Anti il 12 p40 p70

Naïve CD4⁺ T cells were purified from mouse spleen and MLNs using Naive CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Purified naïve CD4⁺ T cells were plated at a density of 2.5 × 10⁵ cells/well at 37 °C in 48-well plates pre-coated with anti-CD3e (Clone: 145-2C11, 1 μg/ml, BD Biosciences) and anti-CD28 (Clone: 37.51, 1 μg/ml, BD Biosciences) antibodies. Activated cells were polarized under Th0 (no supplement), Th1 [IL-12 (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems) and anti-IL-4 antibody (5 μg/ml, BD Biosciences)], Treg [TGF-β (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems), anti-IFN-γ (5 μg/ml, BD Biosciences), anti-IL-12 p40/p70 (5 μg/ml, BD Biosciences) and anti-IL-4 (5 μg/ml, BD Biosciences) antibodies] differentiation conditions in complete RPMI medium or polarized in splenic DC-conditioned medium (overnight culture supernatant of CpG-ODN-stimulated splenic DCs).

Antibodies used in the current study were Anti-CD11b PE (eBioscience M1/70)/APC (BD,M1/70), anti-GR-1 FITC (BD RB6-8C5), anti-Ly6G FITC (RB5-8C5) APC (RB5-8C5), anti-Ly6C PreCP-Cr™5.5 (BD AL-21), anti-IL10 FITC (BD), anti-IL12 (P40/P70) (BD), anti-p-Stat3 PE (BD PY705), anti-NOS2 PE (eBioscience CXNFT), arginase FITC (R&D), anti-CD8 PE (BD), anti-IFNγ FITC (BD).
The numbers of IFN-γ-secreting CD8⁺ T cells were analyzed by flow cytometry after adding Golgi plug (1 μg/mL, BD Pharmingen) for 8 hours. Analysis was performed on a Becton-Dickinson FACScan with CELLQuest software (Becton-Dickinson Immunocytometry System, Mountain View, CA) and Flowjo 10 software.

Splenocytes were incubated with anti-mouse CD16/32 (BD Biosciences; San Jose, CA) at a concentration of 1 ug per million cells for 5 min at 4°C to block Fc receptors. The panel of mAbs used for flow cytometric analyses were anti-Gr1 (RD-8C5), anti-CD11b (M1/70), anti-Ly6C (AL21), anti-Ly6G (1A8), anti-CD11c (N418), anti-F4/80 (BM8), anti-NK1.1 (PK136), anti-TLR2 (6C2), anti-TLR4 (MTS510), anti-TLR5 (85B152.5), anti-IL-10 (JES5-E16E3), and anti-IL-12 (p40/p70), which were purchased from BD Biosciences, eBiosciences, and BioLegend (San Diego, CA). Intracellular staining was performed using BD Cytofix/Cytoperm Kit (BD Bioscience). Data were collected on a Dako CyAN and analyzed using Summit software (Dako; Carpinteria, CA).