Increased frequency of T_h17 cells in freshly isolated mononuclear cells in myeloma. (A) Mononuclear cells were isolated from multiple myeloma (MM) patients (blood, N = 11, bone marrow [BM], N = 4) and from healthy donors (HDs; blood, N = 12; BM, N = 3) and stimulated for 6 hours with PMA and ionomycin and stained for intracellular interleukin-17 (IL-17) and interferon-γ (IFN-γ). Proportion of IL-17–producing T_h17 cells was determined in CD4 population by flow cytometry. A representative dot plot analysis showing percentage of cells that are positive for intracellular IL-17 and IFN-γ within gated CD4 population using matching peripheral blood and BM samples from MM and HDs is shown. (B) Composite results presented as mean value with SEM for T_h17 with in CD4 population. (C) RNA was isolated from CD4 cells from MM patients (N = 3) and healthy donors (N = 3) using QIAGEN kits and quantitative PCR was performed. *P < .05.