Induction of ErbB-2/neu-specific protective and therapeutic antitumor immunity using genetically modified dendritic cells: enhanced efficacy by cotransduction of gene encoding IL-12


Overexpression of ErbB-2/neu occurs in 20–30% of patients with breast cancer and indicates a poor prognosis. The presence of a detectable immune response to ErbB-2/neu in some patients suggests that this oncogene may be a useful target for vaccine therapy. We evaluated whether genetic immunization using dendritic cells (DC) transduced ex vivo with an adenovirus expressing the ErbB-2/neu gene (AdNeuTK) could induce protective and therapeutic immunity against a breast tumor cell line overexpressing ErbB-2/neu. Subcutaneous (s.c.) immunization with the DC vaccine elicited protective immunity in an average of 60% of animals. CTL analysis demonstrated specific cytotoxic activity against breast tumor cells, as well as syngeneic fibroblasts transduced with AdNeuTK. In vivo depletion studies demonstrated both CD4+ and CD8+ T cells were required. In a therapeutic setting, immunization with the DC vaccines could cure mice with pre-established tumors and efficacy was further enhanced by cotransducing DCs with a vector expressing murine IL-12 (AdmIL-12). These studies support DC vaccines as a therapeutic strategy for human breast cancer, while emphasizing the importance of optimizing an immune response by combining tumor antigen presentation with immunostimulatory cytokines.


ErbB-2/neu is a member of the receptor tyrosine kinase (RTK) family of growth factor receptors. Although a specific ligand has not been identified, activation of ErbB-2/neu occurs through homo- and heterodimerization with other Erb family members leading to trans-tyrosine phosphorylation and transduction of a mitogenic signal.12 ErbB-2/neu is amplified and overexpressed in 20–30% of human breast cancers and studies in transgenic mice have demonstrated a direct role for this RTK in the process of malignant transformation.34 Patients with ErbB-2/neu-positive tumors demonstrate a more aggressive clinical course and a poor prognosis.56

ErbB-2/neu has been evaluated as a potential target for the development of cancer vaccines because pre-existent T cell and Ab responses to ErbB-2/neu have been described in breast cancer patients, although not at a level sufficient to prevent tumor outgrowth.78 Indeed, elevated anti-ErbB-2/neu T cell responses have been demonstrated in breast and ovarian cancer patients following immunization with peptides derived from the ErbB-2/neu protein.910 Peptide vaccine-induced T cell responses have also been described in animal models.11 However, despite the promising results from these single peptide immunization studies, whether peptide-specific T cell response can be translated to antitumor immunity has yet to be established. In one study, peptide-specific T cells elicited following immunization could lyse target cells pulsed with the peptides from the vaccine inoculum but the T cells were not able to recognize tumor cells that naturally expressed ErbB-2/neu protein.10 Genetic immunization using a DNA plasmid encoding ErbB-2/neu has been evaluated as a breast cancer vaccine in mouse models.121314 Unlike peptide vaccines, which must be specific for each individual's MHC, expression of plasmid encoded tumor antigens within the host antigen-presenting cells (APC) following vaccination results in the presentation of multiple tumor-associated epitopes in the context of MHC class I and/or class II molecules. Direct intramuscular injection with DNA plasmid expressing ErbB-2/neu induced antigen-specific cellular and humoral immune responses in mice. These animals were partially protected against challenge with ErbB-2/neu-expressing tumor cells1213 and the DNA vaccine could slow tumor development in rat ErbB-2/neu- transgenic mice.14

Recent studies have clearly demonstrated that DNA vaccination requires host dendritic cells (DC) for priming the T cell response, through either direct transfection or antigen transfer from transfected non-hematopoeitic cells. Given that DCs are functionally impaired in cancer patients1516 and in tumor-bearing animals,1718 antigen processing and presentation following genetic immunization may be inefficient in these situations. This impairment can be overcome if the DCs are differentiated ex vivo from precursors present in the peripheral blood.15 The ex vivo cultured DCs are fully functional and can be used as cellular vectors for vaccines.1920

