Generation of cytotoxic T cell against HBcAg using retrovirally transduced dendritic cells.
OBJECTIVE
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 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 one-week interval. Intracellular IFN-gamma 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 x 10(5) 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).


CONCLUSIONS
Retroviral transduction of myeloid DCs progenitors expresses efficiently HBcAg, and genetically modified DCs evoke a higher CTLs response than HBcAg in vivo.
Ding
Chuan-Lin
CL
Department of Microbiology and Immunology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
Yao
Kun
K
Zhang
Tian-Tai
TT
Zhou
Feng
F
Xu
Lin
L
Xu
Jiang-Ying
JY
D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology / Q000637:transplantation; D001854:Bone Marrow Cells / Q000166:cytology; D002454:Cell Differentiation / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology* / Q000637:transplantation; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000276:immunology*; D019694:Hepatitis B, Chronic / Q000276:immunology / Q000628:therapy; D007167:Immunotherapy; D051379:Mice; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology* / Q000276:immunology*; D014161:Transduction, Genetic
D006512:Hepatitis B Core Antigens