Viral Hepatitis
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 15, 2003; 9(7): 1512-1515
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1512
Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells
Chuan-Lin Ding, Kun Yao, Tian-Tai Zhang, Feng Zhou, Lin Xu, Jiang-Ying Xu
Chuan-Lin Ding, Kun Yao, Feng Zhou, Department of Microbiology and Immunology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Tian-Tai Zhang, Department of Medicine, Affiliated Hospital of Lanzhou Medical College, Lanzhou 730000, Gansu Province, China
Lin Xu, Jiang-Ying Xu, Gene Center of Nanjing Military Medical College, Nanjing 210099, Jiangsu Province, China
Author contributions: All authors contributed equally to the work.
Supported by a grant from Key Lab Programs of Jiangsu Province, No. k2030
Correspondence to: Professor Kun Yao, Department of Microbiology and Immunology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
Telephone: +86-25-6662901 Fax:+86-25-6508960
Received: January 14, 2003
Revised: March 1, 2003
Accepted: March 5, 2003
Published online: July 15, 2003

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 granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin-4(IL-4) for 6 d. 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 one-week interval. Intracellular IFN-γ and IL-4 of immunized mice lymphocytes were analyzed. Generation of CTLs in lymphocytes 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 3 × 105 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 DCs in vitro in the presence of rmGM-CSF 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.

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