Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor

Abstract

The present study uses an in vivo murine tumor model expressing the human HER-2/neu antigen to evaluate the potential vaccine using dendritic cells (DCs) infected with adenovirus AdVHER-2. We first investigated whether infected DCs (DCHER-2) engineered to express HER-2/neu could induce HER-2/neu-specific immune responses. Our data showed that (i) AdVHER2-infected DCHER-2 expressed HER-2/neu by Western blot and flow cytometric analysis, and (ii) vaccination of mice with DCHER-2 induced HER-2/neu-specific cytotoxic T-lymphocyte (CTL) responses, but protected only 25% of vaccinated mice from challenge of 3×105 MCA26/HER-2 tumor cells. Further, to enhance the efficacy of DCHER-2 vaccine, we coinfected DCs with both AdVHER-2 and AdVTNF-α. The infected DCs (DCHER-2/TNF-α) displayed the expression of both HER-2/neu and TNF-α by flow cytometric and ELISA analysis. We next investigated whether DCHER-2/TNF-α could induce stronger HER-2/neu-specific immune responses. We found that DCHER-2/TNF-α displayed up-regulation of immunologically important CD40, CD86, and ICAM-I molecules compared with DCHER-2, indicating that the former ones are more mature forms of DCs. Vaccination of DCHER-2/TNF-α induced stronger allogeneic T-cell proliferation and 36% enhanced HER-2/neu-specific T-cell responses in vitro than DCHER-2 cells. More importantly, it stimulated the significant anti–HER-2/neu immunity in vivo, which protected 8/8 mice from challenge of 3×105 MCA26/HER-2 tumor cells. Therefore, DCs genetically engineered to express both the tumor antigen and cytokines such as TNF-α as an immunoadjuvant are likely to represent a new direction in DC vaccine of cancer.

Main

HER-2/neu is a proto-oncogene encoding a 185-kDa protein with homology to the epidermal growth factor receptor (EGFR) family.1 This transmembrane protein consists of a cystein-rich extracellular domain (ECD) that functions in ligand binding and an intracellular domain (ICD) with kinase activity. It is amplified and overexpressed in several human cancers including breast, ovarian, and gastric carcinomas.2,3 Patients with HER-2/neu-positive tumors are associated with a poor prognosis.4,5 Studies of patients with HER2/neu-expressing tumors have demonstrated the existence of humoral and T-cell responses against HER-2/neu6,7 though insufficient to prevent tumor outgrowth. In addition, HER-2/neu-specific T-cell responses can also be generated in patients8,9 and in animal models10 by immunization with HER-2/neu-derived peptides. Taken together, these studies provide the support for HER-2/neu as a candidate for tumor vaccines.

The current approved HER-2/neu immunotherapy is passive transfer of a humanized HER-2/neu monoclonal antibody (Herceptin), which showed inhibition of tumor growth in only limited population of HER-2/neu patients.11 Although clinical studies using HER-2/neu peptide vaccine induced HER-2/neu-specific CD4+ and CD8+ T cells in patients with breast cancer, it could not result in any tumor rejection.8,9 Therefore, a strategic goal of current HER-2/neu-specific immunotherapy has become the induction of stronger tumor-specific cytotoxic T-lymphocyte (CTL) responses.

Genetic DNA vaccines using plasmid DNA expressing HER-2/neu have been reported to induce both HER-2/neu-specific humoral and T-cell immune responses, which partially protect mice from rechallenge of HER-2/neu-positive tumor cells, and reduce tumor development in rat HER-2/neu transgenic mice.12,13,14 DNA vaccines require host antigen-presenting cells (APCs) to present HER-2/neu antigen for priming T-cell responses. However, The host APCs in patients and tumor-bearing mice are often functionally impaired.15,16,17 As a circumvention strategy aimed at this problem, investigators have begun to employ in vitro cytokine-stimulated bone marrow (BM)-derived dendritic cells (DCs), which are fully functional in priming T-cell responses.18,19