We, and others, have previously shown that ex vivo transduction of DCs with an adenovirus vector (Ad) expressing a defined tumor antigen can result in a high level of transgene expression in the DCs.2122 Immunization with the genetically modified DCs induces protective immunity and regression of established tumors in a number of murine tumor models.21232425 In the current study, we have evaluated a DC/Ad vaccine expressing ErbB-2/neu in a murine model of breast cancer. We demonstrate that immunization with the DC/Ad vaccine is effective in inducing antigen-specific CTL responses leading to protective immunity mediated by CD8+ CTL. More importantly, vaccination with DC/AdNeuTK suppresses the growth of pre-established tumors and this therapeutic effect is further enhanced by cotransduction of the DCs with Ad vector encoding murine IL-12 (AdmIL-12), resulting in a substantial improvement in tumor-free survival.


Expression of rat ErbB-2/neu protein in murine DCs and PTO516 fibroblast cells following AdNeuTK transduction

FACS analysis was performed to determine Ad-mediated ErbB-2/neu gene expression in the ErbB-2/neu-negative cell line PTO516 and cultured primary DCs. As shown in Figure 1, ErbB-2/neu protein was readily detected using specific mAb Ab4 in AdNeuTK-infected cells 24 h after infection, whereas naive cells, or cells infected with AdLacZ, showed only background levels of Ab4 staining. The expression of the ErbB-2/neu gene in AdNeuTK-transduced cell lines was also detected by immunohistochemistry (data not shown).

Figure 1

Expression of rat ErbB-2/neu proteins in PTO516 cells and dendritic cells transduced with AdNeuTK. Cells were transduced with the control virus AdLacZ (MOI 100) or with AdNeuTK (MOI 100) for 24 h. FACS analysis was carried out using mAb Ab4, which recognizes the extracellular domain of rat ErbB-2/neu. Experiments were repeated three times and the results shown were from one representative experiment.

Induction of CTL responses using AdNeuTK-transduced DCs

To determine whether DCs transduced with AdNeuTK would induce an ErbB-2/neu-specific CTL response in vivo, FVB/n mice were immunized with 1 × 106 of DC/AdNeuTK or DC/AdLacZ. Splenocytes were harvested from immunized mice, re-stimulated in vitro with ErbB-2/neu expressing targets, and assayed for specific cell lysis against a group of target cells. A representative experiment is shown in Figure 2. Immunization with AdLacZ-infected DCs did not elicit a measurable ErbB-2/neu-specific CTL response (Figure 2a), while potent CTL activity against ErbB-2/neu-positive cell line NDL and a syngenic fibroblast cell line PTO516 transduced with AdNeuTK was observed in mice immunized with DC/AdNeuTK (Figure 2b). Only a background level of cytotoxic lysis to ErbB-2/neu-negative PTO516 target cells was detected in DC/AdNeuTK-immunized mice (Figure 2b), demonstrating that elicited CTL response by DC/AdNeuTK was ErbB-2/neu specific. The lytic activity against ErbB-2/neu was inhibited by anti-CD3 and anti-CD8 mAbs indicating that the lytic effectors measured in these experiments were CD8+ T cells (Figure 2c). To determine the specific mode of killing, concanamycin A (CMA) and emetin were used to inhibit both perforin- and Fas-mediated CTL killing, respectively.2627 CMA treatment completely abrogated CD8+ T cell-mediated cytotoxicity, while emetin only produced a modest loss of cytotoxic activity (data not shown). These observations are consistent with other reports showing that CD8+ CTL lyse tumor cells predominantly through a cytotoxic granule-dependent pathway.28

Figure 2

Cytotoxic activity of splenocytes from DCAdNeuTK-immunized mice. Splenocytes were harvested from mice 14 days following immunization with DC/AdLacZ (a) or with DCAdNeuTK (b). Five days after restimulation with NDL cells, effector cells were harvested and tested for cytotoxic activity in a 51Cr-release assay using NDL and PTO516 transduced with AdNeuTK as targets. Panel c indicates the percentage of inhibition of lysis against NDL (E:T 90:1) in the presence of anti-CD3/CD8 mAbs or theperforin synthesis inhibitor CMA. The data are representative of three independent experiments.

