title: Dendritic cells engineered to secrete anti-DcR3 antibody augment cytotoxic T lymphocyte response against pancreatic cancerin vitro.
abstract: OBJECTIVE
  To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC)in vitroinduced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb).
  
  
  METHODS
  DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a51Cr releasing test. T cell responses induced by RNA-loaded DCs were analyzed by measuring cytokine levels, including IFN-γ, IL-10, IL4, TNF-α and IL-12.
  
  
  RESULTS
  The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DC-tumor-anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P< 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P< 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P< 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-γ and lower level IL4 than those incubated with DC-total tumor RNA or controls (P< 0.01).
  
  
  CONCLUSIONS
  DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PCin vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.
authors.affiliation: Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu, Department of Gastroenterology, Shenyang General Hospital of PLA, Shenyang 110016, Liaoning Province, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Chen
authors.affiliation: Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu, Department of Gastroenterology, Shenyang General Hospital of PLA, Shenyang 110016, Liaoning Province, China.
authors.forename: Xiao-Zhong
authors.initials: XZ
authors.lastname: Guo
authors.affiliation: Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu, Department of Gastroenterology, Shenyang General Hospital of PLA, Shenyang 110016, Liaoning Province, China.
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Li
authors.affiliation: Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu, Department of Gastroenterology, Shenyang General Hospital of PLA, Shenyang 110016, Liaoning Province, China.
authors.forename: Jia-Jun
authors.initials: JJ
authors.lastname: Zhao
authors.affiliation: Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu, Department of Gastroenterology, Shenyang General Hospital of PLA, Shenyang 110016, Liaoning Province, China.
authors.forename: Wen-Da
authors.initials: WD
authors.lastname: Xu
mesh_terms: D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D019496:Cancer Vaccines / Q000276:immunology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000276:immunology*; D005260:Female; D006801:Humans; D016219:Immunotherapy, Adoptive; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000276:immunology* / Q000628:therapy*; D053323:Receptors, Tumor Necrosis Factor, Member 6b / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D019496:Cancer Vaccines; D053323:Receptors, Tumor Necrosis Factor, Member 6b; C505911:TNFRSF6B protein, human
keywords: Antibody-encoding RNA; Cytotoxic T lymphocyte response; DcR3; Dendritic cell; Pancreatic cancer