Hmgb1-IL-23-IL-17-IL-6-Stat3 Axis Promotes Tumor Growth in Murine Models of Melanoma
Figure 3
IL-23 is critical for the generation of IL-17 in vivo and in vitro. (a) IL-23p19 and IL-23p40 mRNA levels were analyzed by realtime PCR in tumor tissues from tumor-bearing mice one or two weeks after B16-F10 inoculation (). (b) IL-23p19 and IL-23p40 mRNA levels were analyzed by realtime PCR in tumor tissues two weeks after B16-F10 inoculation from WT, WT + Anti-γ δ TCR, and IL-17−/− groups (). (c) Wild-type mice were inoculated with B16-F10 tumor cells and injected i.p. with normal rat-IgG, -IL-23p19 mAb, or -IL-23p40 mAb (100 μg/mouse) on days 0, 1, 6, 10, and 14 (). Then tumor sizes were monitored (). (d) IL-17 relative mRNA expression in tumor tissues from B16-F10 tumor-bearing mice treated with rat-IgG, -IL-23p19 mAb, or -IL-23p40 mAb (). (e) The percentage of cells producing IL-17 in control, anti-IL-23p19, and anti-IL-23p40 treated group (). (f) IL-17-producing γ δ T cells after stimulation with IL-23. IL-17+γ δ T cells were analyzed by flow cytometry after stimulation for 48 hours. Tumor lymphocytes were isolated from tumor-bearing mice 1 week after B16-F10 inoculation. The cells were stimulated with medium, IL-1β (50 ng/mL), IL-23 (50 ng/mL), or the combination for 48 hours. (g) Increase in the secretion of IL-17 from tumor γ δ T cells stimulated with IL-23 in vitro. γ δ T cells were purified from tumor lymphocytes by MACS and were stimulated with medium, IL-1β (50 ng/mL), IL-23 (50 ng/mL) or the combination for 48 hours. After 48 hours of stimulation, the supernatants were collected, and IL-17 concentrations were measured by ELISA kits. The data show means ± SEM of tumor size and are representative of three independent experiments. .