Effect Of Dendritic Cells Pulsed With Tumor Antigen Co-cultured With Cytokine-induced Killer Cells On Cytotoxicity Against Lung Cancer Cells

Cancer cells existed in the peripheral blood are the major reason to cause tumor's relapse and metastasis and patient's death. The eradication of residual cancer cells in the peripheral blood is very significance for preventing tumor's relapse and improve patients' life. Immunologic effector cells as an adjuvant in anticancer immunotherapeutic strategy don't destroy the human's immune system and function ,but can directly kill cancer cells. It has become an important adjuvant in anticancer strategy such as operation, radiotherapy and chemotherapy.Cytokine induced killer cells are the major histocompatibility complex-unrestricted cytotoxic lymphocytes and generated from peripheral blood monocytes by a cytokines including anti-CD3 monoclonal antibody, interleukin-2 and interferon-gamma. CIK cells are the population of heterogeneous effector cells which possess an enhanced cytotoxicity and a higher proliferation rate in all of immunologic effector cells. Dendritic cells are the most powerful of all antigen presenting cells and play a criticalrole in the induction of primary immune response. So it will be effective way that CIK cells co-cultured with DCs pulsed with tumor antigen are applied to the treatment of cancer. In this study, we tested the cytotoxic effect of DCs pulsed with tumor antigen co-cultured with CIK cells on lung cancer cells and determined the feasibility of treatment with them for tumors. It will provide meaningful theoretical and experimental proof for the tumor therapy.ObjectiveTo investigate the cytotoxic effect of dendritic cells pulsed with lung cancer lysate antigen co-cultured with cytokine-induced killer cells on A549 cell strain and lung cancer primary cells, and comparing the cytotoxic activity with dendritic cells co-culture with lymphokine-activated killer cells,only CIK cells and LAK cells, DC-CIK cells show its high cytotoxicity against lung cancer cells. This study provides the useful theoretical and experimental proofs for DC-CIK cells treatment of lung cancer in medicine.MethodsPBMCs were isolated from healthy donors by Ficoll density-gradient centrifugation and were induced to CIK cells, LAK cells and DCs by routine methods. DC cells were pulsed with lung cancer lysate antigen on day 8. After 24 hours of incubation, the morph of DCs were observed by the inverted microscope . Phynotypes of DCs were analysed by flow cytometry after be pulsed and impulsed with lung cancer lysate antigen. According to the study project, DC-CIK cells ,CIK cells, DC-LAK cells and LAK cells were classified as the effector cells, A549 cells and lung cancer primary cells were classified as target cells. The effector cells and target cells were accordingly divided into 8groups. The cytotoxic activity was carried out by LDH release assay at various ratios of effector cells to target cells( 10:1,20:1,50:1). The secretory amount of IL-2,IL-4,IL-12 and IFN-r were evaluated by enzyme linked immunosorbent assay inthe collected supernatants according to the manufacturer's instruction.Results一,Effector cells' morphology and proliferation observation: The isolated PBMCs were allowed to adhere in culture flasks for 2 hours at 37°C 5%CO2 incubation. The adherent cells were induced to generate DCs ,and DCs were pulsed with lung cancer lysate antigen on day 8.After 24 hours of incubation,the cells became bigger,irregular and typical DCs with pseudopodium. The floating cells were separately induced to CIK cells and LAK cells. CIK cells proliferated rapidly and became a lot of large growth-colonies. Otherwise,LAK cells's morph were simple and no obvious growth-colonies were developed. CIK cells posses a higher proliferation rate than LAK cells in the same incubate conditions.二,DCs'phenotype analyses: Dendritic cells on day 9 were stained with various types of FITC or PE conjugated mouse antibodies and analysed by flow cytometry. DCs were phenotyped with the monoclonal markers CD40,CD80,CD86 and HLA-DR.In unpulsed and pulsed DCs, expression of costimulatory molecules CD40,CD80,CD86 and HLA-DR increased from 4.25% to 74.31, from 12.12% to 85.81%, from 5.16% to53.29 and from 38.02% to 90.71% respectively. The phenotype of unpulsed and pulsed DCs showed significant difference (P <0.01) .The results revealed that DCs became obviously mature after DCs pulsed with lung cancer lysate antigen.三,Cytotoxic effects of effector cells by LDH release assay: At various ratios of effector cells to target cells( 10:1,20:1,50:1), cytotoxic effects of DC-CIK cells, DC-LAK cells, CIK cells and LAK cells on A549 cells and lung cancer primary cells were determined by LDH release assay. The results indicated: 1 , At the same ratio ofeffector cells to target cells, DC-CIK cells group showed the stronger cytotoxic effect than other three effector cells group. Significant difference was found in all of effector cells against A549 cells and lung cancer primary cells (P<0.05) .2,At the different ratios, cytotoxic effect of DC-CIK cells group on lung cancer cells showed difference and increased significantly by the rise of ratios of effector cells to target cells. DC-CIK cells group had the strongest cytotoxic effect at the ratio of 50:1. 3,At the same ratio, cytotoxic effect of DC-CIK cells on between A549 cells and lung cancer primary cells showed no significant difference (P>0.05) .四,The secretory amount of cytokines changed in the cytotoxicity assay: The secretory amount of IL-2, IL-4, IL-12 and IFN-r were evaluated at the ratio of effector cells to target cells(50:1). On the same target cells, the amount of IL-12 and IFN-r were secreted higher in DC-CIK cells group than other three effector cell groups (P <0.05) . But the amount of IL-2 were secreted higher in DC-LAK cells group and LAK cells group than other two effector cell groups, the significant difference was found (P<0.05) . In all of cytotoxicity assays, no IL-4 was detected.ConclusionCIK cells cocultured with DCs pulsed with lung cancer lysate antigen had a significantly enhanced cytotoxic activity compared with DC-LAK cells, CIK cells or LAK cells alone. The results could be explained by that DCs secreted some cytokines and co-stimulators to induce specifical CIK cells and promote CIK cells'proliferation. DCs can enhance CIK cells cytotoxic effect by above-mentioned two factors. DC-CIK cells could be applied to the treatment of tumor as a new ,extensive and effective immunotherapeutic effector cells, especially to prevent the tumor's relapse and metastasis after tumor patients'operation.