PBMC Culture and DC Maturation

Thawed (peripheral blood mononuclear cells) PBMCs were resuspended at 10⁷ cells/ml in 24-well tissue culture plates (5 × 10⁶ cells/well) or in a 75-cm² tissue culture flask (7 × 10⁷ cells/flask) containing AIM medium (Life Technologies, Monza, Italy) supplemented with FLT3L (50 ng/ml; Miltenyi Biotec, Bologna, Italy) to mobilize resident DCs, as previously described (23 (link), 24 (link)). After 24 h (day 1), the peptides were added to the cultures, and DC maturation was induced with TNFα (1,000 U/ml, Miltenyi Biotec), IL-1β (10 ng/ml, Miltenyi Biotec), IL-7 (0.5 ng/ml, R&D Systems), and prostaglandin E2 (1 μM, Calbiochem, Milan, Italy). On day 2, (Fetal Bovine Serum) FBS (Euroclone) was added at a final v/v ratio of 10%. Medium was then replaced at days 4 and 7 with fresh RPMI 1640 (Euroclone, Milan, Italy) enriched with 10% FBS, non-essential amino acids (Euroclone) and sodium pyruvate (Sigma-Aldrich, Milan, Italy). Antigen-specific CD8⁺ T cells were characterized on day 10.