Female BALB/c (H-2d) and C57BL/6 (H-2b) mice (8-10 wk old) were purchased from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai, China). All mice were kept under pathogen-free conditions in the animal center of the Shanghai Second Medical University (Shanghai, China).
CT26 (H-2b), a mouse colon adenocarcinoma cell line, and SGC-7901 (H-2b), the gastric cancer cell line were purchased from the Shanghai Cell Biology Institutes, Chinese Academy of Sciences (Shanghai, China). CT26 cell lines grow in BALB/c mice. Both cell lines were cultured in RPMI (Roswell Park Memorial Institute) medium 1640 (GIBCO, USA) containing 10% heat-inactivated fetal calf serum (FCS), 2 mmol/L glutamine, penicillin G (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a humidified incubator supplemented with 5% CO2.
Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, IL-2, and IL-7 were purchased from Becton Dickinson (New Jersey, USA). Phenotypic analysis, fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies against murine cell surface molecules, such as MHCI(H-2Kd), MHC II (I-Ab), CD11b, CD11c, CD40, CD80, CD86, and CD8α, were provided by Pharmigen (CA, USA). Mitomycin C (MMC) was purchased by Jingmei Biothe (Shenzhen, China).
Generation of bone marrow-derived DCs
Primary bone marrow DCs (BM-DCs) was obtained from mouse bone marrow precursor according to a protocol by Zhang et al[16
]. BALB/c mice bone marrow was obtained from tibia and femurs by flushing with the media. Tissue was minced in a single-cell suspension through a nylon mesh. BALB/c mice bone marrow cells were stained sequentially with biotinylated anti-c-kit MAb and Streptavidm MicroBeads (Dynal, Norway). c-kit+
hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator (Milteyi Biotec, Auburn, CA) from the of BALB/c mice bone marrow cells. The cells (6 × 106
) were cultured for 7 d in fresh RPMI medium 1640 containing 10% FCS, GM-CSF (4 ng/mL), and IL-4 (10 ng/mL).
DC immunofluorescence analysis was performed as previously described[16,17
]. In brief, DCs prepared and isolated from BALB/c mice, as described above (2 × 105
-4 × 105
cells), were incubated with FITC-labeled MAbs against CD40, CD11b, CD11c, or CD80 and PE-labeled MAbs against MHCI, MHC II, CD86 or CD8α followed by FACScan analysis (Becton Dickinson, USA). Instrument compensation was set in each experiment using two-color stained samples.
Recombinant adenovirus vector
Recombinant adenoviruses IL-12 (AdVIL-12) ecoding the murine IL-12 gene were donated by Dr. Y. Y. Zhang (Health Science Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China). These recombinant adenoviruses were propagated in 293 cells and purified on a CsCl density gradient. Their titres were determined using a plaque assay on 293 cells. Aliquots of the adenovirus solutions were stored at -80°C.
Adenoviral transduction of DCs and secretion of IL-12
DCs generated on d 8 were plated at 2 × 106 cells/well in 1ml of RPMI 1640 medium containing 10% FCS with AdVIL-12 at a multiplicity of infection (MOI) of 100. After 2 h incubation at 37°C in a humidified incubator supplemented with 5% CO2 with gentle agitation every 20 min, the DCs culture medium was replaced with 2 mL of RPMI 1640 medium supplemented with 10% FCS, and the cells were incubated for another 48 h. AdVIL-12 transduced DCs (AdVIL-12/DCs) were harvested, washed twice with PBS, and used for the tumor cell lysate pulsing in vitro. Immunofluorescence analysis of AdVIL-12/DCs was performed as described above.
DCs (1 × 106) were infected with AdVIL-12 at MOI 100. Viral supernatant was replaced after 2 h with the cell culture medium. The supernatant was collected and tested for mIL-12 by enzyme-linked immunosorbent assay (ELISA) kit (Rockford, IL, USA) at different time intervals (0, 12, 24, 48, 72, 96 h). Moreover, DCs were infected with AdVIL-12 at different MOI (50, 100, 200, 300, 500). The supernatant was collected 48 h later and tested for mIL-12 by ELISA.
Pulsing DCs with tumor cell lysates
DCs were pulsed with freeze-thawed tumor lysate at a 1:3 DCs: tumor ratio. CT26 tumor (6 × 106) were lysed by rapid freezing (liquid nitrogen) and thawing in a 37°C bath three times. The AdVIL-12 transduced DCs (AdVIL-12/DCs, 2 × 106 cells/mL) or bone marrow-derived DCs (2 × 106 cells/mL) were incubated in six-well plates in the presence of CT26 tumor lysates (6 × 106 cell equivalents/mL) in RPMI Medium 1640 containing 10% FCS, GM-CSF (4 ng/mL), and IL-4 (10 ng/mL) for one day at 37°C and 5% CO2. These CT26 tumor cell lysate-pulsed (TP) AdVIL-12/DCs or DCs were used for vaccination after one day.