DCs are one of the most potent APCs. Because of the critical roles of DCs in the generation of primary immune responses,20 an important avenue of investigation is their potential for modulating immunologic functions such as the induction of tumor immunity. It has been reported that DCs pulsed with MHC class I–restricted tumor peptides were able to induce CTL-dependent antitumor immune responses in vitro and in vivo.21,22 It has also been reported that DCs engineered to express tumor antigens by adenovirus-mediated gene transfer can elicit tumor-specific CTL responses.23,24 Furthermore, Lapointe et al25 have demonstrated that these engineered DCs can generate T cells recognizing multiple MHC class I and class II tumor epitopes. Of perhaps more importance to cancer immunotherapy, Labeur et al26 have recently shown that the induction of antitumor immunity by DC vaccines is correlated with the maturation stage(s) of the DCs. In our previous report, we have also shown that DCs infected with adenovirus expressing transgene TNF-α underwent augmented cellular maturation.27 These mature DCs further induced strong T-cell proliferation/activation and efficient antitumor immunity in animal models.

In this study, we constructed a recombinant adenovirus AdVHER-2 expressing HER-2/neu tumor antigen and investigated the anti–HER-2/neu immune responses by using AdVHER-2–infected DCs in animal models. In addition, we further compared the vaccine efficiency by using DCs infected with AdVHER-2 alone and DCs coinfected with two adenoviruses AdVHER-2 and AdVTNF-α encoding two genes, HER-2/neu and TNF-α, respectively.

Materials and methods

Cell lines, antibodies, cytokines, and animals

A mouse colon cancer cell line MCA26 (H-2Kd) and a human breast carcinoma cell line T-47D were obtained from the American Type Culture Collection (ATCC, Rockville, MD), and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS). 293 cell line (adenoviral E1 transformed human embryonic kidney cells) was also obtained from ATCC and maintained in minimum essential medium with Earle's salts (EMEM; Gibco) supplemented with 10% FCS. Monoclonal rat anti-mouse H-2Kd, Iad, FasL, CD3, CD4, CD8, CD11b, CD11c, CD25, CD40, CD80, CD86, and ICAM-1 antibodies, and the mouse anti-human HER-2/neu antibody were all purchased from Pharmingen (San Diego, CA). The FITC-conjugated goat anti-rat and anti-mouse IgG antibodies were purchased from Bio/Can Scientific (Mississauga, Ontario, Canada). Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were purchased from Endogen (Woburn, MA) and R & D Systems (Minneapolos, MN), respectively. Female C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice were obtained from Charles River Laboratories (St. Laurent, Quebec, Canada). All mice were housed in the animal facility of the Sas-katoon Cancer Center, and all animal experiments were carried out according to the guidelines of the Canadian Council for Animal Care.

Transfection of MCA26 cells with the expression vector pcDNA-HER2

Total RNA was extracted from T47-D tumor cells using RNeasy Mini Kit (Qiagen, Mississauga, Canada). A 2.0-kb cDNA fragment coding for the open reading frame of HER-2 molecule (amino acids 1 to 689) was cloned by reverse transcription polymerase chain reaction (RT-PCR) from a cDNA library of T47-D cells using PFU polymerase (Fig 1A). The cloned HER-2/neu cDNA fragment includes the signal peptide (amino acids 1–21) and the full extracellular (amino acids 22–652) and transmembrane (amino acids 653–675) domains, as well as a short, partial intracellular (amino acids 676–690) domain of HER-2/neu molecule. Two PCR primers were used, namely the sense primer (5′-CAGTG AGCAC CATGG AGCTG G-3′) and the antisense primer (5′-CAGTC TCCGC ATCGT GTACT TCC-3′) with Super Script™ preamplification system (Gibco) according to the manufacturer's instructions. The cloned cDNA fragment of HER-2/neu was ligated into the pCR2.1 vector (Invitrogene, Carlsbed, CA) to form the pCR2.1/HER-2. The HER-2/neu gene sequence was then verified by the dideoxy nucleotide sequencing method. The HER-2/neu gene fragment was further cloned into the plasmid pcDNA3.1 (Invitrogen, San Diego, CA) to form the expression vector pcDNA/HER-2. Twenty million MCA26 cells were resuspended in 0.7 mL phosphate-buffered saline (PBS) and mixed with 0.3 mL PBS containing 10 μg pcDNA/HER-2 DNA. Tumor cells were transfected with pcDNA/HER-2 using a Bio-Rad gene pulser (Bio-Rad Lab, Mississauga, Ontario, Canada) with parameters of 300 V and 125 μF capacitance. The transfected cells were selected for growth in medium containing 4 mg/mL G418 (Geneticin; Gibco). The selected clone MCA26/HER-2 was used for analysis of HER-2/neu expression by flow cytometry.