Protection against tumor challenge following immunization with AdNeuTK-transduced DCs

To determine if the immune response elicited by the DC/AdNeuTK vaccine was protective against tumor cells expressing ErbB-2/neu, mice were immunized s.c. with either DC/AdNeuTK or DC/AdLacZ and challenged 14 days later with NDL cells. As shown in Figure 3, non-immunized animals developed tumors by 20 days after tumor challenge. Complete protection against tumor challenge was observed in 60% of mice immunized with DC/AdNeuTK, lasting for the duration of the experiment (90 days). This protective response was specific for ErbB-2/neu because the NDL tumor grew progressively in mice immunized with DC/AdLacZ and mice vaccinated with DC/AdNeuTK succumbed to challenge with a different breast cancer tumor line that was transformed by the polyoma virus middle T antigen and does not overexpress ErbB-2/neu (data not shown).

Figure 3

Induction of protective immunity by immunization with DCAdNeuTK. FVB/n mice were immunized s.c. with 1 × 106 DC/AdNeuTK or DC/AdLacZ as described in Materials and methods. Mice were challenged s.c. with 7 × 106 NDL cells 14 days after immunization. Tumors were measured every 5 days for 90 days. Data are pooled from five experiments with four to five mice for each group. P < 0.001 (Fisher's exact test), DCAdNeuTK compared with DCAdLacZ and naive.

Involvement of CD4+ and CD8+ T cells in protective antitumor immunity

The importance of CD4+ in antitumor immunity and CTL priming has been well established through recent studies. Based on our knowledge of antigen processing, we hypothesized that a DC/Ad vaccine expressing a membrane-bound protein should provide epitopes for activating both CD4+ and CD8+ T cells, resulting in a highly potent vaccine.29 To investigate the importance of CD4+ and CD8+ T cells in the immune response to our vaccine, FVB/n mice were injected with either mAbs against CD4+ and CD8+ T cells. As demonstrated in Figure 4a, depletion of either CD4+ or CD8+ T cells during the DC/AdNeuTK priming phase (days 1 to 14) following vaccination completely abrogated the protective immune response, indicating that both CD4+ and CD8+ T cells are essential for initiating the immune response, consistent with reports from others.293031 Interestingly, only CD8+ T cells were required for tumor rejection as evident from Figure 4b, where depletion was initiated immediately before tumor challenge. These data confirm in vitro studies demonstrating that while CD8+ T cells are necessary to lyse directly target cells expressing appropriate antigen, CD4+ T cells play a pre-requisite role in the priming phase of a CD8+ T cell response.

Figure 4

In vivo depletion of CD4+ or CD8+ T cells in FVB/n mice during DCAdNeuTK immunization and NDL challenge. (a) FVB/n mice were injected i.p. with anti-CD4 (GK1.5) or anti-CD8 (53–6.72) mAbs 2 days before DCAdNeuTK immunization, and then every 3rd day until day 14 when mice were ready for NDL challenge. (b) mAbs were given 2 days before tumor challenge and then every 3rd day until most control animals (non-immunized mice) developed palpable tumors. The results are representative of two experiments with five to six mice for each group.

Suppression of pre-established tumors by DC/AdNeuTK vaccination

To evaluate whether pre-existing subcutaneous tumors can be suppressed by DC/Ad vaccination, mice bearing day 3 tumors were treated with a single s.c. injection of 1 × 106 DC/AdNeuTK or DC/AdLacZ. A separate control group of animals was left untreated. As shown in Figure 5, unimmunized mice and mice immunized with DC/AdLacZ had a large tumor burden 20 days following tumor implantation, and those mice had to be killed within 30–40 days because their tumor volumes were above 800 mm3. In contrast, treatment with DC/AdNeuTK resulted in substantially reduced tumor burden until day 40 (P < 0.01, DCAdNeuTK compared with naive and DCAdLacZ) and 30% of treated mice were tumor free at the termination of the experimental period.

