of each PCR product was electrophoresed on 2% agarose gel. The gels were stained with ethidium bromide solution at a concentration of 1 µg/ml

Transcription

1 of each PCR product was electrophoresed on 2% agarose gel. The gels were stained with ethidium bromide solution at a concentration of 1 µg/ml for 30 min and destained in distilled water for 15 min. For quantification, the gels were illuminated by ultraviolet lamp, and the intensity of each band was quantified with a densitometer. By the way, the intensity of each recombinant BCG cdna fluorescent band was normalized with the corresponding β-actin cdna fluorescent band and compared to those of DNA standards by using the digital ChemiGenius image analysis system (Syngene, Cambridge, UK). To further determine the relative expression levels of five mycobacterial subunits derived from multi-rbcg in vivo, total RNA was extracted using TRIzol (Gibco BRL, Grand Island, NY, USA) from lysates of MBT-2 implants transfected with multi-rbcg. A two-step semi-quantitative RT PCR method was performed to measure the expressions of Ag85A, Ag85B, Mpt64, PstS3 and ESAT6, respectively. These cdna quantities were selected empirically so that PCR amplification did not reach a plateau by 25 cycles. Ten microliters of each RT PCR product was electrophoresed on 2% agarose gels. The intensity of each fluorescent band was normalized in the specified gene s linear amplification range using the corresponding internal control gene for β-actin. The quantity and base pair size of the PCR generated fragments were also estimated relative to DNA ladder standards using the digital ChemiGenius image analysis system (Syngen). 2.5 Determination of the Antitumor Effects of Recombinant BCG and 50

2 Interleukin-12 DNA Vaccines on Cellular Immune Responses and T-helper 1 Immune Response Lymphocyte proliferation assay. The procedures have been described by Peter Andersen et al. (Andersen P et al., 2002) In briefly, MBT-2 bladder cancer implantation CH 3 /HeN mice were immunized with multi-rbcg using electroporative immunogene therapy or anesthetized by intraperitoneal injection of attenuated BCG vaccine as described above. To determine whether recombinant BCG lymphoproliferation was induced in immunized animals, spleen cells were harvested 1 week after multi-rbcg electroporative immunization. The spleenic T lymphocytes were enriched with nylon wool columns, and 100 µl of 2 x 10 6 cells/ml in RPMI culture medium (RPMI 1640 containing 5% fetal bovine serum, 100 nm L-glutamine, 10 nm penicillin-streptomycin, and 5 x 10-5 M 2-mercaptoethanol) was added to each well in 96-well plates. The mycobacterial ESAT6 T-cell epiotope synthetic peptide (amino acid 23-43) was added to each stimulated well at a final concentration of 0.5 µg/ml. Transferrin (120 µg/ml; Sigma) served as a negative control antigen, and concanavalin A (10 µg/ml; Sigma) served as a positive mitogenic control. Control wells received cells only. Cells in all the wells were cultured in a total volume of 200 µl of medium. After 72 h in culture, cells were pulsed with [ 3 H] thymidine (1 µci/well) (Amersham Pharmacia Biotech, Piscataway, NJ) for 18 h. Cells were then harvested with FilterMate (Packard, Meriden, Conn.), and the incorporated radioactivity was 51

3 determined by TopCount (Packard). The stimulation index was calculated as the mean counts per minute of the stimulated wells divided by the mean counts per minute of the control wells. The data are represented as means ± SD of three independent experiments Preparation of cytotoxic T lymphocyte effector cells and cytotoxic assay. Effector cells were derived from splenocytes as precursor of CTLs. The responding splenocytes were obtained from sequentially immunized mice 14 d after electroporative immunogene therapy of multi-rbcg, resuspended in complete RPMI medium (Invitrogen Life Technologies) and cultured at a concentration of splenocytes per culture well together with 10 µg of MBT-2 extracts in a total volume of 2 ml. The effector cells were harvested after 5 d of culture. Two cell lines, both originating from C 3 H mice, were transfected with multi-rbcg and used as target cells. MBT-2 is a transitional cell carcinoma, but Sq-1979 is a squamous cell carcinoma. Target cells were labelled with 100 µci Na 2 ( 51 Cr)O 4 for 1 h at 37 C in RPMI-1640 medium containing 10% FCS and subjected to cytotoxicity assays. Labelled target cells (10 4 /well) were incubated in triplicate with effector cells in a total volume of 200 µl RPMI in 96-well microtitre plates at different effector : target cell ratios. After 12 h co-incubation, specific lysis was determined as: specific lysis (%) = 100 (experimental release spontaneous release)/(maximum release spontaneous release). Spontaneous release varied from 5% to 10%. All experiments were performed in triplicate, and each group consisted of five 52