Figure 1
figure1

Expression of HER-2/neu molecule. A: RT-PCR analysis. RNA was obtained from T47-D and 293 cell lines. The first-strand cDNA was synthesized from RNA using reverse transcriptase; PCRs were conducted using primers sets for HER-2/neu and the control “housekeeping gene,” GAPDH. B: Western blot analysis. Protein extracts were obtained from MCA26, MCA26/HER-2, DCHER-2, and DCpLpA and loaded to each well of a polyacrylamide gel under reducing conditions. The transferred nitrocellulose paper strips were incubated with the mouse anti–HER-2/neu antibody followed by the peroxidase-conjugated goat anti-mouse IgG antibody. Detection of HER-2/neu expression was accomplished by using enhanced chemiluminescence reagent.

Recombinant adenovirus AdVHER-2

The cloned cDNA HER-2/neu fragment was ligated into the pLpA plasmid to form the adenoviral vector pLpA-HER-2. The construction of recombinant adenovirus AdVHER-2 from the vectors pLpA-HER-2 and pJM17 was conducted as previously reported.28 The recombinant adenoviruses AdVLacZ expressing the E. coli β-galactosidase (a marker gene) and AdVpLpA (i.e., with no gene insert used as a control adenoviral vector were previously constructed in our laboratory28). The AdVGFP and AdVTNF-α expressing the green fluorescent protein (GFP) and the mouse TNF-α was obtained from Dr Z Xing, McMaster University, Ontario, and Dr S Tikoo, University of Saskatchewan, Saskatchewan, Canada, respectively. All these E1-deleted replication-deficient recombinant adenoviruses under the control of the cytomegalovirus (CMV) early/immediate promoter/enhancer were amplified in 293 cell line, purified by cesium chloride ultracentrifugation and stored at −80°C.

Preparation of BM-derived DCs

The preparation of BM-derived DCs was previously described.27 Briefly, BM cells prepared from femora and tibiae of normal BABL/c mice were depleted of red blood cells with 0.84% ammonium chloride and plated in DC culture medium [DMEM plus 10% FCS, GM-CSF (20 ng/mL) and IL-4 (20 ng/mL)]. On day 3, the nonadherent granulocytes, and T and B cells were gently removed and fresh media were added, and 2 days later the loosely adherent proliferating DC aggregates were dislodged and replated. On day 6, the nonadherent cells were harvested. The DCs generated in this manner displayed: (i) typical morphologic features of DCs (i.e., numerous dendritic processes) and (ii expression of MHC class I (H-2Kb) and II (Iab) antigens, costimulatory molecules (CD40, CD80 and CD86), and adhesion molecules (ICAM-1, CD11b, and CD11c). These DCs were then used for in vitro AdVHER-2 and/or AdVTNF-α infection.

Adenoviral infection of DCs

To test the amenability of DCs to adenoviral infection, serial dilutions of AdVLacZ stock (2×1010 pfu/mL) were added to triplicate cultures of DCs in 96-well plates (1×105 cells/well) to form different multiplicity of infection (MOI). The cells were incubated with the adenovirus in 293 serum-free medium (Gibco) for 2 hours at 37°C, then the medium was replaced with DMEM/10% FCS and the cells incubated for an additional 24 hours at 37°C. To assess β-galactosidase expression,28 the cells were fixed in formaldehyde/glutaraldehyde, then stained and counterstained with X-gal and nuclear fast red, respectively. The proportions of positive (i.e., blue staining) cells were determined from triplicate wells and taken as the percentage of transduction. Control DCs infected with AdVpLpA (DCpLpA) did not exhibit any intrinsic β-galactosidase activity or false-positive staining. We observed a dose-dependent response to the adenoviral infections, with maximal staining (82%) at an MOI of ≥100. Therefore, an MOI of 100 was selected for infection of DCs with AdVHER-2 and/or AdVTNF-α in this study. DCs infected with AdVHER-2 and AdVHER-2/AdVTNF-α were termed DCHER-2 and DCHER-2/TNF-α, respectively. To enact this, following viral adsorption for 1 h at 37°C in six-well culture plates, the DC culture medium was replaced with DMEM plus 20% FCS. The cells were incubated for an additional 24 hours at 37°C, and then harvested for phenotypic analysis by flow cytometry and for immunization of animals. The culture supernatants of the transfected DCHER-2/TNF-α were assayed for TNF-α by enzyme-linked immunosorbent assay (ELISA) using TNF-α ELISA kit (Endogen, Woburn, MA).

