After incubating at 37°C and 5% CO2 for 48 h, 1 μCi 3H-thymidine (Amersham) was added to each well. The cultures were harvested 18 h later and then processed for measurement of incorporated radioactivity in a liquid scintillation counter. The inhibitors of NO, 200 uM L-NMMA; arginase, 40 uM nor-NOHA (NW-hydroxyl-nor-l-arginine) (Calbiochem); or ROS scavenger, 5 mM NAC (N-acetyl l-cystein) (Sigma) were added at the beginning of the culture. One million of SCs or IHLs were incubated in 1%

FBS PI3K inhibitor 1% BSA in PBS with the relevant Abs. Intracellular cytokine staining [48], nitrotyrosine staining [35], and detection of CD107a (BioLegend) [49] were made as previously described. For iNOS detection, splenocytes were cultured and stimulated with Con A (5 mg/mL) for 48 h. Then, cells were stained with allophycocyanin-anti-CD11b (clone M170) and PE-anti-Gr1, fixed, permeabilized with Cytofix/Cytoperm buffer, and were incubated with rabbit

polyclonal anti-iNOS Ab (BD Bioscience). After washing, samples were examined using BD FACS Canto II flow cytometer (BD Biosciences). The Abs conjugated were allophycocyanin-anti-Ly6G/Ly6C (Gr-1, clone RB6–8C5), PE-anti-Ly6G (clone 1A8), FITC-anti-Ly6C (clone AL-21) (BD Bioscience), allophycocyanin-anti-CD4 (clone GK1.5)(BioLegend), PE-anti-CD8 (clone 53-6.7), PE-anti-IL6 (MP5-20F3), PE-anti-IFNγ (XMG1.2,), PCI-32765 molecular weight PE-anti-IL-17A (clone eBio17B7) (eBioscience), and anti-Phospho-Stat3 (Tyr705)(clone D3A7) (Cell Signaling). Oxidation-sensitive dye DCFDA (Molecular Probes/Invitro-gen), was used to measure ROS production [27]. Cytokine levels were determined by ELISA sandwich for detecting TNF-α, IL6, and IFN-γ (eBioscience) in plasma and in culture supernatants from sorted MDSCs cultured in supplemented RPMI 1640 at 24 h. Splenocytes were cultured

with ConA for 48 h, fixed in 4% paraformaldehyde, blocked with PBS-BSA Etoposide 1% and labeled with allophycocyanin-anti-CD4, PE-anti-CD8, and Alexa Fluor 488-anti-NT and visualized using FV1000 (Olympus) confocal microscope. Sorted CD11b+Gr1+ were put on a slide by the citospin technique and were stained with DNA-binding fluorochrome Hoechst 33 258 (2 ug/mL) and FITC-anti-phosphoSTAT3. Slides were observed with a NIKON ECLIPE Microscope. Purified MDSCs were washed and lysed (1% Triton X-100, 0.5% sodium deoxicholate, 9% SDS, 1 mM sodium ortovanadate, and 10 g PMSF in PBS). Aliquots of tissue lysates, were separated on a 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking, they were incubated with rabbit polyclonal Ab anti-p47phox (Santa Cruz) followed by HRP-anti-rabbit Ab (Sigma) and assayed using the ECL chemiluminescent system. Protein loading was visualized by anti-actin Ab (Santa Cruz). Experimental differences over the controls were analyzed with the Student’s t-test and nonparametric test and differences with p-value of <0.