4 immunized mice Proliferation responses of splenocytes The responding splenocytes were obtained from sequentially immunized mice on days 14 after electroporative immunogenetherapy and treated with a condition medium with complements (Invitrogen Life Technologies), anti-cd4 (GK 1.5) or anti-cd8 (Lyt 2.2) monoclonal antibody (BD PharMingen) to confirm the division of splenocytes, and then the cells were resuspended in complete medium and cultured at a concentration of splenocytes per culture well together with 10 µg of MBT-2 implants expressing multi-rbcg in a total volume of 0.2 ml. After 72 h in culture, cells were pulsed with [ 3 H]thymidine (1 µci per well; Amersham Pharmacia Biotech, Piscataway, NJ) for 18 h and then harvested using a cell harvester. Incorporated radioactivity was calculated as the mean uptake in cpm ± SEM of triplicate cultures Immune phenotyping of murine peripheral blood mononuclear cells (PBMCs) PBMCs were freshly isolated from mice on days 14 after immunization with the multi-rbcg and/or mil-12 and separated by gradient centrifugation of heparinized blood on Ficoll-Paque plus (Amersham Biosciences, Piscataway, NJ). To stimulate T cells (CD3e) and/or NK cells (CD56) expansion of PBMCs by multiple BCG antigens, multi-rbcg expressed tumor implants was excised and collected at the 7 days after immunization. Subpopulations of PBMCs ( cells/ml) 53

5 were co-cultured with 10 µg of homogenized lysates of MBT-2 implants expressing multi-rbcg for 5 days and then incubated with 10 µl of the anti-leu-4 (CD3e) FITC-labeled and anti-leu-19 (CD56) PE-labeled monoclonal antibodies (BD PharMingen), respectively. After cells were incubated on ice for 1 h, they were washed twice and analyzed by flow cytometry (BD Biosciences) Cytometric Bead Array (CBA) assay The Cytometric Bead Array (CBA; BD Biosciences, San Diego, CA, USA) uses flow cytometry to detect mouse cytokines from small volume serum samples. In this study, BD mouse Th1/Th2 cytokine CBA kit was used to measure IL-2, IL-4, IL-5, IFN-γ, and TNF-α protein levels in a single serum sample to distinguish between Th1 and Th2 reactions. Briefly, mouse serum from 4 groups (N = 8 each) was collected at day 0, 7, 14 and 21 after vaccine treatment. Fifty µl/test of serum added to 10 µl/test of mixed CBA beads were incubated at room temperature 2 h, washed, and then incubated with a second cytokine PE-labeled antibody. Beads positive for cytokines present were both PE and FL-3 positive, while beads for cytokines not present were only FL-3 positive. Flow cytometry was carried out using a dual-laser FACSCalibur TM instrument (BD Biosciences, San Diego, CA, USA) and data were displayed using CellQuest (BD Biosciences, San Diego, CA, USA) software and analyzed by BD CBA analysis software. The expected sensitivity was in pg/ml range. The data are the means ± SEM of the serum cytokine levels from the indicated groups. 54