To examine the efficiency of coinfection of DCs with two individual adenoviral vectors, we infected DCs with either AdVGFP or AdVTNF-α, or both of these two viruses at MOI of 100 for each virus. After 24-hour culture as described above, DCs infected with AdVGFP (DCGFP) were subjected to flow cytometric analysis. DCs infected with AdVTNF-α (DCTNF-α) or with both AdVTNF-α and AdVGFP (DCGFP/TNF-α) were fixed and processed using a commercial kit (Cytofix/CytoPerm Plus with GolgiPlug; Pharmingen), and stained with PE-conjugated anti–TNF-α antibody according to the manufacturer's protocols. These transfected DCs were then subjected to flow cytometric analysis.

Flow cytometric analysis

For phenotypic analysis of tumor cells, MCA26 and MCA26/HER-2 cells were stained for 30 minutes on ice with antibodies specific for H-2Kd, Iad, Fas, or HER-2/neu (5 μg/mL each), respectively. For phenotypic analysis of DCs, DCHER-2 and DCHER-2/TNF-α were incubated with antibodies specific for H-2Kd, Iad, CD11b, CD11c, CD40, CD80, CD86, ICAM-1, and HER-2/neu on ice for 30 minutes, respectively. For phenotypic analysis of T cells, naive and MCA26/HER-2–specific T cells as described in the following sections were incubated with antibodies specific for CD3, CD4, CD8, CD25, and FasL on ice for 30 minutes, respectively. These cells were washed three times, then incubated with FITC-conjugated goat anti-rat or mouse IgG antibody (1:60). After three more washes with PBS, these cells were analyzed by flow cytometry. Isotype-matched monoclonal antibodies were used as controls.

Western blot analysis

The expression of HER-2/neu in MCA26/HER-2 and DCHER-2 cells was assessed using Western blot. Briefly, the protein extract was prepared from these cells using the extraction buffer containing 125 mM tris(hydroxymethyl)aminomethane, 0.05% sodium dodecyl sulfate, and 10% β-mercaptoethanol. Cell extracts were harvested from the supernatant after centrifugation at 1000×g for 5 minutes. The supernatants containing the protein samples were electrophoresed through 10% gels and transferred onto nitrocellulose papers; blots were blocked using PBS containing 10% bovine serum albumin (BSA) and incubated with the anti–HER-2/neu antibody followed by the goat anti-mouse IgG antibody conjugated with peroxidase. Detection of HER-2/neu was accomplished by using enhanced chemiluminescence reagent according to the manufacturer's protocol (New England Nuclear Life Science Products, Boston, MA).

Allogeneic mixed lymphocyte reactions

Naive T cells were purified from C57BL/6 mouse splenocytes as nylon wool nonadherent cells.29 The primary mixed lymphocyte reactions MLRs) were performed as previously described.29 Briefly, graded doses of irradiated DCs, DCHER-2, and DCHER-2/TNF-α (3000 rad) were cocultured in 96-well plates with constant numbers (2×105) of allogeneic T cells from C57BL/6 mouse. After 3 days, T-cell proliferation was measured using an overnight [3H]thymidine (1 mCi/mL, Amersham Canada) uptake assay (1 μCi/well). The levels of [3H]thymidine incorporation into the cellular DNA were determined by liquid scintillation counting.

