Generation Of Functional And Mature Dendritic Cells From Peripheral Blood Mononuclear Cells Using Different Cytokine Combinations

[Objective] The present study probed into generation of functional and mature dendritic cells from peripheral blood mononuclear cells using different cytokine combinations, and detected the cultured cells' ability to stimulate allogeneic T cells, in order to explore the impact of cytokines on DCs and find a reliable method to induce and culture DCs.[Method] Healthy person' s heparinized peripheral blood was collected under sterile condition, and mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation. These cells were cultured in 24-well culture plates with RPMJ-1640 medium supplemented with 10% heat-inactivated fetal calf serum, after two hours of culture nonadherant cells were gently removed and fresh medium were added into the culture in the presence of rhGM-CSF (50ng/ml), IL-4(40 ng/ml) , IL- 13(40ng/ml) , PHA (50μg/ml) , alone or in combination in order to generate DC. At 6th day, LPS (20ng/ml) , IL-2(5ng/ml), TNF-α(20ng/ml) were added into the culture to induce DC maturation, cultured cells were analyzed by microscope. Cultured cells labeled with monoclonal antibodies by immunology fluorescence were detected by flow cytometrv to find CD1a + , CD83 + , CD86 + ,CD209+ cells positive rate at 3rd, 6th, 9th day. They were tested for their ability to stimulate allogeneic T-cells in vitro in Mixed Lymphocyte Reaction assays by MTT.[Results] 1. PBMCs were cultured with different, cytokine combination after 6 days, the morphologic features of DCs were observed by microscope, included: iregular shape, increased size cells with pronounced dendritic veils and the cells in suspension. 2. IL-4/GM-CSF-DCs cultured with LPS, 1L-2 and TNF-a expressed CDla, CD83, CD86, CD209, the positive rate was 50. 2%, 48. 2%, 77. 4%, 64. 3% respectively. The expression level was higher than that in DCs cultured with rhGM-CSF, IL-4 or with rhGM-CSF, IL-J3. These expressions paralleled the stimulatory abi1i ty of a]1ogeneicT cells. When stimulator cells/effector cells was 1:10, the ability of stimulating allogeneic T cells was strongest especially in the DCs cultured with rhGM-CSF/IL-4/TNF-a/LPS/IL--2. 3. Compared with DCs cultured with rhGM-CSF, IL-4, generation of functional and mature dendritic cells cultured with rhGM-CSF,1L 13 have no significant changes. 4. When cells cultured with rhGM-CSF/lL-4 or rhGM-CSF/ JL-13 were stimulated with PHA, the cultured cells were observed wi t h cell cluster, and the maturation and function of DCs has also improved.[Conclusions] Our findings suggested that peripheral blood mononuclear cells can be induced into DCs with the aboved different cytokine combination, but peripheral blood .mononuclear colls cultured with rhGM