Dendritic cells (DCs) are professional Ag-presenting cells that are being considered as potential immunotherapeutic agents to promote host immune responses against tumor Ags. In this study, recombinant adenovirus (Ad) vectors encoding melanoma-associated Ags were used to transduce murine DCs, which were then tested for their ability to activate CTL and induce protective immunity against B16 melanoma tumor cells. Immunization of C57BL/6 mice with DCs transduced with Ad vector encoding the hugp100 melanoma Ag (Ad2/hugp100) elicited the development of gp100-specific CTLs capable of lysing syngeneic fibroblasts transduced with Ad2/hugp100, as well as B16 cells expressing endogenous murine gp100. The induction of gp100-specific CTLs was associated with long term protection against lethal s.c. challenge with B16 cells. It was also possible to induce effective immunity against a murine melanoma self Ag, tyrosinase-related protein-2, using DCs transduced with Ad vector encoding the Ag. The level of antitumor protection achieved was dependent on the dose of DCs and required CD4+ T cell activity. Importantly, immunization with Ad vector-transduced DCs was not impaired in mice that had been preimmunized against Ad to mimic the immune status of the general human population. Finally, DC-based immunization also afforded partial protection against established B16 tumor cells, and the inhibition of tumor growth was improved by simultaneous immunization against two melanoma-associated Ags as opposed to either one alone. Taken together, these results support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding tumor-associated Ags.

The identification of tumor-associated Ags (TAAs)2 recognized by CTL, as well as the cloning of genes encoding TAAs, has improved the prospect for cancer immunotherapy (Refs. 1, 2, 3, 4 and reviewed in Ref. 5). On the basis of this knowledge, several investigators have focused on the delivery of TAA-derived proteins/peptides or TAA genes to professional APCs, in particular dendritic cells (DCs), to elicit immune responses capable of eradicating tumor cells.

DCs are potent Ag presenters that express high levels of costimulatory molecules and are capable of activating both CD4+ and CD8+ naive T lymphocytes (6, 7, 8). Results obtained in several animal models have shown that DCs pulsed with defined tumor-associated peptides or with peptides eluted from the surface of tumor cells are capable of inducing an Ag-specific CTL response resulting in protection from tumor challenge and, in some instances, regression of established tumors (9, 10, 11, 12). The same type of approach has also been tested in human clinical trials with encouraging results. For example, Hsu et al. have reported that four B cell lymphoma patients infused with autologous DCs pulsed with tumor-specific Id protein all developed an Id-specific proliferative response, accompanied by complete tumor regression in two patients and partial regression in a third (13). More recently, Nestle et al. reported that melanoma patients treated with autologous DCs pulsed with tumor lysate or a mixture of CTL peptide epitopes developed cell-mediated immunity with objective clinical responses in 5 of 16 patients evaluated (14).

We have favored a gene-based rather than a peptide/protein-based approach to DC immunization, because transduction of DCs with a TAA-encoding transgene offers several potential advantages over peptide-pulsing. First, expression of an entire TAA gene circumvents the need for the identification of specific CTL epitopes within the protein since it allows for processing and presentation of all natural CTL and, possibly, helper epitopes in the context of the host’s MHC type. In addition, TAA expression within DCs provides the cell with a renewable supply of Ag for presentation, as opposed to a single pulse of peptide(s), which eventually decays from the cell surface. As a result, the Ag-presenting activity of genetically modified DCs shows greater persistence (G. Linetie, S. Shankara, R. Doll, L. Eaton, B. Roberts, and C. Nicolette, manuscript in preparation).

The introduction of genetic material into DCs can be achieved with varying levels of efficacy, using techniques such as electroporation, lipid-mediated transfection, calcium phosphate precipitation, and virally mediated gene transfer (15, 16, 17). Adenoviral vectors were selected in this study since we and others have found adenovirus (Ad) to be a highly efficient and reproducible method of gene transfer into DCs (15, 17, 18, 19, 20).

