Fig. 2

ONO-4641-expanded CD11b⁺Gr-1⁺ cells displayed an immunosuppressive phenotype. a Arginase activity detected by CD11b⁺Gr-1⁺ cells sorted from vehicle-treated and ONO-4641 -treated (0.3 mg/kg/day for 7 consecutive days) mice (n = 3 in each group). Data are presented as the mean ± SEM. **P < 0.01. The numbers on the X-axis denote the number of CD11b⁺Gr-1⁺ cells. b, c Representative two-parameter histograms (b) and percentage of cell proliferation (c) determined in the proliferation assay for CD3⁺ T cells co-cultured with an equal number (1 × 10⁵) of vehicle- or ONO-4641-expanded CD11b⁺Gr-1⁺ cells by the carboxyfluorescein succinimidyl ester (CFSE) method. Data shown are as the mean ± SEM, and are representative of three independent experiments performed in triplicate. d Expression of Arg1, Il10, Chi3l3, Mrc1, Retnla, Klf4, Nos2, Tnfa, Il6, and Il1b in CD11b⁺Gr-1⁺ cells sorted from the lungs of vehicle-treated or ONO-4641-treated mice (Day 7) (n = 3–4 in each group). Data are presented as the mean ± SEM. **P < 0.01; ***P < 0.001. e TNF-α levels in the supernatant of the medium, in which MH-S macrophages were cultured with or without an equal number (5 × 10⁵) of vehicle-treated or ONO-4641-expanded CD11b⁺Gr-1⁺ cells (n = 3 in each group). Data are presented as the mean ± SEM. *P < 0.05. f, g Representative two-parameter histograms (f) and quantification (g) of intracellular detection of IFN-γ in CD3⁺ T cells from the spleen after co-culture with or without equal numbers (5 × 10⁵) of vehicle-treated or ONO-4641-expanded CD11b⁺Gr-1⁺ cells. Data shown are representative of three independent experiments performed in triplicate. h Representative immunoblotting of phosphorylated (p)-ERK (Thr202/Tyr204). Total (t)-ERK was used as the loading control. Relative band intensities were determined by densitometry analysis. i Quantification of CD11b⁺Gr-1⁺ cells in the lungs from mice intraperitoneally treated with MEK/ERK inhibitor U0126 (5 mg/kg) or control. At 30 min after treatment, mice were subsequently intragastrically treated with vehicle or ONO-4641 (0.3 mg/kg/day) once a day for 7 consecutive days. (n = 4–5 in each group). Data are presented as the mean ± SEM. *P < 0.05