Immune-suppressive activity of MDSCs in MM. MM-MDSC–mediated T-cell suppression was assessed in autologous MDSC–T-cell cultures by proliferation assays. MDSCs and autologous CD3⁺ T cells were isolated from (A) PBMCs or (B) BMMCs from patients with relapsed MM disease and were cultured in the presence of T-cell stimulators (CD3/CD28 MAbs and IL-2) for 4 days; then T-cell proliferation was measured by 3H-thymidine incorporation assay. The data represent mean ± SD of triplicate cultures. The data shown are representative of 3 different experiments. Statistical significance is indicated (Student t test, 1-tailed distribution, P < .05). (C) MDSC-mediated immune suppression was determined using CFSE flow cytometry analysis within autologous T-cell subpopulations including total CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and CD3⁺CD8⁺CD56⁺ NKT cells in BMMCs from patients with relapsed MM disease. CD11b⁺CD14⁺HLA-DR⁺ APCs, CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺ MDSCs, and autologous CD3⁺ T cells were sorted from BMMCs of patients with relapsed MM disease. Allogeneic healthy donor PBMCs were used as control stimulator cells. T cells labeled with CFSE were cultured in the presence of aCD3/CD28 MAbs and IL-2 for 6 to8 days alone or with healthy allogeneic PBMCs, autologous APCs, or autologous MDSCs. Proliferating T-cell subpopulations were determined using CFSE costaining with MAbs against CD3, CD4, CD8, and CD56. CFSE^low dividing cells represent proliferating total T cells, and CFSE^high nondividing cells represent the suppressed total T-cell population (top, histogram plots). CFSE^low proliferating (large gated box) and CFSE^high nonproliferating (small gated box) T-cell subpopulations within CD4⁺ T cells (second row), CD8⁺ T cells (third row), and NKT cells (bottom row) plots are shown. (D) Mechanisms of MDSC-mediated T-cell suppression were investigated in MDSC-autologous T-cell cocultures using specific inhibitors of MDSC-associated suppressive factors ARG1 (nor-NOHA), iNOS (NMMA), ROS (apocynin), and COX2 (celecoxib). MDSCs and autologous CD3⁺ T cells were isolated from patients with relapsed MM disease, and then cocultured for 4 days in the presence of T-cell stimulators (aCD3/CD28 MAbs, IL-2), with or without inhibitors. T-cell proliferation was measured by 3[H]-thymidine incorporation assay. The percentage of proliferating T cells is demonstrated as T-cell+MDSC with inhibitor relative to T-cell+APC with inhibitor. The blue column indicates APC–autologous T-cell culture, and the red columns indicate MDSC–autologous T-cell culture. The data represent mean ± SD of triplicate cultures and are representative of 3 different experiments. Statistical significance is indicated (Student t test, 1-tailed distribution, P < .05).