Cytotoxic activity assay

Red blood cell–depleted splenic lymphocytes from mice vaccinated with DCHER-2 and DCHER-2/TNF-α were cocultured in 24-well plates with irradiated MCA26/HER-2 cells (40,000 rad), using 5×106 lymphocytes and 2×105 MCA26/HER-2 cells per 2 mL of DMEM plus 10% FCS. After four days, the activated T cells were harvested by Ficoll-Hypaque gradient centrifugation and used as effector cells in a chromium-release assay against radiolabeled MCA26 and MCA26/HER-2 target cells. The target cells (104/well) were incubated for 8 hours in triplicate cultures with effector cells at various effector/target ratios. The percent specific lysis was calculated using the formula:

The spontaneous count per minute (CPM) release in the absence of effector cells was less than 10% of specific lysis; maximal CPM release was effected by adding 1% Triton X-100 to the cells.29

Vaccination of mice with DCHER-2 and DCHER-2/TNF-α

For evaluation of tumor prevention, mice were vaccinated subcutaneously (s.c.) with 1×106 DCHER-2 and DCHER-2/TNF-α cells twice with a 7-day interval. Ten days subsequent to the second vaccination, the mice (n=8 per group) were challenged by s.c injection of 3×105 MCA26 or MCA26/HER-2 cells. Animal mortality and tumor growth were monitored daily for up to 60 days; for humanitarian reasons, all mice with tumors that achieved a size of 1.5 cm in diameter were sacrificed.

Results

HER-2 expression on MCA26/HER-2 and DCHER-2 cells

The mouse colon cancer cell line MCA26 and DCs were transfected and infected with the expression vector pcDNA-HER-2 and the adenoviral vector AdVHER-2, respectively. The HER-2/neu expression of transfected MCA26/HER-2 and infected DCHER-2 cells was then examined by Western blot and flow cytometry. As shown in Figure 1B and Figure 2, MCA26/HER-2 and DCHER-2 cells displayed significant amount of cellular and cell-surface HER-2/neu expression, whereas the original MCA26 tumor cells and the control adenovirus-infected DCpLpA cells showed no expression of HER-2/neu molecule.

Figure 2
figure2

Flow cytometric analysis of HER-2/neu expression. The expression of HER-2/neu in MCA26, MCA26/HER-2, DCHER-2, and DCpLpA cells (solid lines) was analyzed by flow cytometry using the mouse anti–HER-2/neu and the FITC-conjugated goat anti-mouse IgG antibodies. Isotype-matched monoclonal antibodies (dotted lines) were used as controls.

Coinfection of DCs with AdVHER-2 and AdVTNF-α

To enhance DC maturation, we coinfected DCs with AdVHER-2 and AdVTNF-α to generate genetically modified DCHER-2/TNF-α cells. The expression of the cell surface HER-2/neu on DCHER-2/TNF-α cells was similar to that on DCHER-2 cells (data not shown). To examine TNF-α expression of DCHER-2/TNF-α, we conducted ELISA assays. Our preliminary data showed that the concentrations of TNF-α in the supernatants of DCpLpA and DCHER-2/TNF-α cells were approximate 0.1 and 8 ng/mL, respectively. To further examine the efficiency of cotransfection with two individual adenoviral vectors, we coinfected DCs with AdVGFP and AdVTNF-α simultaneously. As shown in Figure 3, around 90% of DCGFP and DCTNF-α cells expressed GFP and TNF-α, respectively; and nearly 60% of DCTNF-α/GFP cells expressed both TNF-α and GFP, indicating that expression of two transgenes in individual DC by coinfection of two different adenoviral vectors is efficient.

Figure 3
figure3

Flow cytometric analysis of double transgene expression. DCs were infected with either AdVGFP or AdVTNF-α at MOI of 100 (1×107 DCs vs 1×109 pfu of either AdVGFP or AdVTNF-α), or both of these two adenoviruses at MOI of 100 for each adenovirus (1×107 DCs vs 1×109 pfu of AdVGFP and 1×109 pfu of AdVTNF-α). The infected DCs were stained by PE-conjugated anti–TNF-α antibody as described under Materials and methods. The values of mean fluorescence intensity in (i) upper left, (ii) lower right, and (iii) upper right panels represent the percentage of DCs stained with (i) red (PE), (ii) green (GFP), and (iii) both green (GFP) and red (PE) fluorescences, respectively. They also represent the DCs expressing (i) the transgene TNF-α, (ii) the transgene GFP and (iii) both TNF-α and GFP, respectively.