It has been reported that immunization of mice with DCs transduced with Ad vector encoding a model Ag (e.g., OVA and β-galactosidase (β-gal)) gives rise to a specific CTL response and provides protective and/or therapeutic immunity against tumor cells stably expressing the same Ag (17, 18, 19, 20). In this study, we investigated the ability of Ad vector-transduced DCs to induce protective immunity against endogenous tumor Ags, as opposed to foreign model Ags introduced exogenously.

The B16 melanoma tumor model was used as a test system to evaluate and characterize the immunizing activity of DCs transduced with Ad vectors encoding melanoma-associated Ags (MAAs). The B16 tumor cell line expresses the murine homologues of human MAAs such as gp100, tyrosinase-related protein (TRP)-1, TRP-2, and melanoma Ag recognized by T cells 1 (MART-1) (21, 22, 23). Accordingly, DCs derived from murine bone marrow were transduced with Ad vector encoding the human and/or murine version of two known MAAs, gp100 and TRP-2, and were tested for their ability to induce a CTL response and provide immunity against B16 melanoma tumor cells. The impact of various factors, such as the dose of DCs, nature of the MAA, CD4 activity, and preexisting immunity to Ad, on the development of antitumor immunity was investigated.

Female wt C57BL/6 mice and C57BL/6 CD4 knockout (KO) mice were purchased from Taconic (Germantown, NY) and were used at 8–12 wk of age. Syngeneic SV40-transformed fibroblasts (SVB6KHA) have been described elsewhere (24) and were a gift from Dr. Linda Gooding (Emory University, Atlanta, GA). The YAC-1 NK cell target derived from the A/Sn mouse strain and the C57BL/6-derived EL4 lymphoma cell line were both purchased from the American Type Culture Collection (ATCC, Manassas, VA). The B16.F10 melanoma cell line syngeneic to C57BL/6 mice was obtained from the National Cancer Institute (Bethesda, MD). For injection, B16.F10 cells (1.5–2 × 104 cells) were resuspended in PBS and delivered to the abdomen s.c. in a 100-μl volume. Tumor size was measured with electronic digital calipers three times per week, starting around day 10. Tumors ≥3 mm2 in size were scored as positive.

All recombinant Ad vectors used were derived from Ad serotype 2, from which the E1 region was deleted and replaced with an expression cassette containing a CMV promoter driving expression of the transgene. The vectors encoding β-galactosidase (Ad2/β-gal-4) and hugp100 (Ad2/hugp100v1) contained intact E3 and E4 regions (25, 26). A second version of Ad2/hugp100 (Ad2/hugp100v2), as well as the vectors encoding enhanced green fluorescent protein (Ad2/EGFP) and murine gp100 (Ad2/mgp100) or vector lacking a transgene (Ad2/EV), possessed an intact E3 region with an E4 region modified by removal of all open reading frames and replacement with the E4 open reading frame 6 and protein IX moved from its original location (27). Finally, the Ad vector encoding murine TRP-2 (Ad2/mTRP-2) contained an intact E4 region but was deleted for E3. The E2 region was left intact in all vectors.

Adenoviral particles were gradient purified as previously described (27), and titers were determined by end-point dilution on 293 cells using FITC-conjugated anti-hexon Ab (28).

DCs were prepared from bone marrow essentially as described by Inaba et al. (29). Briefly, bone marrow was flushed from the tibias and femurs of C57BL/6 mice and depleted of erythrocytes with commercial lysis buffer (Sigma, St.Louis, MO). Bone marrow cells were then treated with a mixture of Abs (PharMingen, San Diego, CA) directed against CD8 (clone 53-6.7), CD4 (clone GK1.5), CD45R/B220 (clone RA3-6B2), Ly-6G/Gr-1 (clone RB6-8C5), and Ia (clone KH74), followed by rabbit complement (Accurate Chemical and Scientific, Westbury, NY) to deplete lymphocytes, granulocytes, and Ia+ cells. The remaining cells were cultured for 6 days in six-well plates in RPMI 1640 medium (Life Technologies, Grand Island, NY), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 10% FCS, and 100 ng/ml recombinant mouse GM-CSF (Genzyme, Cambridge, MA). Loosely adherent DCs were then collected, replated in 100-mm dishes, and cultured in the same medium for another 24 h after removal of contaminating nonadherent cells. This final DC population was then collected for FACS analysis and transduction with Ad vector.

