Study On Dendritic Cells-mediated Anti-HBV Immunity By Silencing Murine B7-DC Gene In Dendritic Cells

Hepatitis B virus is a noncytopathic DNA virus and is the pathogen of acute and chronic hepatitis.Studies show that CD8+ T cells display poor proliferation and effector functions in chronic HBV infected patients.It indicates that enhancement of the responses of virus-specific T-cell may be a way of terminating chronic HBV infection.Recently,dendritic cells(DCs) are being considered as one of the efficient means to treat tumor or chronic viral infections.Thus,DC-based therapeutic vaccines against HBV is feasible.Many studies have revealed that the molecules of B7 family provide signals that are critical for both stimulating and inhibiting T cell activation.B7-DC is the fifth member of B7 family.It has been identified as a second ligand for PD-1,which is a negative regulator of T cell activation.B7-DC can express selectively on dentritic cells.Thus,blockade of B7-DC/PD-1 interactions between dentritic cells and T cells may induce stronger T cell response.RNA interference is a novel gene regulatory mechanism that uses dsRNA molecules to shut down gene expression at the posttranscriptional level through mRNA degradation,and it is one of the practical methods to block the gene expression at the present time.In the present study,the total RNA was extracted from murine immature bone marrow-derived dendritic cells and the extracellular fragment of B7-DC cDNA was amplified by RT-PCR.The recombinant plasmid pET32a(+)-B7-DC_ECD was constructed by cloning the extracellular fragment of B7-DC cDNA into the prokaryotic expression vector pET32a(+).After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, pET32a(+)-B7-DC_ECD was transformed into E.coli BL21 and the interest protein was expressed under the induction of IPTG and was analyzed by SDS-PAGE and Western blot.The results showed that a 582bp of extracellular fragment B7-DC cDNA was obtained and the sequence was confirmed correct by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21.Meanwhile,we have investigated whether the vectors carrying short hairpin RNA targeting against murine B7-DC gene could silence the expression of B7-DC and analyzed the function of gene-modified dendritic cells.Two shRNA plasmid vectors against murine B7-DC were constructed and transfected into murine bone marrow-derived dendritic cells.The data suggested that B7-DC mRNAs of dendritic cells were both downregulated significantly after transfection with shRNA plasmids.The inhibition rates were 61.4%and 45.9%for shRNA1-B7-DC and shRNA2-B7-DC,respectively.MLR(mixed lymphocyte reaction) assay showed that DCs transfected by shRNA plasmid induced markedly higher allogeneic lymphocyte proliferation than DCs transfected by pAS vector plasmid and untreated DCs at all dilutions.Gene-modified DCs were pulsed with HBV specific peptides and the immunization was performed in HBV transgenic mice.The data demonstrated that the specific CTL activities increased by silencing of B7-DC compared with HBV specific peptide-pulsed DCs transfected with pAS or HBV specific peptide-pulsed DCs at the E/T ratio of 50:1.These results indicated that blockade of B7-DC on DCs augmented the cytotoxicity activity induced by immunization with peptide-pulsed DCs.Furthermore,the sera were used to detect the level of HBsAg and HBV DNA.The data showed that blockade of B7-DC on DCs could induce stronger anitviral immunity elicited by peptide-pulsed DCs and the level of serum HBsAg and HBV DNA decreased significantly,suggesting that silencing of B7-DC is of potential value in DCs-based therapy on HBV infection.