Figure 5

Suppression of pre-established subcutaneous tumors by administration of DC/AdNeuTK or DCAdNeuTK/AdmIL-12. FVB/n mice were inoculated s.c. with 7 × 106 NDL tumor cells on day 0. Three days later, animals (eight mice per group) were treated s.c. with a single injection of 1 × 106 DC/AdNeuTK, DC/AdNeuTK/AdmIL-12, or DCAdLacZ, respectively. This experiment was repeated with similar results, P < 0.01 (Student's t test), DCAdNeuTK or DCAdNeuTK/AdmIL-12 compared with DCAdLacZ and naive.

Since IL-12 has been shown to be critical in DC function both as a paracrine factor to enhance CTL and Th1 cell activation and as an autocrine factor for DC maturation,3233 we evaluated whether cotransduction of DCs with AdmIL-12 and AdNeuTK could further enhance antitumor immunity in our therapeutic model. To determine whether cotransduction would suppress gene expression from one of the vectors, DCs were transduced with either AdLacZ, AdmIL-12 or both and gene expression was evaluated 48 hours later. DCs transduced with AdmIL-12 alone at a MOI of 100 or cotransduced with AdmIL-12 and AdLacZ, at a MOI of 100 each, produced 160–170 ng/106 cells, while IL-12 levels from DCs transduced with AdLacZ alone were less than 0.07 ng/106 cells (Table 1). We also examined the expression of LacZ in DCs transduced with either AdLacZ alone or AdLacZ in combination with AdmIL-12 by X-gal staining. In both groups, more than 90% of DCs expressed LacZ 48 h after infection at a MOI of 100 (Table 1). These data demonstrate the flexibility of the DC/Ad vaccine in that multiple vectors can be used to transfer both tumor antigen and adjuvant cytokines.

Table 1 IL-12 production in supernatants from transduced DCs and β-gal staining

In the therapeutic model, no tumors were apparent in mice treated with DCs co-transduced with AdNeuTK and AdmIL-12 at day 40 after tumor cell inoculation (Figure 5) and more than 66% of treated mice remained tumor free for the duration of the experiment (Table 2), demonstrating an adjuvant effect of IL-12 in DC cancer vaccination (P < 0.05, DCAdNeuTK/AdmIL-12 versus DCAdNeuTK). These results were a combined effect of both ErbB-2/neu and IL-12 expression in the DC inoculum because DCs transduced with AdmIL-12 alone had no inhibitory effect on tumor growth (Table 2). Thus, in this model, the ability to combine multiple Ad vectors in a single DC inoculum can confer increased therapeutic properties not offered by the basic DC/Ad vaccine expressing tumor antigen alone.

Table 2 Long-term survival following treatment with DC/AdNeuTK vaccines


In this study we have evaluated a novel vaccination strategy for breast cancer using genetically modified DCs. The results show that immunization of FVB/n mice with DCs transduced with an Ad vector encoding modified ErbB-2/neu (DC/AdNeuTK) can elicit antigen-specific CTL responses and protect mice against a challenge with tumor overexpressing ErbB-2/neu. More importantly, the DC/AdNeuTK vaccine could successfully cure mice of pre-established tumors and the therapeutic activity could be further enhanced by cotransducing DCs with AdNeuTK and AdmIL-12, demonstrating the flexibility of the DC/Ad system.

Clinical studies involving immunization with peptides derived from the ErbB-2/neu protein could activate antigen-specific CD4+ and CD8+ T cells in breast cancer patients, yet generation of a cellular response against ErbB-2/neu peptide has not correlated with tumor rejection.910 Refinement of the peptide vaccines is hampered by the lack of defined tumor-rejection peptides from ErbB-2/neu, so alternative strategies are needed. DCs are the most effective APC and genetic modification of DCs with tumor antigen-encoding genes should result in multiple epitope presentation in the context of MHC class I and/or class II molecules; therefore this approach bypasses the need for peptide identification. We have chosen to use Ad vectors for DC gene transfer because these viruses can transduce DCs with high efficiency and easily allow the introduction of multiple vectors into the same DC population. In addition, we have previously demonstrated that the DC/Ad vaccine could overcome the Ad-associated hepatic toxicity and was efficacious in mice with pre-existing anti-Ad immunity, suggesting the potential of the DC/Ad system as a clinical vaccination regimen.2134

