Mediators of Inflammation / 2013 / Article / Fig 3

Research Article

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. .
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