The Comparison Of Inducing Specific T Cell Response By Fusion Of DCs With Leukemia Cells And Freeze-thawed Leukemia Antigen-loading DCs

AIM: To optimize the choice of antigen and antigen-loading protocols and to provide laboratory date for the design of DC-based vaccine with clinical safety and efficacy through comparing the potency of in vitro leukemic antigen-specific CTL induction by fusion of DCs with leukemia cells and freeze-thawed leukemia antigen-loading DCs.METHODs: 200 milliliters of blood was collected from health donors, heparinized and then subjected to HES-Ficoll two-step isolation to collect mononuclear cells(PBMC). After washed four to five times using RPMI-1640 to remove platelets, PBMC were resuspended with 10%FBS-RPMI1640 and seeded in 24-well plates at the cell density of 2×10⁹/L. After incubated 2 hours at 37℃, 5%CO₂, the nonadherent cells were washed out for CTL cultivation and the adherent population were collected and then cultured in CM containing rhGM-CSF(8×10⁵IU/L)and rhIL-4(8×10⁵IU/L).The medium was replenished with cytokines every 3 days. On day 5, cells were divided into four groups: Group A were induced to fuse with K562 cells or blasts from CML patients by 500g/L PEG-100mL/L DMSO; group B were loaded with lysates from corresponding cells with equal quantities; group C were co-cultured with K562 cells or CML cells; and groupD were cultured alone without antigen loading as control. Before fusion, K562 cells were labeled using a red fluorescent linker dye PKH26. After fusion, flow cytometry were used to detect hybrids labeled with both PKH26 and FITC-conjugated anti-HLA-ABC mAb dyes and assess the efficiency of fusion. On day 6, TNF-a were added to induce the terminal maturation of DC. On day 8, cells in each group were co-cultured with autologous T cells respectively for five days. MTT coloremetry was used to measure proliferative response in autogenous mixed lymphocyte culture and antigen-specific MHC-restricted cytotoxicity against DC loaded with antigens derived from K562 or CML cells in each group.RESULTs: DC induced by GM-CSF+IL-4 and matured by TNF-a had classical morphologic characteristics. They expressed CD83 and CDla, two specific markers for DC, along with the up-regulation of costimulatory molecules CD80, CD86, and HLA-DR. Through convert microscope we detected that mediated by PEG-DMSO membrane fusion were firstly taken place between K562 cells and DC to format binucleate or plurinuclear cells. Then the two nuclei in giant cells began to fuse with each other to generate K562-DC Hybrids. The efficiency of DC-leukemia cell hybrids formation inPEG-DMSO-mediated cell fusion ranged from 17.33% to 29.94%. For an stimulator-to-responder ratio of 50:1, stimulation index(SI) for the proliferating response of T cells induced by cells in group A, B, C and D were 3.12±0.11, 3.16±0.12, 3.18±O.1O and 3.08±0.03 respectively (P>0.05). However, the percentage of CD8+ Tc cells in group A and group B increased dramaticly, with (32.63±4.76)% before culture, (63.25±6.23)% and (69.32±5.69)% respectively after proliferating, whereas the percentage of CD4+ Th cells in both groups decreased relatively, leading to the decreases of CD4+-to-CD8+ ratio. Comparing CTL activities against K562 lysates-primed DC, U937-pulsed DC and k562 targets in four groups, we found that the cytotoxicity against K562 lysates-primed DC induced by K562-DC hybrids were much more potent than that induced by lysates-loaded DC at the same E:T ratio(P<0.05). Furthermore, both of the two groups showed killing activity against HLA-ABC K562 targets, suggesting that a small portion of CTL activity was non-MHC-restricted. Little non-antigen-specific cytotoxicity was showed against U937-pulsed DC. In addition, in one case, primary CML cells instead of K562 cells were used to fuse with DC or their freeze-thaw lysates were used to prime DC, aslo resulting in the superior activity of leukemia-DC hybrids in antigen sepcific killing.CONCLUSION: Immune-competent mature DC could be induced from the CD14+ monocytes in PB effectively with the triple cytokine combination of GM-CSF, IL-4 plus TNF-a. The efficiency of cell fusion could be assessed effectively and quickly though the measurement of double fluorescently-labeled cells by flow cytometry. DC primed with freeze-thaw lysates and DC-leukemic hybrids can both stimulate the directional proliferation of autogenous CD8+ Tc cells. However, compared with freeze-thaw lysates loaded DC, DC-leukemic hybrids showed higher efficiency in antigen presenting and the stimulation of leukemia-specific CTL, which may provide a promising immunotherapeutic protocol for leukemias.