To limit the possibility of deleterious consequences, the transforming activity of the ErbB-2/neu molecule used in our vaccine has been inactivated by a single amino acid substitution (lysine to alanine), unlike other reports where the entire intracellular domain was removed.35 By virtually retaining the entire ErbB-2/neu molecule, we have maximized the potential repertoire of ErbB-2/neu peptides in the DC/Ad vaccine. Chen et al12 showed that plasmid DNA encoding a truncated rat ErbB-2/neu that lacked the intracellular domain could induce protective immunity against ErbB-2/neu-expressing mammary tumors as effectively as plasmid encoding the full-length ErbB-2/neu oncogene, arguing that the intracellular domain offered no advantage. Evidence from clinical trials, however, demonstrated that CD4+ T cells recognizing peptides from both extracellular and intracellular domains of human ErbB-2/neu protein can be induced by peptide vaccination and it has been suggested that the intracellular domain may represent a better immune target given its limited exposure to the external environment.9 Conceptually, vaccination with the full-length DNA through ex vivo targeting of the DCs should have the advantage of presenting the complete repertoire of ErbB-2/neu epitopes in association with MHC class I and class II molecules, maximizing the number of peptide epitopes available for T cell recognition. T cell reactivity against a broad range of target peptides should also reduce the incidence of immune escape by antigen-loss variants.

The DC/Ad vaccine described in this article was highly effective in inducing CTL activity and antitumor immune response against ErbB-2/neu-expressing tumors in a CD4-dependent manner. We observed the complete loss of tumor protection when CD4+ or CD8+ T cells were depleted during the immune priming phase (days 1 to 14 following vaccination) before tumor challenge, suggesting that both CD4+ and CD8+ T cells are required for the induction of antitumor immune response. When mice were treated with anti-CD4 or anti-CD8 mAb immediately before tumor challenge, only the depletion of CD8 cells resulted in abolition of protective immunity. These in vivo results strongly support recent arguments emphasizing a critical role for CD4+ T cells in the induction of CD8+ CTL.36 Additionally, our in vivo studies concur with our in vitro CTL analysis where we observed cytotoxic activity in spleens from DC/AdNeuTK-immunized mice that was mediated by CD8+ T cells through the classic perforin-dependent pathway. Our results provide evidence for the first time that genetic immunization with modified ErbB-2/neu oncogene can activate both CD4+ and CD8+ T cells which directly correlate with protective antitumor immunity.

The efficacy of the DC/AdNeuTK vaccine was also evaluated in a therapeutic setting using mice with pre-established NDL tumors. A single treatment with DC/AdNeuTK significantly suppressed tumor growth and resulted in long-term survival in 30% of the mice. This therapeutic effect was further improved by cotransduction of DCs with AdNeuTK and AdmIL-12, leading to a delay in tumor growth in 100% of treated mice and 66% of these animals remained tumor free for more than 3 months. Our results agree with the observations that transfection with the IL-12 gene dramatically enhances the efficacy of DC-based antitumor and anti-bacterial vaccines in murine models.3738 High levels of IL-12 production can be achieved through transduction of the DCs with Ad vector encoding IL-12 gene and it is expected that paracrine secretion of IL-12 at the site of antigen presentation would induce a more potent immune response. Moreover, enhanced antitumor immunity by cotransduced DCs may be attributed to IL-12-induced DC maturation since up-regulation of B7–2 and CD40 was consistently observed in our cultured DCs following AdmIL-12 transduction (data not shown). This incorporated DC vaccination approach may be particularly useful in the scenario where a given tumor antigen is subject to self tolerance. As such, we expect that it will be more difficult to induce protective antitumor immunity in rat ErbB-2-transgenic mice than our current transplant tumor model, even using the DC/Ad vaccine to present ErbB-2 antigens, because tolerogenic response to rat ErbB-2 is relatively stringent in transgenic background. Whether cotransduction of DCs with AdNeuTK and AdmIL-12 can break self tolerance in rat ErbB-2-transgenic mice to achieve prevention of spontaneous tumor development is under testing in our laboratory.

