Hil 4

Peripheral blood mononuclear cells (PBMCs) were isolated from patients' peripheral blood using Ficoll-Hypaque (Solarbio, Beijing, China) and cultured in RPMI 1640 containing 5% autologous plasma, 10 ng/mL hGM-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany), and 10 ng/mL hIL-4 (Miltenyi Biotec). The immature DCs were infected with lentivirus on day 5, and polybrene (transfection enhancer) was added. Fresh medium was replaced after 24 h of transfection, and poly I : C was added on day 6 to promote the expression of endogenous genes. The mature DCs were then collected on day 7. The maturation status of DCs was observed through a microscope (Leica Microsystems Inc., Wetzlar, Germany). The expression of CD80, CD83, CD86, and human leukocyte antigen (locus) DR (HLA-DR) in DCs were measured by flow cytometry (Beckman Coulter). To assess DC maturation, flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to detect secreted cytokines. MG-7Ag expression in DCs was detected by quantitative PCR (qPCR) and gel electrophoresis.

The NKT cell hybridoma 2E10 was cultured as described (25 (link)). Bone marrow-derived DCs from B6 mice were prepared as described (23 (link), 26 (link)), where after 6 days of culture in a complete RPMI-1640 medium (ThermoFisher Scientific) supplemented with 5 ng/mL mGM-CSF (R&D), DCs were purified with AutoMACS and anti-mouse CD11c microbeads (Miltenyi Biotec). Human DCs were prepared as described (27 (link)), where CD14⁺ monocytes were purified from PBMNCs with a MACS LS column and anti-human CD14 microbeads (Miltenyi Biotec) and cultured for 6 days in a DendriMACS GMP medium containing 800 U/mL hGM-CSF and 250 U/mL hIL-4 (all from Miltenyi Biotec). Human umbilical cord blood derived mononuclear cells were prepared by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare), and NKT cell cultures were performed as reported (23 (link)) with a minor modification, where the culture medium consisted of 50% AIM-V medium (ThermoFisher Scientific), 45% RPMI-1640, 5% heat-inactivated fetal bovine sera (Sigma), 1 × NEAA, 1 mM sodium pyruvate, 55 µM 2-ME, 2 mM l-glutamine, and 100 U/mL penicillin/streptomycin (all from ThermoFisher Scientific) and supplemented with 100 U/mL hIL-2 (Shionogi, Japan).