Dendritic Cell Vaccination for Anti-Tumor Immunity

Dendritic cells (DC) were prepared from C57BL/6 bone marrows as previously described (33 (link)). Briefly, bone marrow cells were flushed with ice-cold PBS and erythrocytes lysed using ACK buffer (8.7 g/L NH₄Cl, 1g/L KHCO₃, 0.05 mM EDTA, pH 7.3). Cells were cultured in DMEM 20% heat-inactivated FBS (Gibco), 1mM sodium piruvate, 2 mM glutamine and antibiotics (1 U/ml penicillin, 1 μg/ml streptomycin) (Invitrogen), in presence of 20 ng/mL mrGM-CSF and 10 ng/mL mrIL-4 (ImmunoTools). Cells were cultured for 5 days, with addition of GM+IL-4 every 2 days. Immature DCs were pulsed during 5 h with TRAMP-C1 lysates (4 freeze/thawing steps), and then matured with 2 ng/mL CpG-1826 (phosphorothioate 5'-TCCATGACGTTCCTGACGTT-3', IDT) and 20 μg/mL Poly-U (Sigma-Aldrich) during an overnight culture. To obtain experienced anti-tumor lymphocytes, wild type or Lgals1-/- mice were s.c. immunized with tumor lysate-pulsed dendritic cells. At day 7 pot-immunization, lymphocytes obtained from axillary, brachial and inguinal lymph nodes were used for immune reconstitution of nude mice.