Factors regulating MDSC suppressive activity. (A) Cells collected from the tumor site or spleens of EL-4 tumor-bearing mice were stimulated with PMA, labeled with 1 µM dichlorodihydrofluorescein diacetate (DCFDA), and stained with APC-conjugated anti–Gr-1 antibody and PerCP-conjugated anti-CD11b antibody. DCFDA fluorescence was measured in Gr-1⁺CD11b⁺ population by flow cytometry. Cumulative results from four independently performed experiments are shown. In all panels, * denotes statistically significant difference (P < 0.05) from naive mice. (B) RNA was extracted from Gr-1⁺CD11b⁺ cells isolated from spleens or tumor of the same mice and expression of gp91^phox and p47^phox was measured in quantitative real-time PCR (qRT-PCR). The experiment was performed in triplicates and repeated twice with the same result. (C) MDSCs from spleen and tumor ascites were stimulated with 30 ng/ml IFN-γ for 48 h and expression of inos was measured in qRT-PCR (left). The same cells were mixed at the indicated ratio with 2 × 10⁵ splenocytes stimulated with anti-CD3/CD28 antibodies. After a 48-h incubation, culture medium was collected and assayed for nitrites (right). Experiments were performed in triplicates and repeated three times with the same results. #, statistically significant (P < 0.05) differences between MDSC isolated from spleen and tumor at the same time point. (D) Arginase 1 gene expression (left) and enzymatic activity (right) were evaluated in MDSC from tumor site and spleen. All experiments were performed in triplicates and repeated three times with the same results. *, statistically significant differences (P < 0.05) from naive mice; #, statistically significant (P < 0.05) differences between ascites and spleen of the same mice. (E) Suppressive activity of MDSC from gp91^phox knockout mice on IFN-γ production by transgenic 2C T cells after stimulation with either specific peptide (left) or anti-CD3/CD28 antibodies (right). Each experiment was performed in triplicates. Two experiments with the same results were performed. *, statistically significant differences (P < 0.05) from naive mice. (F) MDSCs isolated from spleens or tumor site were incubated at a 1:4 ratio with naive syngeneic splenocytes stimulated with anti-CD3/CD28 antibodies in the presence of iNOS (0.5 mM L-NNMA) and arginase (0.5 mM nor-NOHA) inhibitors. IFN-γ production was measured in ELISPOT assay and cell proliferation by incorporation of ³H-thymidine. Three experiments with the same results were performed. Error bars show mean and SD.