Effect of ex vivo culture duration on phenotype and cytokine production by mature dendritic cells derived from peripheral blood monocytes

BACKGROUND: To generate clinical-grade dendritic cells (DCs) ex vivo for immunotherapy trials, peripheral blood monocytes are typically cultured in granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then matured using one or more agents. Duration of the initial DC culture is one important variable that has not been systematically evaluated for its effect on the characteristics of the final mature DC product. STUDY DESIGN: DCs were generated from elutriated peripheral blood monocytes by incubation in medium containing 2000 units per mL each of GM-CSF and IL-4 for 3 to 7 days, followed by maturation with lipopolysaccharide and interferon-γ (IFN-γ). DC yield, viability, flow cytometric phenotype, and cytokine production were evaluated. RESULTS: The percentage yield and viability of mature DCs were similar after GM-CSF/IL-4 culture for 3 or 7 days. In either case, mature DCs expressed abundant CD80, CD86, CD83, and CCR7, but 3-day DCs expressed these antigens in a more consistent and homogeneous manner. Mature 3-day DCs produced much more IL-12 and less IL-10 after restimulation with CD40L-LTK than 7-day DCs. The former were also more effective in presenting immunogenic peptides to CD8 T cells. Analogous changes in cytokine production were observed in mature DCs prepared using lower concentrations of GM-CSF/IL-4 or when the alternative maturation cocktails poly(I:C)/IFN-γ and soluble CD40L/IFN-γ were used. CONCLUSION: Extended initial culture of DCs in GM-CSF/IL-4 does not affect yield or viability of subsequently matured DCs, but can adversely affect their ability to homogeneously express high levels of functionally important surface molecules such as CD83 and CCR7 and to produce IL-12.