In conclusion, these studies support the use of the DC-based vaccine as a therapeutic strategy to target both CD4+ and CD8+ T cells and emphasize the flexibility of this system for optimizing an immune response by combining tumor antigen presentation with immunostimulatory cytokines. The high efficiency of Ad-mediated gene transfer and ease of Ad construction/production make this a practical and promising approach for the development of novel immunotherapeutic strategies for human tumors overexpressing ErbB-2/neu.

Materials and methods


Female FVB/n (H-2q) mice, 6 to 8 weeks old, were purchased from the Charles River Laboratories (Wilmington, DE, USA) and housed under standard conditions specified by the Canadian Council on Animal Care at McMaster University.

Cell lines

PTO516 is a kidney fibroblast cell line derived from a normal FVB/n mouse. The NDL cell line was generated from a tumor that developed on a transgenic mouse harboring a mutated rat ErbB-2/neu transgene under the control of the MMTV promoter.39 These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat inactivated fetal bovine serum (FBS). All cell culture reagents were purchased from GIBCO (Grand Island, NY, USA).

Adenoviral vectors

The full-length cDNA for rat ErbB-2/neu was used to construct the Ad vector. For safety, a point mutation was introduced in codon 758 converting a lysine to an alanine residue at the putative ATP binding site. Previous studies demonstrated that this single amino acid replacement inactivated the kinase activity of ErbB-2/neu.35 The kinase-inactive open reading frame for rat ErbB-2/neu (NeuTK) was isolated from the plasmid pJ4 Neu N and subcloned into the Ad5 shuttle vector, pMH5. The shuttle plasmid was then cotransfected with the rescue vector pBHG10 into 293 cells to generate the AdNeuTK vector. The construction of AdmIL-12 and AdLacZ has been previously described.4041

Flow cytometric analysis

To measure ErbB-2/neu expression, PTO516 cells and DCs were infected with AdNeuTK at a multiplicity of infection (MOI) of 100 (AdLacZ was used as a control) for 24 h. Cells were washed in PBS and incubated with anti-ErbB-2/neu mAb Ab-4 (Oncogene Research Products, Boston, MA, USA) at 4°C for 40 min. These cells were washed twice, then incubated at 4°C for another 40 min with fluorescein-conjugated goat anti-mouse IgG Ab. The fluorescence intensity was measured using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Preparation of bone marrow DCs and infection with Ad vectors

Bone marrow DCs were generated as previously described.21 Briefly, erythrocyte-depleted bone marrow cells were collected and cultured in complete medium (CM) (RPMI 1640 containing 10% FBS, 50 μM 2-ME, 2 mM glutamine, 0.1 mM nonessential amino acids, and 100 μg/ml penicillin and 100 U/ml streptomycin), supplemented with 10 ng/ml murine GM-CSF and 10 ng/ml murine interleukin-4 (Schering-Plough Research Institute, NJ, USA). On day 2 of culture, non-adherent cells were gently removed, and fresh medium was added and maintained for an additional 3 days. At 5 days of culture, non-adherent cells and loosely adherent proliferating aggregates were collected for analysis and immunizations. A panel of selected phenotypic markers including MHC class II, B7–2, CD40, and CD11c, was used to characterize cultured DCs by flow cytometry (data not shown).

Transduction of DCs with Ad vectors was performed on day 4 of culture. DCs were infected with AdNeuTK (DC/AdNeuTK), AdmIL-12 (DC/AdmIL-12) or AdLacZ (DC/AdLacZ) at a MOI of 100 and cultured for 24 h. In some experiments, DCs were infected with AdNeuTK in combination with AdmIL-12, or AdmIL-12 in combination with AdLacZ. Ad-transduced DCs were purified over metrizamide (>90% purity) and washed three times with PBS.