DCHER-2/TNF-α cells up-regulate expression of CD40, CD86 and ICAM-1

We then analyzed the impact of AdVTNF-α or AdVHER-2 infection on the expression of a number of cell surface markers (MHC class I and II antigens, CD11b, CD11c, CD40, CD80, CD86, and ICAM-1) in DCs. As shown in Figure 4, infection of AdVHER-2 led to a mild up-regulation of CD40, CD86, and ICAM-1 expression on DCHER-2 cells. However, the DCHER-2/TNF-α cells that expressed the TNF-α gene in trans were more markedly affected, with 2.3- to 3.2-fold greater expression of CD40, CD86, and ICAM-1 being evident in these cells, relative to the untreated DCs. The expression of MHC class I and II antigens, CD11b, CD11c, and CD80 remained unchanged on these DCs (data not shown).

Figure 4
figure4

Comparison of the phenotypic changes by flow cytometry. The expression of CD40, CD86 and ICAM-1 in DCHER-2, DCHER-2/TNF-α and uninfected DCs (solid lines) was analyzed by flow cytometry using the rat anti-CD40, CD86 and ICAM-1 antibodies and the FITC-labeled goat and rat antibody. The values of mean fluorescence intensity are shown in the right upper corners. Isotype-matched monoclonal antibodies (dotted lines) were used as controls. One representative experiment of two is shown.

DCHER-2/TNF-α cells induce enhanced T-cell proliferation in vitro

DCs are potent stimulators of primary mixed lymphocyte reaction (MLR) and are able to induce the proliferation of allogeneic CD8+ T cells in vitro.30 Just as stimulation of antigen-specific T-cell responses by DCs is strongly af-fected by their maturational status,26 so too is stimulation of primary MLRs by these cells. Thus, we next compared the abilities of our DC populations to stimulate primary MLRs against allogeneic CD8+ T cells. We found that DCHER-2/TNF-α, which had been coinfected with AdVHER2/neu and AdVTNF-A, induced stronger allogeneic T-cell proliferative responses in vitro than DCHER-2 (P<.05) and DCs (P<.01) (Fig 5).

Figure 5
figure5

DC expressing a TNF-α transgene induce stronger mixed lymphocyte reactions. Irradiated DCHER-2/TNF-α (black triangles), DCHER-2 (black circles), or DCs (open circles), 1×104 cells/well and reciprocal dilutions thereof, were cocultured for 3 days with 1×105 allogeneic C57BL/6 T cells. The overnight [3H]thymidine uptake seen on day 4 is expressed as the mean of three determinations. Background proliferation of DCs or T cells alone was always below 4000 cpm. One experiment of two is shown.

DCHER-2/TNF-α significantly enhance tumor-specific T-cell responses in vitro

Next, we addressed the specific antitumor effector functions induced by vaccination of the mice with DCHER-2/TNF-α. Spleen lymphocytes from immunized mice were cocultivated with irradiated MCA26/HER-2 tumor cells for 4 days and harvested for phenotypic analysis by flow cytometry. As shown in Figure 6, these T lymphocytes comprising both CD4+ and CD8+ T cells displayed high-level expression of FasL and CD25 (IL-2R), indicating that they are activated T cells. To assess the cytotoxic activity, we conducted a chromium-release assay by using these activated T cells as effector cells and the 51Cr-labeled MCA26 and MCA26/HER-2 tumor cells as target cells. As shown in Figure 7, activated T cells from mice vaccinated with DCHER-2/TNF-α showed significant killing activity to MCA26/HER-2 cells. At an effector:target (E:T) ratio of 50, the specific killing activity for activated T cells from mice with vaccination of DCHER-2/TNF-α and DCHER-2 was 53% and 39%, respectively. T cells from mice vaccinated with DCHER-2/TNF-α cells displayed substantially (36%) enhanced CTL activity relative to analogous cells from animals vaccinated with the DCHER-2. This CTL activity was immunologically specific, in as much as none of these populations showed cytotoxic activities against the HER-2/neu-negative MCA26 tumor cells, and T cells from the mice vaccinated with uninfected DCs had only little activity against the MCA26/HER-2.

Figure 6
figure6

Phenotypic analysis of activated T cells from DCHER-2/TNF-α-vaccinated mice. Naive T cells and activated T cells from DCHER-2/TNF-α-vaccinated mice were analyzed by flow cytometry using the rat anti-CD3, CD4, CD8, CD25, and FasL antibodies and the FITC-labeled goat anti-rat antibody (solid lines). Isotype-matched monoclonal antibodies (dotted lines) were used as controls. One representative experiment of two is shown.

