Study On The Cytokines Expression Profiles Of CIK In Vitro

Schmidt and Lanier found CD₃⁺CD₅₆⁺ cells from peripheral blood mononuclear cells induced by cytokines including IL-1, IL-2, IFN-γ in 1986 for the first time, and then be titled as cytokine induced killer cells or CIK cells. Nowadays, CIK has been applied in clinical trials for malignancy therapy. And many new alternatives for cancer therapy have developed, such as DC, CIK and coculture of DC and CIK.Our lab have found that the supernatant of CIK cells cultured in vitro could block the E6 cells being infected by SARS virus. They can be induced in the presence of cytokines including IL-1,IL-2,IFN-γ and monoclonal antibody against CD3 from its precursor lymphocytes. CIK cells have a double positive phenotype characters—CD3⁺CD56⁺,they belong to T lymphocytes with NK-like surface markers CD56⁺. CIK cells have been regarded as a novel choice to cure malignant tumors and acute leukaemia or other diseases. Activated CIK cells could exert a broad antitumor function and clear the minimal focus in patients with leukaemia and malignant tumor. The effector pathways include secreting granules with bacteriolysin and enzymes, ligand and receptor pathways, such as Fas-FasL and TNF related pathways. Granules with perforing proteins play the most important role in their antitumor effect. We supposed that cytokine expressed or secreted by CIK cells might function a lot in the process of deleting tumor cells and normal cells attacked by malignancy and virus. We made a study on the cytokines expression profiles of CIK cells to learn about what is the relationship between cytokines and antitumor or antivirus to set up a new project in clinical trials to cure patients infected with virus.Ficoll was used to separate non-adhesive lymphocytes from peripheral blood monocytes of healthy donors and then they were induced to CIK cells with the cell phenotype characters of CD3⁺CD56⁺ in the presence of IL-1, IL-2, IFN-γ and monoclonal antibody against CD3.The flask was coated with monoclonal antibody against CD3 to maintain high amplification ability before CIK cells were transferred into it. The cell surface markers were detected by flow cytometric analysis in order to check the purity and quality of CIK cells for making the further study. Thesurvival rate was determined dyeing by taipanlan and the cytotoxicity to tumors of CIK cells were analyzed by MTT assay. We made another study on cytokines expression profiles using semi-quantitative RT-PCR method during CIK cells were cultured in vitro from day6 to day30. The results were normalized to human GAPDH. Then we proceeded to quantifying some important and interest cytokines by Real-time RT-PCR using SYBR Green I and cloning a novel cytokines(IFN-λ2) which might help learn the antitumor and antiviral mechanisms; of CIK cells or find some novel cytokines able to interdict virus to infecting.We succeeded to acquiring the cytokines expression profiles in the level of mRNA.And the results showed that CIK was a heterogeneous population with different cellular phenotypes. The percentage of CD3⁺CD56⁺ positive cells came up to 80.21% as the dominant effector cells. In addition, CIK cells could widely express in vitro various kinds of inflammatory-related cytokines such as IFNs, ILs, TNFs and TGF-β1. Supernatant of CIK cells possess strong cytotoxicity on SARS-infected E6 cells. In conclusion, cytokines might play a key role in anti-tumor and anti-infection immune reaction. CIK or DCIK would be an promising approach in clinical trails to inhibit or cure patients infected with viruses via various cytokines secreted or expressed by CIK.