Generation Of Dendritic Cells From Chronic Myeloid Leukemia Cells Using Clinical Reagent IFN-α Combination With GM-CSF

AIM:Dendritic cells (DCs) had been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate optimization of DCs-based clinical vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients inducing by clinical reagents IFN-αcombination with GM-CSF.Methods: Bone marrow mononuclear cells (BMMNCs) were obtained from 3-5 mL heparinized bone marrow of CML patients by Ficoll-hypaque density gradient centrifugation. The mononuclear cell fraction was washed four to five times with RMPI164. BMMNCs were resuspended with RPMI1640 containing 10% human serum and seeded in 24-well plates at 3-5×106/mL with 1mL per well. The plates were incubated at 37℃and 5% CO2 for 2 h. And then, the non-adherent cells were decanted. The adherent cells were cultured in RPMI-1640 medium supplemented with either GM-CSF for research (800 U/mL) plus IL-4 for research (500 U/mL) or GM-CSF for injection (600 ng/mL) plus IFN-αfor injection (200 U/mL). Half medium exchange was performed every 3 days with fresh cytokine-supplemented medium. After 7~9-day, the morphologic features of CML-DCs were observed by inverted microscope and immunophenotypes were analyzed by flow cytometry. The quantity of CML-DCs was counted through full automatic blood analyzer after 7~9-day. The immune function of CML-DCs was detected through allogeneic mixed lymphocyte reaction (allo-MLR) and cytotoxicity assay of antileukemic cytotoxic T lymphocytes (CTL).Results: The 5-day or 7~9-day culture in the presence of different cytokines was successful in 8 patients as evidenced by the significantly up-regulation of CD80, CD86, CD83 and HLA-DR compared to pre-cultured (P<0.05). The percentage of DCs generated in the group B(10.51±4.65)% differed from that of DCs generated in the group A(7.28±2.95)% by expressing CD83 at significantly higher levels after 5 days cultured (P<0.05). There was not significant distinction of expressing CD83 between two groups on 7~9 day. The quantity of CML-DCs in the group A (0.382±0.126)×106/mL was not evidently different with that in the group B (0.357±0.101)×106/mL after 7~9-day. Allo-MLR in the group B was remarkably higher than that in the group A (P<0.05). CTL generated by CML-DCs displayed leukaemia-specific and anti-leukemia characteristics.Conclusions: In vitro, the quality of clinical reagent cultured CML-DCs proved to be comparable to classical method (GM-CSF+IL-4) culture indicating their feasibility for clinical vaccination protocols for CML patients.