Figure 7
figure7

DC expressing a TNF-α transgene induce more robust tumor-specific cytotoxic T-cell responses. Splenic lymphocytes were harvested from mice that had been vaccinated with DCHER-2/TNF-α (open circles), DCHER-2 (black circles), or uninfected DCs (open triangles) were cocultured with irradiated MCA26/HER-2 cells (40,000 rad) for 4 days, then used as effector cells in a chromium-release assay with 51Cr-labeled MCA26/HER-2 tumor cells. To confirm that T-cell cytotoxicity was HER-2/neu tumor specific, we also included MCA26 cells (black triangles) as a target control and activated T cells from DCHER-2/TNF-immunized mice as effector cells. Each point represents the mean of triplicates. The experiment was repeated once with consistent results.

DCHER-2/TNF-α strongly induce protective tumor-specific immunity in vivo

To examine whether DCHER-2/TNF-α cells were also capable of inducing enhanced antitumor immunity in vivo, we vaccinated mice twice with DCHER-2 and DCHER-2/TNF-α, and 10 days later challenged the animals with 3×105 MCA26/HER-2 tumor cells. As shown in Figure 8, MCA26/HER-2 tumor cell challenges were invariably lethal within 6 weeks postimplantation for the PBS vaccination control mice. Vaccination with DCHER-2 can only protect 25% (2/8) of the mice from tumor growth after challenge of MCA26/HER-2. However, vaccination of DCHER-2/TNF-α showed enhanced antitumor immunity, with 8/8 mice protected from challenge of 3×105 MCA26/HER-2 tumor cells. More importantly, vaccination with DCHER-2/TNF-α can only protect mice from challenge of HER-2/neu-positive MCA26/HER-2, but not HER-2/neu-negative MCA26 tumor cells, because 8/8 immunized mice died within 6 weeks post implantation of MCA26 tumor cells. These data further confirm that the antitumor immunity of mice vaccinated with DCHER-2/TNF-α is HER-2/neu tumor specific.

Figure 8
figure8

Vaccination of mice with DCHER-2/TNF-α induces stronger antitumor immunity than vaccination with DCHER-2. BALB/c mice (n=8) were vaccinated twice with DCHER-2 and DCHER-2/TNF-α, then s.c. challenged, 10 days subsequent to the second vaccination, with 3×105 MCA26/HER-2 or MCA26 tumor cells. Control mice were treated with PBS and challenged with 3×105 MCA26/HER-2 tumor cells. The survival time of each mouse was monitored daily. The animal experiment was repeated once with similar results.

Discussion

In this study, we constructed a recombinant adenovirus AdVHER-2 expressing the human HER-2/neu antigen. To limit the possibility of deleterious consequences, the part of ICD with the most transforming activity of HER-2/neu molecule was deleted.31 We then infected DCs with this adenoviral vector and investigated the anti-HER2/neu immune responses by DCHER-2 vaccination in an animal model using a murine tumor cell line MCA26/HER-2 engineered to express HER-2/neu antigen. We chose to use Ad vectors for gene transfer into DCs because these viruses can infect DCs with high efficiency and easily allow introduction of multiple adenoviral vectors into a single cell. Our data showed that vaccination of mice with AdVHER-2–transfected DCHER-2 induced (i) HER-2/neu-specific CTL response with tumor-specific killing of 39% at E:T ratio of 50, and (ii) HER-2/neu-specific antitumor immunity capable of protecting 25% mice from challenge of 3×105 MCA26/HER-2 tumor cells.

