Construction Of Human PD-L1 Gene Transfected Cell Line And Preparation Of Its Monoclonal Antibody

PD-L1, a 290 aa type I transmembrane protein, belongs costimulatory molecules B7 superfamily. PD-L1 is broadly expressed on T, B cells, DCs, Macrophages and a range of nonhematopoietic cells. PD-L1 and its receptor deliver inhibitory signals that play immunoregulatory roles in T cell activation, tolerance, and immune-mediated tissue damage, which is cruicial in the maintaining of immune homeostasis. The PD-1/PD-L1 pathway plays important roles in the occurrence and development of autoimmunity disease, transplant rejection, chronic viral infections and tumor immunity. PD-L1 is highly expressed on many different types of tumors and was significantly associated with their pathological stages and prognosis. PD-1/PD-L1 pathway is a promising target for anti-cancer therapy.Here, we constructed a gene transfected cell line that stably expressing the human PD-L1 and observed its biological function on the proliferation and apoptosis of activated Jurkat cells. We established the hybridoma cell line stably secreting anti-PD-L1 m Ab by using the gene transfected cell line. L929/PD-L1 as immunogen and examined the biological characteristics of the anti-human PD-L1 m Ab. Part 1: Construction of human PD-L1 gene transfected cell line and its biological function on the proliferation and apoptosis of activated Jurkat cellObjective: To construct a gene transfected cell line that stably expressing the human PD-L1 and identify its biological function.Methods: Human PD-L1 gene was subcloned into retroviral expressing vector p EGZ-Term-R. The recombinant plasmid together with its helper virus vector was cotransfected into the 293 T cells. The L929 cells were infected with the supernatant of the transfected 293 T cells, and then were selected with G418. The G418 resistant cells were harvested for screening PD-L1 expression by flow cytometry. L929/PD-L1 cell was cocultivated with PHA stimulated Jurkat cell. The biological effect on proliferation and apoptosis of Jurkat cell was analyzed by cell counting and flow cytometry.Results: Gene transfected line L929/PD-L1 that stably expressed the human PD-L1 was established. Membrane PD-1 expression on Jurkat cell was induced by PHA administration. L929/PD-L1 inhibited the proliferation and induced the apoptosis of activated Jurkat cell.Conclusion: Gene transfected cell line that stably expressed the human PD-L1 is established successfully. The PD-L1 on the cell line inhibited the proliferation and induced the apoptosis of activated Jurkat cell. It confirmed the role of PD-1/PD-L1 pathway in negative immunological regulation. It also provided a valuable tool to study the biological function of human PD-L1 molecule. Part 2: Preparation of anti-human PD-L1 monoclonal antibody and analysis its biological characteristicsObjective: To prepare mouse anti-human PD-L1 monoclonal antibody to explore the roles of PD-1/PD-L1 pathway in immunopathogenesis.Methods: 6-8 weeks BALB/c female mice were immunized with PD-L1 transfected cell L929/PD-L1 that stably expressed human PD-L1 molecule. The spleen cells of immunized mouse were fused with myeloma cells P3X63Ag8, and then were cultured with HAT and HT selective medium. The positive hybridoma cells were screened with L929/PD-L1 cells by flow cytometry. The positive clones were repeatedly subcloned, until the hybridoma cell line sustainably and stably secreting specific anti-PD-L1 m Ab was obtained. The specific recognition of m Ab with the membrane protein PD-L1 was further determined by western blotting analysis. MAb Ig class was detected with Rapid Qualitative Test Strip. Competitive inhibition assay was used to analyze the recognition epitope of the m Ab.Results: A hybridoma cell line stably secreting anti-PD-L1 m Ab was obtained, which named 6G4. The m Ab 6G4 was proved to be Ig M with κ light chain. The m Ab 6G4 was revealed to recognize the PD-L1 molecule on the cells. Competition assay showed that m Ab 6G4 and m Ab MIH1 recognized the different epitopes.Conclusion: A hybridoma cell line stably secreting anti-human PD-L1 m Ab was obtained, which could be used to immunostaining and flow cytometry or western blot for analyzing PD-L1 molecule. It provide an useful tool to explore the roles of PD-1/PD-L1 pathway in immunopathogenesis.