10 μg/ml of GolgiStop (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the cells. The cells were washed with PBS and stained with FITC-conjugated anti-mouse CD4 (0.125 μg per million cells) at 37°C for 20 min. The cells were washed, fixed, permeabilized with Fixation/Permeabilization Buffer and intracellular-stained with PE-conjugated anti-mouse IL-17 (0.05 μg per million cells) and FITC-conjugated anti-mouse IFN-γ (0.25 μg per million cells) (eBioscience, San Diego, CA, USA) for 30 min at 4°C, and analyzed with a flow cytometer (FACSCalibur, Becton Dickinson, Mountain View, CA, USA).

CH (MW 50–190 KDa), TPP, OVA, and poly I:C were purchased from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated anti-mouse IFN-γ, PE-conjugated anti-mouse CD40, and anti-OVA-specific (SIINFEKL/H-2Kb) antibodies and mouse TNF-α, IL-1β, IL-6, and IL-12p70 ELISA Ready-SET-Go kits were purchased from eBioscience (San Diego, CA, USA). A FITC-conjugated anti-mouse CD11c antibody and PE-conjugated anti-mouse CD8a, CD80, CD86, MHC class I, and MHC class II antibodies and a mouse IFN-γ ELISA kit were purchased from Biolegend (San Diego, CA, USA). RPMI 1640 and fetal bovine serum (FBS) were acquired from Biowest (Nuaille, France). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from JW Creagene (Gyeonggi, South Korea). HPV-16 E7 peptide (MW 2.4 kDa, AGQAEPDRAHYNIVTFCCKCDS)37 was purchased from AnyGen Co. (Seoul, South Korea). All other materials were of analytical grade and used without further purification.

After cell isolation, cocultures were set up as mentioned above. Additionally, 50 ng/ml PMA (InvivoGen, San Diego, USA) was added from the beginning, 750 ng/ml ionomycin (Sigma-Aldrich, St. Louis, USA) for the last 4 h, and 1 μg/ml GolgiPlug (BD Biosciences) was added 2 h before cell harvesting. Culture supernatants were harvested and stored at −20°C for IFN-γ ELISA. Cells were fixed in 1 ml Fix/Perm (eBioscience, Hatfield, UK) for 60 min at 4°C. After incubation with permeabilization buffer (eBioscience), cells were stained intracellular with PE-conjugated anti-mouse-Abs (IL-2, clone: JES6-5H4/IFN-γ, clone: XMG1.2/TNF-α, clone: MP6-XT22) from BD Biosciences and with PE-conjugated anti-mouse-IL-13 (clone: eBio13A) and FITC-conjugated anti-mouse-IFN-γ (clone: XMG1.2) all eBioscience.