For analysis of surface markers, DCs were first incubated with unlabeled Abs (PharMingen) specific for the MHC class I (clone AF6-88.5) and class II (clone AF6-120.1) molecules, the costimulatory molecules B7.1 (CD80; clone IG10) and B7.2 (CD86; clone GL-1), the adhesion molecule ICAM-1 (CD54; clone 3E2), the integrin CD11c (clone 3E2), and the myeloid surface marker CD13 (clone R3-242). The cells were then counterstained with FITC-conjugated Abs specific for the primary Ab. FACS analysis of the stained cells was performed on an EPICS Profile Analyzer from Coulter (Palo Alto, CA).

Transduction of DCs with Ad vector was conducted in six-well plates with 4 × 106 DCs/well in a 3-ml volume of RPMI 1640 medium containing 10% FCS and 100 ng/ml GM-CSF. Virus was added to the wells at a multiplicity of infection (MOI) of 500, and the DCs were collected after 18–24 h of incubation. For injection, transduced DCs were washed and resuspended in a 100-μl volume of PBS and delivered either s.c. to the abdomen or i.v. into the tail vein as specified in the text.

To test for the ability of DCs to induce a MLR, varying numbers of bone marrow-derived C57BL/6 DCs (102, 103, 104) were used to stimulate 2 × 105 allogeneic BALB/c T lymphocytes isolated from spleen cells by passage through a commercial T cell purification column (R&D Systems, Minneapolis, MN). Untransduced DCs, as well as Ad2/EGFP-transduced DCs, were tested for MLR induction. In assays of Ag-specific proliferation, 2 × 105 column-purified (R&D Systems) T lymphocytes from naive C57BL/6 mice were incubated with 104 syngeneic DCs that were either untransduced or transduced with wt Ad2, Ad2/EGFP, Ad2/β-gal-4 or Ad2/mTRP-2. All assay cultures were performed in triplicate in round-bottom 96-well plates in a total volume of 200 μl. The cells were cultured for 5 days at 37°C/ 5% CO2 and pulsed with 1 μCi/well [3H]thymidine (New England Nuclear, Boston, MA) for the last 18 h of incubation. Cells were then harvested onto glass fiber filters with a 96-well plate cell harvester (Skatron Instruments, Sterling, VA), and cell-associated radioactivity was measured by scintillation counting (LS6800 Scintillation Counter from Beckman, Fullerton, CA).

To evaluate levels of CTL activity, spleen cells from mice in the same treatment group (three mice/group) were pooled and stimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2 vector at an MOI of 100 for 24 h. Cells were cultured in 24-well plates containing 5 × 106 spleen cells and 0.8–1.5 × 105 stimulator fibroblasts per well in a 2-ml volume. Cytolytic activity was assayed after 6 days of incubation. Target cells consisted of B16 melanoma cells, EL4 lymphoma cells, YAC cells (NK cell target), and fibroblasts, untransduced or transduced with virus at an MOI of 100 for 48 h. Targets were treated with 100 U/ml recombinant mouse IFN-γ (Genzyme) for 24 h (except for YAC cells), labeled with 51Cr (51-Cr; New England Nuclear) overnight (30 μCi/105 cells) and plated in round-bottom 96-well plates at 5 × 103 cells/well. Effector cells were added at various E:T cell ratios in triplicate. In specified instances, effector cells were incubated with a 50-fold excess of unlabeled “cold” YAC cells for 1 h before the addition of 51Cr-labeled target cells to inhibit nonspecific lysis by NK cells (30). The total reaction volume was kept constant at 200 μl/well. After 5 h of incubation of effector and target cells at 37°C/5% CO2, 25 μl of cell-free supernatant was collected from each well and counted in a MicroBeta Trilux Liquid Scintillation Counter (Wallac, Gaithersburg, MD). The amount of 51Cr spontaneously released was obtained by incubating target cells in medium alone. Spontaneous release from target cells was typically below 20%. The total amount of 51Cr incorporated was determined by adding 1% Triton X-100 in distilled water, and the percentage lysis was calculated as follows: % lysis = [(sample cpm − spontaneous cpm)/(total cpm − spontaneous cpm)] × 100.

