Effect of bortezomib on DC immunostimulatory capacity. (A) Adherent monocytes were incubated in GM-CSF and IL-4–containing medium. At day 6 of culture, cells were stimulated for 24 hours with bortezomib at the indicated concentrations. Thereafter, cells were harvested, washed, and used as stimulators in MLRs in the absence of bortezomib. Proliferation was measured at day 5 by thymidine incorporation. Results are presented as means of triplicates with SDs. (B) Adherent monocytes were incubated with GM-CSF and IL-4 to induce differentiation to DCs. At day 6, cells were treated for 24 hours with bortezomib at the indicated concentrations (in ng/mL). LPS (100 ng/mL) was added during the last 16 hours of incubation. Cells were then harvested and used as stimulators in MLRs in the absence of bortezomib. Means of triplicates with SDs are presented. (C) DCs were generated from adherent monocytes by GM-CSF and IL-4. At day 6 of culture cells were incubated for 24 hours with or without 10 ng/mL bortezomib. LPS was added to the medium during the last 16 hours as a maturation stimulus. Thereafter, cells were harvested and washed, and 10⁶ DCs/well were incubated for 48 hours in 24-well plates with 10⁶ allogeneic CD3⁺ PBMCs in the absence of bortezomib. Cells were then harvested and analyzed by flow cytometry by gating on the CD3⁺ lymphocyte population. Solid histograms represent the matched isotype controls.