Increased frequency of MDSCs in patients with MM. The presence and the frequency of MDSCs were quantitated in the PB and the BM of patients with MM (Table 1) and in healthy donors by multiparameter flow cytometry. PBMCs and BMMCs were obtained from patients with newly diagnosed (n = 4), relapsed (n = 4), and relapsed/refractory (n = 9) MM, as well as from healthy donors (n = 5), and then stained for MDSCs using fluorochrome-labeled antibodies against CD11b, CD14, HLA-DR, CD33, CD15, and isotype controls. MDSCs were immunophenotyped as the CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ population and quantitated as a percentage of gated cells by acquiring a minimum of 10 000 live events per sample. (A) Representative multiparameter dot plots of MDSCs within the CD11b⁺CD14^-HLA-DR^-/low population (gated, left) are shown as CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ population (gated, right) in PBMCs from healthy donors (top row), as well as PBMCs (middle row) and BMMCs (bottom row) from patients with relapsed MM. (B) Shown is the frequency of CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ MDSCs in PBMCs from healthy donors compared with PBMCs and BMMCs from patients with MM (left). The frequency of MDSCs in PBMCs from healthy donors compared with BMMCs from patients with newly diagnosed, relapsed, or relapsed/refractory MM are also shown (right). The data represent the percentage of MDSCs in PBMCs or BMMCs. Statistical significance is indicated (Student t test, 1-tailed distribution, P < .05). (C) Shown are representative cytospin images of CD11b⁺CD14⁺ myeloid cells (left) and CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ MDSCs (right) with Wright-Giemsa staining in BMMCs from patients with relapsed MM. Photomicrographs show myeloid cells, identified by the mononuclear or polymorphonuclear cell nuclear staining (blue) using light microscopy (20 × 0.30 objective magnification) (Leica DM IL; Bannockburn, IL) and analyzed using a Leica DFC490 camera and Leica Application Suite version 2.8 software. (D) Intracellular expression of the inhibitory molecules ROS and ARG1 in MDSCs within MM-BMMCs are demonstrated by histogram plots. The x-axis represents ROS or ARG1, and the y-axis represents the number of positive cells within the MM-BMMCs. The negative control is shown (gray dotted line), as are the intracellular expression of ROS or ARG1 in CD11b⁺CD14⁺HLA-DR⁺ myeloid cells (blue line) and in CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ MDSCs (red line) within BMMCs of patients with relapsed MM disease. The data shown are representative of 3 different experiments. hPB, PB from healthy donors; NewDx-MM, newly diagnosed patients; Rel-MM, relapsed patients; Rel/Ref-MM, relapsed/refractory patients.