The Potential Of Dendritic Cells-derived Exosomes In The Immunotherapy Of Chronic Hepatitis B Infection

AIMTo evaluate the anti-HBV immunocompetence of dendritic cells-derived exosomes loaded with Hepatitis B core antigen in vitro.METHEDSPeripheral blood monouclear cells (PBMCs) were isolated from the whole blood of chronic hepatitis B virus carrier by density gradient centrifugation on Ficoll-Hypaque,the adherent cells were cultured in the culture medium containted recombinant human IL-4 and recombinant human GM-CSF to generate DCs.The DCs were then pulsed with HBcAg ,matured by TNF-α,and analyzed by flow cytometry. Exosomes secreted by DCs were purifed by ultracentrifugation ,then identified by electron microscope and western blotting. We used ELISPOT(enzyme-linked immunospot assay),CCK-8(cell counting kit-8) and flow cytometry to evaluate the anti-HBV immunocompetence of the DEXs in vitro.RESULTMature DCs pulsed with HBcAg had a high expression of HLA-DR and costimulatory molecules CD80/CD86(were63%,92.8% and 95%).Small 30-100nm vesicles displaying a cup-shaped morphology characteristic of exosomes are observed by electron microscope , and western blotting show that they express HLA-DR and CD86. ELISPOT results show that mature DEXs loaded with HBcAg have the capacity to induce IFN- p ?roduction without addition of DCs ,and it is greater than HBcAg (897 vs 22 sfc/2×105PBMC) .When coculture with imDCs,only a tiny amount of mDEXs can significantly enhance the IFN- p ?roduction capacity induced by imDCs(raise from 749 to 1394 sfc/3×105PBMC) . The same tendency can be seen in the autologous lymphocyte proliferation test(the ODs of mDEX groups, HBcAg group and mDCs group were1.591,1.542 and 1.700).After 7 days, coculture with mDEXs,the percentage of CD3+CD8+T of PBMCs raise from 28.6% to 37.4%,while the HBcAg group is 34%,and the mDCs group is 39.4%.CONCLUSIONSThe immunocompetence of DEXs pulsed with HBcAg is stronger than HBcAg ,but weaker than mDCs pulsed with HBcAg .Cocultured with only a tiny amount of mDEXs can significantly enhance the immunocompetence of imDCs.