The presence of gp100-specific effector cells in immunized mice was also assessed in an enzyme-linked immunospot (ELISPOT) assay (31). Briefly, spleen cells from mice immunized with DCs transduced with Ad2/hugp100 or Ad2/EV were stimulated with either a known MHC class I-restricted CTL peptide epitope from hugp100 (Ref. 32 ; hugp10025–33-KVPRNQDWL), the homologous epitope from mgp100 (mgp10025–33-EGSRNQDWL), or an irrelevant H-2b-binding CTL epitope from OVA (Ref. 17 ; OVA257–264-SIINFEKL). The peptides were synthesized by Quality Controlled Biochemicals (Hopkinton, MA) and were >90% pure by reverse phase HPLC. Peptide-stimulated spleen cells, as well as unstimulated spleen cells, were plated in the wells of 96-well nitrocellulose filter plates (2.5–5 × 104 cells in 100 μl) coated with rat anti-mouse IFN-γ capture Ab (clone RMMG-1 from Biosource International, Camarillo, CA) and were incubated for ∼48 h at 37°C/5% CO2. The cells were then removed by washing with PBS, and the presence of IFN-γ produced by spleen cells was detected by the addition of biotinylated rat anti-mouse IFN-γ (clone XMG1.2 from PharMingen), followed by alkaline phosphatase-conjugated streptavidin (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The number of stained spots corresponding to IFN-γ-producing cells was enumerated under a dissecting microscope.

To generate a cohort of mice with preexisting immunity against Ad, animals were instilled intranasally with 109 infectious units (i.u.) of wt Ad2, followed by a second instillation with 108 i.u. 14 days later. Eyebleeds were collected 1 day before the administration of DCs, and serum titers of Ad-specific Abs were assessed by ELISA. Serial 2-fold dilutions of serum were added to the wells of 96-well plates coated with heat-inactivated Ad2. Bound virus-specific Abs were detected by the addition of HRP-conjugated goat anti-mouse IgG, IgM, and IgA (Cappel, Durham, NC). The titer was defined as the reciprocal of the highest dilution of serum that produced an OD490 ≤ 0.1.

DCs derived from mouse bone marrow exhibited the veiled dendrite morphology typical of DCs (Fig. 1) and displayed a characteristic set of DC surface markers (33) as determined by FACS analysis (Table I). The cells expressed high levels of the MHC class I and class II molecules, the costimulatory molecules B7.1 and B7.2, the ICAM-I adhesion molecule, the integrin CD11c and the CD13 myeloid surface marker. Exposure of DCs to recombinant Ad2-based vector at a MOI of 500 reproducibly resulted in a transduction efficiency of 90% or greater as determined by the percentage of DCs exhibiting fluorescence following transduction with Ad vector encoding EGFP (Ad2/EGFP). Transduction did not affect the distribution of DC surface markers significantly except for a reproducible increase in levels of MHC class I molecules (Table I).

FIGURE 1.

Morphology of bone marrow-derived DCs (×200 magnification).

FIGURE 1.

Morphology of bone marrow-derived DCs (×200 magnification).

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Table I.

FACS analysis of DC surface markersa

DC SampleB7.1B7.2MHC IMHC IIICAM-ICD11cCD13
Untransduced 80 77 41 70 96 81 80 
Transduced 84 83 85 79 94 71 74 
DC SampleB7.1B7.2MHC IMHC IIICAM-ICD11cCD13
Untransduced 80 77 41 70 96 81 80 
Transduced 84 83 85 79 94 71 74 
a

Results shown are representative of seven separate experiments and are expressed as the percentage of bone marrow-derived DCs staining positive for each marker. DCs were untransduced or transduced with Ad2/β-gal-4.

Bone marrow-derived DCs were functionally active in vitro, as indicated by their ability to induce proliferation of allogeneic T lymphocytes in a mixed lymphocyte reaction. This stimulatory activity was not impaired by transduction with Ad vector (Fig. 2,A). Moreover, DCs transduced with Ad vectors encoding various transgenes were found to induce proliferation of naive syngeneic T lymphocytes, most likely due to processing and presentation of transgene products and/or Ad proteins by the DCs (Fig. 2 B).

