The effect of lenalidomide and bortezomib on MDSCs in the MM microenvironment. (A) Lenalidomide’s and bortezomib’s effects on MDSC frequency in both MM PBMCs (n = 14) and BMMCs (n = 17) are shown. MM-PBMCs and MM-BMMCs were cultured with or without lenalidomide (1 μM) and bortezomib (5 nM) for 16 hours, and MDSCs were determined by flow cytometric immunophenotyping. The data shown are mean ± SEM for the percentage of gated MDSCs in the minimum of 10 000 events per sample. NS indicates the lack of statistical significance (Student t test, 1-tailed distribution, P < .05). Representative 2-parameter dot plots demonstrate CD33⁺CD15⁺ MDSCs (gated small box, right) within the CD11b⁺CD14^-HLA-DR^-/low gated cell population (left) in (B) PBMCs and (C) BMMCs from patients with MM that were cultured in the absence (top) or presence of lenalidomide (middle) or bortezomib (bottom). (D) Lenalidomide’s and bortezomib’s effects on MM-MDSC–mediated T-cell suppression were assessed in autologous MDSC–T-cell cultures in MM-BMMCs by proliferation assays. MDSCs and autologous CD3⁺ T cells were isolated from patients with relapsed MM disease, and then cocultured for 4 days with T-cell stimulators (aCD3/CD28 MAbs, IL-2) in the presence and the absence of lenalidomide (1 μM) or bortezomib (5 nM). T-cell proliferation was measured by 3H-thymidine incorporation assay. The percentage of proliferating T cells is demonstrated relative to T cells alone. 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). (E) Lenalidomide’s and bortezomib’s effects on MDSC-mediated immune suppression were determined in autologous T-cell–MDSC cocultures. MDSCs and autologous CD3⁺ T cells were sorted from the BMMCs of patients with MM. T cells were labeled with CFSE and stimulated with aCD3/CD28 Abs and IL-2, and then cultured for 6 to 8 days alone or with autologous MDSCs in the presence or the absence of lenalidomide (1 μM) and bortezomib (5 nM). Proliferating T-cell subpopulations, including total CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and CD3⁺CD8⁺CD56⁺ NKT cells 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 T-cell population (top, histogram plots). Shown are plots of 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). Len, lenalidomide; Bort, bortezomib.