As a further strategy to enhance the efficacy of DC vaccine, DCs have been genetically engineered to express cytokines such as IL-12 and GM-CSF. In both mouse and human studies, IL-12 expression by engineered DCs can augment priming to antigens delivered in a variety of ways to the cells, such that these DCs were powerful catalysts for the induction of tumor-specific CD4+ Th cells and CD8+ CTLs.32,33 Engineering DCs expressing GM-CSF also reportedly increase therapeutic antitumor immunity in vivo.34 Recently, it has been further reported that the antitumor efficacy of DC vaccination using DCs engineered to express human tumor antigens can be greatly enhanced by cotransduction of DCs with the above cytokine genes as immunoadjuvants.35,36

The maturation processes of DCs are efficiently regulated, such that these cells can achieve different states of activation/maturation and thereby different functional properties,26,37 depending on the precise nature of the signals they receive from their microenvironment. A number of cytokines can significantly affect DC function at various levels, including their viability, morphology, migration, expression of MHC and accessory molecules, and binding and processing of antigen peptides.38 For example, TNF-α is known to have substantial effects on DC maturation with a strong T-cell stimulatory potential.39,40 The maturation of DCs is correlated with the induction of antitumor immunity by DC vaccines.26 More recently, we have demonstrated that DCs infected with adenovirus expressing transgene TNF-α underwent augmented cellular maturation.27 These mature DCs further induced strong CD8+ CTL cytotoxicity in vitro and substantially more effective antitumor immunity in vivo in animal models.

To enhance the efficacy of DCHER-2 vaccination, we further constructed DCHER-2/TNF-α expressing both HER-2/neu and TNF-α by coinfection of DCs with AdVHER-2 and AdVTNF-α. We found that DCHER-2/TNF-α cells displayed a significant amount of cell surface HER-2/neu molecule and secreted a significant amount of TNF-α (8 ng/mL) in the supernatant of infected DC cultures. These DCHER-2/TNF-α cells also displayed up-regulation of immunologically important CD40, CD86 and ICAM-I molecules compared with DCHER-2 cells, indicating that the former ones are more mature forms of DCs. Vaccination of DCHER-2/TNF-α induced stronger allogeneic T-cell proliferation and 36% enhanced HER-2/neu-specific CTL responses in vitro than DCHER-2 cells. More importantly, it could stimulate the significant anti–HER-2/neu immunity in vivo, which protected 8/8 mice from challenge of 3×105 MCA26/HER-2 tumor cells, but not HER-2/neu-negative MCA26 tumor cells.

It was originally reported that plasmid DNA encoding a truncated rat HER-2/neu lacking the ICD could induce antitumor immunity that was as strong as the one induced by the plasmid encoding the full-length HER-2/neu.12 This indicates no advantage of ICD in induction of protective immunity. Evidence from clinical trials, however, demonstrated that CD4+ T cells derived from peptide vaccine recognized peptide epitopes from both ECD and ICD of HER-2/neu,8,41 suggesting ICD may represent a better immune target given its limited exposure to the external environment. It has also been reported that only vaccination with DNA encoding ICD or ICD protein of HER-2/neu can elicit antitumor immunity, but not with DNA encoding ECD or ECD protein.14 Conceptually, vaccination with DNA encoding the full length of HER-2/neu or full-length HER-2/neu protein should have the advantage of presenting both MHC class I and class II epitopes and, therefore, induced enhanced anti–HER-2/neu immune responses. Therefore, we assume that vaccination of DCs infected with new adenovirus expressing the full-length HER-2/neu protein with a single amino acid substitution (lysine to alanine),42 which can inactivate the transforming activity of ICD, may further enhance the efficiency of DC vaccine strategy as described in this study.

Taken together, we demonstrated that CTL-mediated anti–HER-2/neu immunity could be induced in animal model by vaccination of DCs infected with adenovirus AdVHER-2 expressing the human HER-2/neu antigen. More importantly, we further demonstrated that vaccination of DCHER-2/TNF-α cells coinfected with adenoviruses AdVHER-2 and AdVTNF-α could significantly enhance HER-2/neu-specific CTL responses in vitro and antitumor immunity in vivo. Therefore, DCs genetically engineered to express both the tumor antigen and cytokines such as TNF-α as an immunoadjuvant may represent a new direction in DC vaccine of cancer.

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Acknowledgements

This study was supported by a research grant (3-782) from Saskatchewan Cancer Agency.

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Correspondence to Jim Xiang.

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Chen, Z., Huang, H., Chang, T. et al. Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor. Cancer Gene Ther 9, 778–786 (2002). https://doi.org/10.1038/sj.cgt.7700498

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Keywords

  • engineered dendritic cell vaccine
  • recombinant adenovirus
  • HER-2
  • alpha necrosis factor
  • antitumor immunity

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