Myeloma MILs produce IL-17. (A) Intracellular IL-17 staining. MILs and PBLs from either myeloma patients or normal donors were stimulated for 6 hours with PMA and ionomycin in the presence of Golgi-Stop. Cells were stained with CD3⁺, and intracellular staining was then performed for IL-17 and IFN-γ. Data are gated on CD3⁺ cells. (B) CD4/CD8/IL-17 staining. BM from myeloma patients with (n = 3) and without (n = 3) bone disease as well as from normal donors (n = 3) was stimulated for 6 hours with PMA and ionomycin in the presence of Golgi-Stop. Intracellular staining was performed for IL-17 and IFN-γ on either CD4 or CD8 T cells. Shown is the percentage of T cells expressing the respective cytokine. (C) Normal marrow plasma fails to induce a Th17 profile. Myeloma PBLs were cultured for 5 days in medium alone (no plasma), 50% normal BM plasma, or 50% normal BM plasma supplemented with the indicated cytokines at the following concentrations: IL-6, 10 ng/mL; TGF-β, 5 ng/mL; and IL-17, 10 ng/mL. After a 5-day incubation, the cells were stimulated for 6 hours with PMA and ionomycin and stained for CD3 and intracellular IL-17. Data are gated on CD3⁺ cells. (D) IL-17 produced by the myelomatous marrow microenvironment skews PBLs toward a Th17 profile. Myeloma PBLs were incubated for 5 days in the presence of medium alone, 50% myeloma BM plasma with or without anti–TGF-β 10 μg/mL, anti–IL-6 10 μg/mL, or both. Cells were cultured for 5 days as described in “Methods” and stained for intracellular IL-17.