PLCγ2^−/− MDSCs are more potent immune suppressors than WT MDSCs in vitro. (A and B) T cell proliferation assay. PMN- or MO-MDSCs isolated from WT and PLCγ2^−/− mice were co-cultured for 3 d with CFSE-labeled splenocytes from OT-1 transgenic mice (1:10, 1:5, and 1:1 ratios) and stimulated with SIINFEKL peptide (10 pM) in antigen-driven (A) or with anti-CD3 (10 µg/ml) in mitogen-driven (B) experiments. Bar graphs show mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01. Representative flow cytometric analysis of CD8⁺ T cell proliferation (shown as CFSE dilution) in the presence of WT (dashed line) and PLCγ2^−/− (solid line) MDSCs is also shown. (C and D) Suppressive mechanisms of PMN- and MO-MDSCs isolated from WT and PLCγ2^−/− mice. Level of ROS (mean fluorescence intensity, MFI) in PMN and MO subsets in response to PMA stimulation (300 nM for 30 min) was measured using DCFDA staining and FACS. NO₂ release in supernatant of 10⁵ MDSCs subfractions was assayed by a standard Greiss reaction after LPS stimulation (1 µg/ml for 24 h; D). Mean ± SD from three independent experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.