Isolation and expansion of OCT4/Sox2 specific cytotoxic T lymphocytes in vitro

e14578 Background: The main reason for tumor MDR (multidrug resistance ,MDR) is the presence of CSC(cancer stem cells,CSC). In the CSC of lung cancer, OCT4 and Sox2 are closely related to the tumor MDR. Therefore, in the current study, we introduced a method for the generation of OCT4+/Sox2+ CSC specific CTL(cytotoxic T lymphocytes,CTL) through co-culture with CD154+ feeder layer cells. Methods: Lenti/CD154 was constructed. The NIH3T3 cells were incubated with Lenti/CD154 (103 vp per cell) for 24 h at 37 °C and 5% CO2. Then the NIH3T3 cells were subsequently maintained in fresh complete DMEM medium. Lenti/GFP was used as a control. On the 6th day post infection, the infection rate was determined by flow cytometry after incubation with CD154-PE antibody. The feeder layer cells were co-cultured with CD40B cells and the biomarkers of CD40B cells were determined by flow cytometry. The CTL were treated with OCT4 plus Sox2 antigen and then the biomarkers of CTL were examined by flow cytometry and IL-2 and IFNγ were determined by ELISA. Results: The rate of CD154+ NIH3T3 cells was 99.7%, which was higher than that of uninfected-cells (14.8%). After co-culture with CD154+ NIH3T3 cells, the rates of CD40B expressing CD80, CD86 and HLA-A were significantly increased. OCT4 plus Sox2 treatment further increased the CD3, CD4 and CD8 expression and elevated the secretion of IL-2 and IFNγ. After co-culture of CTL with MDR PC9-drived cancer stem cells (CSC) and parental PC9 cells at different ratio, CTL significantly killed the tumor cells compared with control. Conclusions: The generation and expansion of OCT/Sox2 specific CTL is a feasible method for the treatment of lung cancer.