Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells

查看全文 作　　者：Chuan-[1]LinDing;[1]KunYao;Tian-[2]TaiZhang;[1]FengZhou;[3]LinXu;Jiang-[3]YingXu

机构地区：[1]DepartmentofMicrobiologyandImmunology,NanjingMedicalUniversity,Nanjing210029,JiangsuProvince,China;[2]DepartmentofMedicine,AffiliatedHospitalofLanzhouMedicalCollege,Lanzhou730000,GansuProvince,China;[3]GeneCenterofNanjingMilitaryMedicalCollege,NaniinR210099,JianRsuProvince,China

出　　处：《World Journal of Gastroenterology》索引2003年第9卷第7期,共4页

基　　金：a grant from Key Lab Programs of Jiangsu Province,No.k2030 摘　　要：AIM: Cytotoxic T lymphocytes (CTLs) play an important role in resolving HBV infection. In the present study, we attempted to evaluate the efficiency of bone marrow-derived dendritic cells (DCs) transduced with recombinant retroviral vector bearing hepatitis B virus (HBV) core gene and the capability of generating CTLs against HBcAg by genetically modified DCs in vivo.METHODS: A retroviral vector containing HBV core gene was constructed. Replicating DC progenitor of C57BL/6 mice was transduced by retroviral vector and continually cultured in the presence of recombinant mouse granulocytemacrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(IL-4) for 6 days. LPS was added and cultured for additional two days. The efficiency of gene transfer was determined by PCR, Western blot and FACS. Transduced DCs immunized C57BL/6 mice subcutaneously 2 times at an oneweek interval. Intracellular IFN-γ and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphooytes stimulated with mitomycin C-treated EL4-C cell which stably expresses HBcAg was determined by LDH release assays.RESULTS: Recombinant retroviral expression vector (pLCSN) was positively detected by PCR as well as enzyme digestion with EcoRI and BamH I. Retroviruses were generated by pLCSN transfection packing cell and the virus titer was 3x10s CFU/ml. Indirect immunofluorescence and FACS showed that HBV core gene was expressed in murine fibroblasts. Transduced bone marrow cells had capability of differentiating into DCsc in vitro in the presence of rmGMCSF and rmIL-4. The result of PCR showed that HBV core gene was integrated into the genome of transduced DCs.Western blot analysis showed that HBV core gene was expressed in DCs. The transduction rate was 28 % determined by FACS. Retroviral transduction had no influence on DCs expressions of CD80 and MHC class Ⅱ. HBcAg specific CTLs and Th1 type immune responses could be generated in the mice by using transduced DCs as antigen presenting cells (APCs).CONCLUSION: Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo. 关键词：细胞毒性T淋巴细胞乙型肝炎病毒感染树突状细胞粒细胞-巨噬细胞集落刺激因子白细胞介素-4

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Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells

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