Dendritic Cell Differentiation and Maturation

Monocyte isolation and DC differentiation were performed as described previously.⁴ (link) Briefly, monocytes were obtained using the adherence method, where PBMCs were plated in adherent plastic plates (Sarstedt, Nümbrecht, Germany) in medium (X-Vivo 15 medium, Lonza, Basel, Switzerland) supplemented with 5% human serum, 2 mM L-glutamine and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 1,000 U/mL (100 ng/mL) IL-4, and 800 U/mL (50 ng/mL) Granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from STEMCELL Technologies) and incubated in a CO₂, 37°C incubator for 7 days. On day 4, the medium was replaced with fresh medium supplemented with IL-4 and GM-CSF. On day 7, DCs were matured with maturation medium containing 1,000 U/mL (100 ng/mL) IL-4, 800 U/mL (50 ng/mL) GM-CSF, 10 ng/mL tumor necrosis factor alpha (TNF-α) (STEMCELL Technologies), 1 μg/mL PGE2 (Sigma), 10 ng/mL IL-1β (Feldan, Quebec City, QC, Canada), and 100 ng/mL IL-6 (Miltenyi Biotec, Bergisch Gladbach, Germany) and loaded with the desired peptide (1 μg/mL LMP2_426–434, [CLGGLLTMV] or 1 μg/mL WT1_37–45 [VLDFAPPGA], both from JPT Peptides (Berlin, Germany). Last, DC medium was supplemented with interferon γ (IFN-γ; 1,000U/mL, Feldan) for the last 24 h of maturation.