| Objective:1. To establish two cultured systems of dendritic cell (DC) in vitro. one isinterferon-alpha (IFN-α) induced DC (IFN-DC) which were consistent with IFN-αactivated donor lymphocytes infusion (aDLI), the other is traditional interleukin-4-inducedDC (IL-4-DC);2. To compare the differences of biological characteristics and functions ofdendritic cell in these two cultured systems;3. To clarify the association between theIFN-DC and the enhanced anti-leukemia effects in aDLI treatment.Methods:1. The two cultured systems of IFN-DC and IL-4-DC: G-CSF-primedperipheral blood stem cells (PBSC) were collected from healthy donors, peripheral bloodmononuclear cells (PBMC) were isolated by Ficoll-Paque density gradient centrifugation.CD14+monocytes were purified by microbead technology and then divided into twogroups by different cultured systems: The control group (IL-4-DC group): IL-4-DC weregenerated by culturing monocytes in medium containing granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interleukin-4(IL-4) for5days, and then topromote maturation of DC by adding tumor necrosis factor-α (TNF-α). After7days ofculturing, IL-4-DC were gathered; The experimental group (IFN-DC group): IFN-DC weregenerated by culturing monocytes in medium containing GM-CSF and IFN-α, after3daysof culturing, IFN-DC were gathered.2. To compare the differences ofbiological characteristics of two groups: The immunophenotype of DC (CD14, CD83,CD56, CD11c, CD123, CD209, CD40, CD80, CD86, HLA-DR) were analyzed by flowcytometry; Mixed lymphocyte reaction (MLR) was used to evaluate the ability oflymphocyte proliferation; The mRNA expression of dendritic cell-lysosomal associatedmembrane protein (DC-LAMP) was detected by Real Time RT-PCR. The migratorycapacity was measured by transewell migration assay, The mRNA expression of chemokine receptor of CCR7was detected by real time RT-PCR;3. To evaluate thefunction of dendritic cell: a. Cytotoxic capacity: LDH release assay was used to detect thecytotoxicity effect of DC on K562; b. Specific cytotoxicity effect of lymphocyte triggeredby DC: Cytotoxicity effect of lymphocyte on SHI-1after co-culture with DC which pulsedwith freeze-thawed tumor lysate were detected by using LDH release assay.Results:1. Cells grown in GM-CSF/IFN-α and GM-CSF/IL-4/TNF-α displayed finecytoplasmic projections consistent with a DC phenotype;2. The results of flow cytometryanalysis showed that IFN-DC and IL-4-DC expressed CD14at levels lower than thestarting monocyte population, and expressed high levels of CD83, CD11c, CD123, CD209,CD40, CD80, CD86, and HLA-DR.3. IFN-DC and IL-4-DC represent two distinct DCpopulations, there were differences of immunophenotype between the two groups:CD14(81.55±1.23%vs6.80±1.80%, p=0.000), CD209(44.08±5.02%vs95.02±5.76%,p=0.002), CD40(94.29±3.69%vs70.53±2.43%, p=0.001), CD80(30.67±3.82%vs83.38±12.71%, p=0.005), CD86(85.34±5.25%vs70.16±8.25%, p=0.023);4. The results oftransewell migration assay showed that IFN-DC showed a higher migratory capacitycompared to IL-4-DC, The migration index was (3.55±0.45) vs (2.53±0.18), p<0.05.furthermore, the results of real time RT-PCR showed that the mRNA expression of CCR7of IFN-DC was higher than IL-4-DC, p<0.05;5. the results of real time RT-PCR showedthat the mRNA expression of DC-LAMP of IFN-DC was higher than IL-4-DC, p<0.05;6.the results of MLR showed that there was a a trend of IFN-DC showed a slightly strongercapacity in stimulating lymphocytes compared with IL-4-DC at all DC-T cell ratios;7. Thelymphocytes of two group displayed a significant cytotoxicity on SHI-1after co-culturewith DC which pulsed with freeze-thawed tumor lysate by using LDH release assay.Conclusion:1. This study successfully established two cultured systems of IFN-DCand IL-4-DC in vitro;2. IFN-DC and IL-4-DC represent two distinct DC populations;3.IFN-DC showed a higher migratory capacity and antigen-presenting capacity compared toIL-4-DC;4. There was a a trend of IFN-DC showed a slightly stronger capacity instimulating lymphocytes compared with IL-4-DC at all DC-T cell ratios, and thelymphocytes displayed a significant specific cytotoxicity on SHI-1after co-culture withIFN-DC which pulsed with freeze-thawed tumor lysate;5. The strong GVL effect of aDLImay be due to the generation of IFN-DC. |
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