The Study Of Anti-tumor Response By Transfecting PcDNA3.1-HLA-A~*0201 To Yunnan Xuanwei Lung Adenocarcinoma (XWLC-05)

【Objective】:The aim of this study to explore HLA-A2 molecule restrict the cytoxic T lymphocyte-mediated anti-tumor response in vitro.XuanWei Lung Adenocarcinoma XWLC-05 which transfected HLA-A*0201 be specifically recognized by TCR(264scTCR/multimer),to identify the superficial P53_264.272/HLA-A2 complex on the cells.Further taking advantage of the Ag peptide which HLA specific molecule restricted induced T cells to visualize and kill the target cells,based on the high frequency gene locus HLA-A*0201 of Yunnan lung cancer which having been analysed HLA gene polymorphism of Yunnan lung cancer cells.【Method】:With PCR-SSP to screen HLA-A*0201 negative XWLC-05 and HLA-A2(+)and HLA-A2(-)PBMC from healthy donors.Lung cancer cells were transfected with PcDNA3.1-HLA-A*0201 and acquired stable positively transfeced cells clone by G418.Fostered the wild P53264-272 peptide with the positively transfected cells,the binding of the positively transfected cells to wild P53264-272 peptide was confirmed by FCM.PBMCs were stimulated by IL-2.CD8⁺ T and CD4⁺ T cells from the PBMCs were separated by immunomagnetic beads.Finally, cytotoxicity ofT cell were tested by MTT.【Results】:We acquired the stable cells transfected with PcDNA3.1-HLA-A*0201. The HLA-A2 expression on the surface of the transfected cells was tested by FCM and immunofluorescence staining.The recognition of TCR multimer to P53_264.272/HLA-A2 complex was confirmed by FCM.We found that by combining CD8⁺ and CD4⁺ T cells together has resulted in significantly higher cytotoxicity compared to CD8⁺ T cells alone.【Conclusion】:In vitro,transfection of PcDNA3.1-HLA-A*020t to HLA-A*02 negative tumor cells could induce HLA-A2 restricted,CD8-mediated antitumor response.HLA-A2 expression on the cells surface could be achieved by transfecting HLA-A*0201 gene into HLA-A*02 negative cells.This HLA-A2 protein may present the wild P53 peptide,and eventually induced HLA-A2 restricted,CD8-mediated antitumor response.