DC/Ad vaccination and in vivo depletion of T cell subsets

FVB/n mice were immunized with 1 × 106 AdNeuTK-infected DCs in 200 μl PBS injected subcutaneously (s.c.) in the hind flank. Control animals received either PBS or DCAdLacZ. Mice were challenged s.c. with 7 × 106 NDL cells in 200 μl PBS at day 14 after immunization in the opposite hind flank. Tumor size was estimated by determining the longest diameter and average width and calculating the volume assuming a prolate spheroid.

Immunodepletion of CD4+ T cells (hybridoma GK 1.5; American Type Culture Collection (ATCC), Rockville, MD, USA) or CD8+ T cells (hybridoma 53.6.72; ATCC) was performed by intraperitoneal injection of 100 μl of ascites diluted in a volume of 500 μl of phosphate-buffered saline (PBS). Two antibody depletion protocols were assessed. The first protocol (priming phase depletion) was initiated 2 days before immunization and then every 3rd day until day 14 after immunization, at which point animals were challenged with 7 × 106 NDL cells by s.c. injection in the left hind flank. In the second protocol, antibody treatment was initiated 2 days before tumor challenge then every 3rd day until most control animals (immunized with PBS and challenged with NDL cells) developed palpable tumors. The efficiency of specific antibody depletion of CD4+ and CD8+ T cells in spleens was more than 90% by flow cytometry (data not shown).

Suppression of pre-established subcutaneous tumors by DC/Ad vaccination

FVB/n mice were inoculated s.c. with 7 × 106 NDL cells 3 days before the initiation of immune therapy. Animals were either left untreated, or treated with s.c injection of DC/AdNeuTK or DC/AdLacZ. To determine whether the cotransduction of DCs with AdNeuTK and AdmIL-12 can enhance antitumor immunity, one group of tumor-bearing mice were treated with two vector-transduced DCs and DCs transduced with AdmIL-12 alone was also included as a control. The tumor size was measured weekly and mice were killed when tumors became ulcerated or when tumor volume reached 800–1000 mm3.

Cytotoxicity assays

Splenocytes were isolated from FVB/n mice 14 days after immunization with the DC/Ad vaccine and restimulated in vitro with irradiated NDL cells (5000 rad) at a responder to stimulator ratio of 50:1. Five days later, effector cells were harvested and mixed with 51Cr-labeled target cells at various E:T ratios. NDL and PTO516 transduced with AdNeuTK were used as target cells for ErbB-2/neu-specific CTL assays and PTO516 cells alone were used as a non-ErbB-2/neu expressing control. The mixtures were incubated in 96-well plates at 37°C for 4 h. In inhibition experiments, a 10 μl aliquot of purified anti-CD3 mAb (PharMingen, Ontario, Canada) or ascites containing mAb specific for CD4+ (GK1.5; ATCC) or CD8+ T (53.6.72; ATCC) cells was added to the wells. Effector cells were also preincubated with 100 nM of concanamycin A (CMA; Sigma) or 5 nM of emetin (Sigma, St Louis, MO, USA) for 2 h selectively to prevent perforin (CMA)- or Fas (emetin)-mediated cytotoxicity.2627 The percentage of specific 51Cr release was evaluated as (c.p.m. experimental – c.p.m. background/c.p.m. maximum – c.p.m. background) × 100%.

Cytokine analysis and X-gal staining

Supernatants were collected from DC cultures 48 h after infection with AdmIL-12, AdLacZ, or AdmIL-12 plus AdLacZ. IL-12 production was quantified using ELISA kits from R&D Systems (Minneapolis, MN, USA). Cells harvested at the same time were stained with X-gal as previously described.21 Bright blue cells in each sample were counted and expressed as a percentage of the total cells.


Differences between groups were evaluated by Student's t test or Fisher's exact test. Values of P were considered significant at 0.05.


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We thank Duncan Chong, Xueya Feng and Chunyan Li for their expert technical assistance, and Dr Jonathan Bramson for critical reading of the manuscript. This work was supported in part by funds from the Breast Cancer Society of Canada, the Medical Research Council of Canada (MRC), the Hamilton Health Science Corporation and the St Joseph's Hospital.

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Correspondence to Y Wan.

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  • dendritic cell
  • ErbB-2/neu
  • adenovirus
  • IL-12
  • breast cancer
  • immunotherapy

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