ABSTRACT
Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.
Subject(s)
Animals , Female , Humans , Mice , /genetics , Adenoviridae/genetics , Apoptosis/genetics , Dendritic Cells/virology , Prostate-Specific Antigen/genetics , /immunology , Adenoviridae/immunology , Apoptosis/immunology , Cytotoxicity, Immunologic/immunology , Dendritic Cells/immunology , Dendritic Cells/pathology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , /immunology , /immunology , Phenotype , Prostate-Specific Antigen/immunology , Recombinant Proteins/genetics , Transduction, Genetic/methodsABSTRACT
PURPOSE: The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells. MATERIALS AND METHODS: Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry. RESULTS: The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs. CONCLUSION: The results indicated the recombinant mouse 4-1BBL can effectively activate DCs.