Molecular signature of MDSCs in MM. Immunomodulatory effects of lenalidomide and bortezomib on the expression of MDSC-associated immune suppressive/inflammatory molecules were assessed in MM-BM using intracellular staining flow cytometry analysis. BMMCs from patients with relapsed MM disease were stimulated with LPS (1 μg/mL) and cultured for 16 hours with or without lenalidomide (1 μM) and bortezomib (5 nM). (A) Intracellular expression of immune-suppressive inflammatory molecules COX2 and COX1 were evaluated in MDSCs within BMMCs using anti-COX2 (PE) and anti-COX1 (fluorescein isothiocyanate ) MAbs. Representative 2-parameter dot plots indicate intracellular expression of COX2 (top row) and COX1 (bottom row) in MDSCs, gated as CD11b⁺CD14^-HLA-DR^-/lowCD33⁺CD15⁺. The data represent the percentage of positive cells per the minimum of 10 000 events per sample. The data shown are representative of 3 different experiments. (B) Intracellular expression of inhibitory cytokines (IL-6, IL10) and stimulatory cytokines (IFN-γ, GM-CSF), as well as surface expression of the GM-CSF receptor, were evaluated by flow cytometry in MM-BM MDSCs. MDSCs were gated as indicated above, and the percentage of expression of cytokines and receptors were demonstrated in representative 2-parameter dot plots. The data shown are representative of 3 different experiments. SSC, side-scattered light.