IL-6 is dispensable for the suppressive activity of MDSC on primary CD4+ T-cell activation. IL-6 is dispensable for the suppressive activity of MDSC on primary CD4+ T-cell activation. A, Cells from spleens and lymph nodes were harvested from IL-6+/+ or IL-6−/− mice 20 days after MCA205-OVA inoculation and analyzed for MDSC. Anti-IL-6 antibody (Ab) treatment was carried out 4 days before harvesting. Representative plots (left) and the number of Gr-1+CD11b+MDSC (right) are shown. NS, no significance. B, Total RNAs were isolated from purified Gr-1+MDSC in tumor-bearing IL-6+/+ or IL-6−/− mice, and mRNA expression of the indicated genes were analyzed. Data are normalized to the corresponding levels of GAPDH and are shown as relative expression and are expressed as a relative expression (mean ± SEM) compared with the average value of the IL-6+/+ MDSC (n = 4). WT, wild-type. C, the levels of ROS in IL-6+/+ or IL-6−/− MDSC from tumor-bearing mice or control Gr-1+ cells from tumor-free mice were determined using the intracellular ROS-reactive fluorescent dye, Deep Red. Representative histograms and the mean fluorescence intensity (±SEM) are shown. D and E, naïve OT-II cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies or OVA peptide-pulsed dendritic cell. The indicated numbers of MDSC were added to the culture. T-cell proliferation (D) and IFN-γ production (E) were measured by [3H] thymidine uptake and ELISA, respectively. Results shown are the mean ± SEM of triplicate assay. cpm, count per minute. Hirotake Tsukamoto et al. Cancer Immunol Res 2013;1:64-76 ©2013 by American Association for Cancer Research