AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiv...AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.展开更多
基金Supported by Natural Science Foundation of Shanghai,No.11ZR1427100
文摘AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.
