负载泛素化乙肝病毒核心抗原的树突状细胞诱导 CTL 的增强作用。
Enhancement of CTLs induced by DCs loaded with ubiquitinated hepatitis B virus core antigen.
机构信息
Department of Infectious Diseases, Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China.
出版信息
World J Gastroenterol. 2012 Mar 28;18(12):1319-27. doi: 10.3748/wjg.v18.i12.1319.
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-10 and 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 II. DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-γ 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 HBcAg-specific cytotoxic T lymphocytes.
CONCLUSION
LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Th1 and generated HBcAg-specific CTLs.
目的
研究通过慢病毒载体转染树突状细胞（DC）表达泛素化乙型肝炎病毒核心抗原（LV-Ub-HBcAg）能否诱导体外乙型肝炎病毒核心抗原（HBcAg）特异性细胞毒性 T 淋巴细胞（CTL）反应。
方法
将重组 LV-Ub-HBcAg 转染到高度易感的 293T 细胞中，以获得高病毒滴度。从 BALB/c 小鼠分离骨髓来源的 DC，用重组粒细胞-巨噬细胞集落刺激因子和重组白细胞介素（IL）-4 培养。加入 LV-Ub-HBcAg、编码乙型肝炎病毒核心抗原的 LV-HBcAg、LV 或脂多糖诱导 DC 成熟，并通过流式细胞术分析 DC 表型。酶联免疫吸附试验（ELISA）检测上清液中 IL-12 的水平。用细胞计数试剂盒-8 检测 T 淋巴细胞的增殖。用不同的 LV 培养和诱导 DC 成熟，并与同种异体 T 细胞共培养，通过 ELISA 检测上清液中 T 细胞分泌的 IL-2、IL-4、IL-10 和干扰素-γ的水平。用流式细胞术分析增殖 T 细胞的细胞内细胞因子，用乳酸脱氢酶释放试验测定特异性 CTL 活性。
结果
LV-Ub-HBcAg 诱导的 DC 分泌更多的 IL-12，并上调 CD80、CD86 和主要组织相容性复合体 II 的表达。不同 LV 致敏的 DC 有效地促进细胞因子分泌；LV-Ub-HBcAg 诱导的 IL-2 和干扰素-γ水平高于 LV-HBcAg。与 LV-HBcAg 转导的 DC 相比，LV-Ub-HBcAg 转导的 DC 更有效地刺激 T 淋巴细胞增殖，并产生 HBcAg 特异性细胞毒性 T 淋巴细胞。
结论
LV-Ub-HBcAg 能有效诱导 DC 成熟。成熟的 DC 能有效地诱导 T 细胞向 Th1 极化，并产生 HBcAg 特异性 CTL。
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