Construction of recombinant adenovirus encoding TIPE-2
The recombinant adenovirus vector encoding TIPE-2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the TIPE2 cDNA was cloned into the shuttle vector pDC315 and sequenced. The desired replication-deficient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque purification, the adenovirus containing the TIPE-2 gene was amplified and purified from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at -80 ◦C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
Dendritic cell generation
Briefly, PBMCs were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS. The adherent cells were cultured for 5 days in RPMI-1640 containing 1000 U/ml of granulocyte-macrophage colony-stimulating factor (R&D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R&D Systems, Inc.), and were the cultured for an additional 2 days in the presence of 1000 U/ml of tumor necrosis factor α (R&D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis.
FACS analysis of DC phenotypes
DCs were collected and resuspended in cold FACS buffer (phosphate- buffered saline with 0.2% BSA and 0.09% sodiumazide). Cells were immunostained with FITC-conjugated mouse anti-human CD11c, CD83, and CD86 antibodies (eBioscience, USA). The appropriate FITC isotype antibody (eBioscience, USA) was used as a control. A total of 1×106 cells were incubated for 4h at 4◦C with antibodies. The cells were washed once with FACS buffer and then resuspended and phenotyped on a FACScan (Becton-Dickinson, USA).
Adenovirus-mediated gene transfer.
In a 24-well plate, DCs were seeded at a density of 5×104 cells/well and incubated for approximately 24 h until 50-70% confluent in flasks with DMEM supplemented with 10% (v/v) FBS and 1% penicillin- streptomycin at 37 ◦ C in a humidified atmosphere of 5% CO2. Virus was added to the wells at an MOI of 200 and DCs were harvested after 24 h of incubation.
Western blot assay
For Western blot assay, proteins of the cell extracts were separated SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with 5% non-fat milk in PBS and then with anti-TIPE-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at room temperature. After washing, the membranes were incubated with an alkaline phosphatase-conjugated goat antimouse IgG antibody (Amersham Biosciences, Little Chalfont, UK) for 1 h at room
temperature. Immunoreactive bands were detected using the ECL Western blot analysis system (Amersham Biosciences).
Coculture of DCs and T cells by one-way MLRs assay
DCs were prepared using the method described above. Single cell suspensions were treated with mitomycin C (50μg/ml; Sigma-Aldrich) for 20min at room temperature and were then washed twice with RPMI 1640. These cells were used as the stimulator cells in the assay. Responder CD4 + or CD8 + T cells were harvested by negative isolation using magnetic beads according to the manufacturer’s protocol (Dynal T Cell Negative Isolation kit; Invitrogen Life Technologies). The purity of the CD4 + or CD8 + T cells was determined by FACS (BD Biosciences); only CD4 + or CD8 + T cell populations with >95% purity was used in this study. Responder (2×105) and stimulator (4×105) cells were added to round-bottom 96-well plates to a final volume of 200μl RPMI1640 with 10% FCS. Each experiment was performed in triplicate.
Cells activated in an MLR were allowed to incubate for up to 3 days before harvesting. [3H]Thymidine(0.5μCi/well; MPBiomedicals) was added 24h before harvesting (SkatronInstruments) using Type A filter mats (Perkin-Elmer Life and Analytical Sciences) and a beta plate scintillation mixture (Perkin-Elmer). Disintegrations per minute were determined using a liquid scintillation counter (1205 Betaplate; Perkin-Elmer).
ELISA assay for quantitating cytokine production
X-ray-irradiated DC (2×104) and the T cells (1×105) were seeded into wells of 96-well flat-bottom culture plates in RPMI 1640 10% fetal bovine serum (Invitrogen Life Technologies) for 3 days. Supernatants were removed and further analyzed for cytokine production (IL-2 and IFN-γ) with ELISA. Plates were read at 405 nm using a Labsystems Multiskan enzyme-linked immunosorbent assay reader.
Flow cytometry for detecting activation marker expression
FCM assay was used to characterize activation marker expression of T cells. CD4 + or CD8 + T cells were cocultured with DCs for 3 days. The
cells were washed with PBS and then stained with anti-CD3-PE,
anti-CD25-FITC and anti-CD69-APC at 4 ◦C for 1h, and then washed by PBS. CD25 and CD69 activation markers were measured using a FCM Calibur flow cytometer (BD company). Data were analyzed with CellQuest software.
The statistical significance of differential findings between experimental groups and controls was determined by Student’s t-test and considered significant if two-tailed P-values were <0.05.