Figure 1: IL-6 produced from B16 cells were the main STAT3 activators to T cells. (A): Cytokine profile of CD4+/CD8+ T cells from DLNs of B16 melanoma–bearing mice on day 16 or control naïve mice with multiple cytokine ELISA analyses by stimulation with PMA plus ionomycin in vitro. (** p < 0.01). (B): In vitro suppressive function by B16-conditioned supernatant on IFN-γ-production in CD4+/CD8+ T cells. Purified T cells from naïve mice were co-cultured for 24 h with medium containing NIH3T3-conditioned or B16-conditioned supernatant and PMA plus ionomycin in the absence or presence of anti-IL-6 or anti-IL-10 neutralizing mAbs (100 ng/ml). Data are representative of two individual experiments. Data from only one representative experiment are shown. (** p < 0.01). (C): pSTAT3 expression in CD4+/CD8+ T cells from naïve mice following 12 h of co-cultivation in medium containing NIH3T3-conditioned or B16-conditioned supernatant. Cells were cultured in the absence or presence of rR9-fusion proteins (rR9-GFP or rR9-GRIM19; 1 μM/ml), anti-IL-6, anti-IL-10 neutralizing mAb (100 ng/ml), or rIL-12 (1 ng/ml). Expression of pSTAT3 was analyzed by flow cytometry by using anti-pSTAT3 mAb. Gray lines represent isotype IgG. Data are representative of three individual experiments. Data from only one representative experiment are shown. (D): Analyses of IL-6/IL-10 concentrations in B16-conditioned supernatant by ELISA analyses in the absence or presence of rR9-fusion proteins (1 μM/ml). Data are representative of two individual experiments. Data from only one representative experiment are shown. (** p < 0.01, * p < 0.05). (E): In vivo kinetics of pSTAT3 expression in CD4+/CD8+ T cells from DLNs or spleen after inoculation of (2×105) B16 cells into abdominal skin on day 0. Expression of pSTAT3 was analyzed by using anti-pSTAT3 mAb on days 3, 5, 7, and 14. Gray lines represent the control antibody, isotype IgG. Data are representative of three individual experiments.