Investigation Of Biological Activity And Anti-neoplastic Effects Of Cytokine-induced Killer Cells In Vitro

Part 1 The biological characteristics of cytokine-induced killer cells in vitroObjective:To investigate the biological activity of cytokine-induced killer (CIK) cells in vitro.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry, respectively. Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay. Interleukin-12 (IL-12), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) levels released by DC-CIK cells were quantified by enzyme-linked immunosorbent assays (ELISA).Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. At first, its amplification was very slow, and it increased quickly at the 4th-8th day, it reached its peak amplification at the 9th-10th day, at approximately 100-fold.The main effectors cells in this population were CD3+CD8+ cells and CD3+CD56+cells. DC-CIK cells were cytotoxic to B95 cells, Jhhan cells and M07e cells, with the highest cytotoxicity towards B95 cells. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were very high. Conclusion:DC-CIK cells induced with cytokines displayed powerful amplification and strongly killing activities in vitro. It suggested that DC-CIK cells induced with cytokines may play killing activities through Thl pathway in vitro, as a result of high secretion of Thl cytokines, such as IL-12, IFN-y, and TNF-α. Part 2 The mechanism of LFA-1/ICAM-1 mediated anti-neoplastic effects of cytokine-induced killer cellsObjective:To investigate the molecular mechanism underlying LFA-1/ICAM-1 mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. When treated with LFA-1 monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay, while RT-PCR and Western-blot were used to determine mRNA and protein expressions of GATA-3 and T-bet in DC-CIK cells, respectively. IL-12, IFN-γand TNF-αlevels released by DC-CIK cells were quantified by ELISA.Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human LFA-1 monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under 20μg/ml LFA-1 monoclonal antibody in comparison with the control group (P<0.05), while it also resulted in a decrease of the cytotoxicity towards Jhhan cells and M07e cells, however there was no statistically significant differences (P> 0.05;P> 0.05). It led to a highest elevation of GATA-3 mRNA and protein levels (P<0.05; P<0.05)and a highest decrease of T-bet mRNA and protein levels (P<0.05; P<0.05)under 20μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were lowest under 20μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group (P<0.05;P<0.05;P<0.05).Conclusion:GATA-3 and T-bet were implicated in the LFA-1/ ICAM-1 mediated anti-neoplastic effects of DC-CIK cells via activation of the Thl pathway, with high secretion of Th1 cytokines, such as IL-12, IFN-γ, and TNF-α. Part 3 The mechanism of T-bet mediated anti-neoplastic effects of cytokine-induced killer cellsObjective:To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. When treated with T-bet monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay. FCM was used to detect CD4+CD25+Treg cells, while RT-PCR and Western-blot were used to determine mRNA and protein expressions of Foxp3 and GATA-3 in DC-CIK cells,respectively. IL-12, IFN-γand TNF-a levels released by DC-CIK cells were quantified by ELISA.Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human T-bet monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under 10μg/ml T-bet monoclonal antibody in comparison with the control group (P<0.05), while it also resulted in a decrease of the cytotoxicity towards Jhhan cells and M07e cells, however there was no statistically significant differences (P> 0.05;P> 0.05). Treatment with 10μg/ml T-bet monoclonal antibody resulted in a highest increase in the proportion of CD4+CD25+Treg cells in B95 cells group in comparison with the control group (P<0.05), and it also resulted in an increase in Jhhan cells group and M07e cells group, however there was no statistically significant differences (P> 0.05;P> 0.05). It led to a highest elevation of Foxp3 and GATA-3 mRNA and protein levels(P<0.05;P<0.05;P<0.05;P<0.05)under 10μg/ml T-bet monoclonal antibody in B95 cells group in comparison with the control group. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were lowest under 10μg/ml T-bet monoclonal antibody in B95 cells group in comparison with the control group (P<0.05;P<0.05;P<0.05).Conclusion:Foxp3 and GATA-3 were implicated in the T-bet mediated anti-neoplastic effects of DC-CIK cells via activation of the Thl pathway and suppression of the Th2 and Treg pathways.