Objective:Dendritc cells are the potent initiators of immune responses. Targeting antigen to dendritic cells is an attractive method to improve the potency of DNA vaccines. The aim of this study is to confirm the functional activity of human cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) to target a plasmid-encoded antigen to human dendritic cells in vitro. Methods:The plasmid pGJGLU/GFP contains the signal peptide and the extracellular domains of human CTLA-4, the Fc region of human IgG1, the GLU region of GTF-I of Streptococcus mutans, with the skeleton of pEGFP/N1. The COS-7 cells were transiently transfected with sofastTM transfection reagent when the cell density was 60-80%. The CHO cells were transfected with lipofecta-mine reagent and stably expression cell line was constructed. The protein secreted from the transfected cells was detected with ELISA and western blotting. Human dendritic cells were cultured and harvested on 7 days. The concentrated supernatant from pGJGLU/GFP and pEGFP/N1 transfected cells were incubated with human dendritic cells. Before incubation, one group of cells was blocked of B7 molecules, which bind avidly to CTLA-4 extracellular region. Flow cytometer analysis was performed to detect the protein bound to human dendritic cells. Results:Specific vesicles with green protein could be observed in the COS-7 and CHO transfected cells with pGJGLU/GFP and pEGFP/N1. Fusion protein was detected only in the supernant from pGJGLU/GFP transfected cells by ELISA and Western blotting. Flow cytometric analysis demonstrates that GFP-CTLA-4–Ig-GLU protein encoded by pGJGLU/GFP binding to human dendritic cells. When B7 molecules on human dendritic cells were blocked, no protein was detected as showed by the comparable flurosence intensity with negative control. Conclusions:Protein encoded by plasmid fused to genes encoding human CTLA-4 could bind to human dendritic cells. Acknowledgments:This study was supported by grant No. 30330660 from the natural Science Foundation of China.

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