Tumor model and DC-based vaccination
In the established tumor model, CT26 tumor cells (5 × 105 cells per mouse) were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice (10/each group). Tumor-bearing mice were injected with 1 × 106 DCs vaccination: CT26 TP AdVIL-12/DCs or CT26 TP DCs on d 3 and 10. As a positive control, tumor bearing mice were treated in parallel with unstimulated DCs, CT26 tumor lysate alone, or PBS alone. Tumor growth was assessed by calipers (mean of 2 perpendicular diameters) every 2-3 d. Tumor volume was estimated by the formula 4/3 πr3.
In the protective anti-tumor experiment, each group consisting of 10 naïve BALB/c mice were injected s.c. in their abdominal walls with 1 × 106 DCs vaccination: CT26 TP AdVIL-12/DCs or CT26 TP DCs on d 0, 7, and 14. Control mice were injected with unstimulated DCs, CT26 tumor lysate alone, and PBS alone. Each mouse was challenged s.c. with a lethal dose of 2 × 106 CT26 cells on d 21. Survival differences among the groups receiving different vaccinations were evaluated following the primary challenge with CT26 cells.
Assays for cytotoxic T lymphocyte activity and interferon gamma secretion in the immunized mice
To confirm that tumor-specific cytotoxic T lymphocytes (CTLs) had been generated in the immunized mice, splenic CD3+ T cells from tumor-free mice that survived the CT26 tumor challenge through d 61 following DC vaccination, were magnetically isolated using CD3 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany). T cells (1 × 106 cells) from surviving mice were restimulated ex vivo by culturing in IMDM containing 10% FCS and MMC-treated CT26 tumor cells (1 × 105). Five days later, the MMC-treated CT26 tumor cells and the T cells were collected for measuring CTL activity and IFNγ secretion. The restimulated effector T cells (2 × 105 in 100 μL per well) were added to the wells containing target CT26 or SGC-7901 tumor cells (5 × 104 in 100 μL per well) in 96-well plates. Supernatant from each well was collected after 20 h and was measured cytolytic activity against target CT26 tumor cells and SGC-7901 tumor cells with a Cytotoxicity Detection Kit (LDH; Boehringer Mannheim, Mannheim, Germany), IFNγ production was determined with the mouse IFNγ ELISA kit (Endogen, Woburn, MA, USA). In some experiments, the target CT26 tumor cells (1.5 × 105) were incubated with a MAb to MHC classImolecules (anti-H2Db/H2Kb; 50 g/mL) or with control antibody (anti-H2Dd; 50 g/mL) at 37°C for 30 min before the addition of restimulated effector T cells to evaluate the specificity of CTL activity.
Assays for cytotoxic T lymphocyte activity ex vivo
To confirm that tumor-specific cytotoxic T lymphocytes (CTLs) can be generated ex vivo, splenic CD3+ T cells (1 × 106 cells/mL) were magnetically isolated from naïve BALB/c mice using CD3 microBeads. These cells were cultured in RPMI medium 1640 containing 10% FCS, then primed ex vivo in the presence of cytokines including IL-2 and IL-7 (5 ng/mL per) at d 0, 7, and 14 with CT26 TP AdVIL-12/DCs or CT26 TP DCs at a 1:20 stimulator to responder cell ratio. Unpulsed DCs and AdVIL-12/DCs were used as controls. Fresh medium containing IL-2 and IL-7 was exchanged every four days. The primed T cells were effector cells, CT26 or SGC-7901 tumor cells were target cells. On d 21, target cell suspension was added into 96 well plates, and effector cells were titrated to the dilutions target cells by serial dilutions (E-T mix, E:T, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1). Supernatant from each well was collected after 20 h in order to measured cytolytic activity against target CT26 and SGC-7901 tumor cells, which was done with a cytotoxicity detection kit as mentioned above.
Differences were evaluated using Statistical Package for Social Science 11.0 (SPSS11.0). Statistical analysis was performed using a Student’s t test. Survival differences among mice groups were evaluated with a log-rank test of the Laplan-Meier survival curves. Statistical tests were two-tailed. P values < 0.05 were considered to be statistically significant.