6 2.5.6 Characterization of tumor infiltrating lymphocytes. MBT-2 implants were collected 14 days after electroporative immunogenetherapy with multi-rbcg and/or mil-12. Total RNA was extracted from the implants ( cells/ml) with TRIzol Reagent (Invitrogen Life Technologies). Lymphocyte cell-surface marker expression was determined from 10 µg of total RNA by multi-probe ribonuclease protection assay (RPA). The RiboQuant Non-Rad RAP system (BD PharMingen, San Diego, CA, USA) was used for efficient synthesis of high specific-activity riboprobes from the mcd-1 Template set (BD PharMingen) following the manufacturer s directions Immunohistochemistry of CD4, CD8a, CD3e, CD19 and F4/80. Seven days after immunogene therapy, quadriceps femoris muscle and tumor tissue samples were collected from the experimental groups of mice. Samples were either fixed in the phosphate-buffered formalin for paraffin-embedding immunohistological analysis, snap-frozen in liquid nitrogen for FITC fluorescent staining and fluorescent protein histological analysis, or immediately homogenized for Western blot analysis to determine the level of Bcl-2 protein expression in bladder tumor. To distinguish between cellular and humoral immune responses, simultaneous immunohistochemistry was used to detect the effects of treatment on immune cell responses. Samples of the tumor tissue were collected from the experimental groups of mice seven days after vaccination. Detection of CD4, CD8a, CD3e, CD19 and F4/80 were 55

7 assessed in paraffin-embedded sections by the avidin-biotin-peroxidase complex method described by Morimur et al (Morimura MO et al., 2001). The 4-µm-thick paraffin embossed tissue sections were deparaffinized and then treated with 3.0 % hydrogen peroxide (Sigma-Aldrich, St. Louis, MO, USA) to block endogenous peroxidase activity. Subsequently, antigen retrieval procedures for formalin-fixed paraffin sections were performed by immersion of the slides in 1 x ChemMate buffer for Antigen retrieval (S2031, Dako, Kyoto, Japan) and autoclaving at 700 W for 10 min. The protocol for Dako LSAB2 Kit, Peroxidase (Dako, Kyoto, Japan) was followed for each sample. The sections were blocked with 5% FCS/PBS for min and then incubated at room temperature with the primary antibody (purified rat anti-mouse CD4, clone L3T4, BD PharMingen, CA, USA; purified rat anti-mouse CD8a, clone Ly-2, BD PharMingen, CA, USA; purified Armenian hamster anti-mouse CD3e, clone 145-2C11, BD PharMingen, CA, USA; purified rat anti-mouse CD19, clone 1D3, BD PharMingen, CA, USA; purified rat anti-mouse F4/80, clone CI : A3-1, SeroTec, Oxford, UK) diluted 1 in 25 in DAKO antibody diluent with background reducing components (S3022, Dako, Kyoto, Japan), followed by the secondary antibody (Biotinylated mouse anti-hamster antibody, BD PharMingen, CA, USA ; Biotin conjugated goat anti-rat antibody, AP136B, Chemicon, CA, USA) diluted 1 in 50 in DAKO antibody diluent for 90 min. Then samples were exposed to streptavidin-biotin-peroxidase complex, and diaminobenzidine tetrachloride (Dako, Kyoto, Japan) was used as a chromogen. Counterstaining was performed with Mayer's hematoxylin. The percentages of cell immunopositive for CD4, CD8a, CD3e, CD19 and F4/80 were calculated by counting the cells in a

8 and 400-power field. Three investigators blinded to the treatment graded CD4, CD8a, CD3e, CD19 and F4/80 expressions on the following scale: (-), no cells positive; (+), 1-25% of cells positive; (++), 26-50% of cells positive; (+++), % of cells positive. Their counts did not differ significantly. 2.6 Determination of the Antitumor Efficacy of Recombinant BCG and Interleukin-12 DNA Vaccines on Xenografted Murine Bladder Cancer Assessment of apoptosis by TUNEL assay. Fourteen days after electroporative immunogene therapy, five further paraffin-embedded sections of the tumor tissue samples from the experimental groups of mice were resectioned and cut in the middle at the site of the original tumor inoculation. The tissue was then fixed in 10% buffered formalin and routinely evaluated on 4-µm-thick sections stained with H & E for histopathological analysis. The proportion of viable bladder tumor cells in each tumor at five randomly-selected areas was calculated using an image analyser (MAC Scop, Nagano, Japan), and the overall effect of multi-rbcg electroporative immunogene therapy was evaluated. TUNEL staining was performed using an ApopTag Peroxidase In Situ Apoptosis Detection kit (Intergen Company, Purchase, NY, USA). The manufacturer s instructions were followed with some modifications as described by Gujral et al (Gujral et al., 2001). Briefly, the 10-µm-thick paraffin tissue sections were 57