FIGURE 2.

In vitro assessment of the functional activity of bone marrow-derived DCs. A, In a mixed lymphocyte reaction, increasing numbers of DCs derived from C57BL/6 bone marrow (1 × 102-1 × 104 DCs) were used to stimulate 2 × 105 allogeneic BALB/c T lymphocytes. Untransduced DCs, as well as DCs transduced with Ad vector encoding green fluorescent protein (Ad2/EGFP), were tested. The levels of proliferation induced were measured by tritiated thymidine incorporation after 5 days of culture. Results shown represent the mean cpm of triplicate wells ± SEM. The background proliferation of untransduced DCs and Ad2/EGFP-transduced DCs incubated alone was highest with 1 × 104 DCs, with cpm values of 2867 ± 681 and 3392 ± 367 cpm, respectively. Results from one representative experiment are shown. A decrease in the levels of proliferation induced by the highest concentration of untransduced DCs (1 × 104) was observed in two of three experiments, and the reason for this phenomenon is unclear. B, To assess primary Ag-specific proliferation, naive C57BL/6 T lymphocytes (2 × 105/well) were incubated with syngeneic untransduced DCs or with DCs transduced with wt Ad2 or Ad2 vectors expressing various transgenes (104 DCs/well). Proliferation levels were assessed after 5 days of culture. Background proliferation of transduced DCs incubated alone was as follows: wt Ad2, 5004 ± 42; Ad2/EGFP, 1198 ± 293; Ad2/β-gal-4, 3059 ± 1137; Ad2/mTRP-2, 920 ± 208; and untransduced DCs, 2867 ± 681 cpm. The induction of Ag-specific proliferation by transduced DCs was observed in three of three separate experiments.

FIGURE 2.

In vitro assessment of the functional activity of bone marrow-derived DCs. A, In a mixed lymphocyte reaction, increasing numbers of DCs derived from C57BL/6 bone marrow (1 × 102-1 × 104 DCs) were used to stimulate 2 × 105 allogeneic BALB/c T lymphocytes. Untransduced DCs, as well as DCs transduced with Ad vector encoding green fluorescent protein (Ad2/EGFP), were tested. The levels of proliferation induced were measured by tritiated thymidine incorporation after 5 days of culture. Results shown represent the mean cpm of triplicate wells ± SEM. The background proliferation of untransduced DCs and Ad2/EGFP-transduced DCs incubated alone was highest with 1 × 104 DCs, with cpm values of 2867 ± 681 and 3392 ± 367 cpm, respectively. Results from one representative experiment are shown. A decrease in the levels of proliferation induced by the highest concentration of untransduced DCs (1 × 104) was observed in two of three experiments, and the reason for this phenomenon is unclear. B, To assess primary Ag-specific proliferation, naive C57BL/6 T lymphocytes (2 × 105/well) were incubated with syngeneic untransduced DCs or with DCs transduced with wt Ad2 or Ad2 vectors expressing various transgenes (104 DCs/well). Proliferation levels were assessed after 5 days of culture. Background proliferation of transduced DCs incubated alone was as follows: wt Ad2, 5004 ± 42; Ad2/EGFP, 1198 ± 293; Ad2/β-gal-4, 3059 ± 1137; Ad2/mTRP-2, 920 ± 208; and untransduced DCs, 2867 ± 681 cpm. The induction of Ag-specific proliferation by transduced DCs was observed in three of three separate experiments.

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After confirming the functionality of DCs in vitro, their ability to induce a CTL response against an MAA was evaluated in vivo. DCs were transduced with an Ad vector encoding hugp100 (Ad2/hugp100v1), a differentiation Ag that is expressed by most melanomas but is also present in normal melanocytes and pigmented cells of the retina. Ad2/hugp100v1-transduced DCs (5 × 105) were administered i.v. to C57BL/6 mice, and, 15 days later, spleens were collected for assessment of CTL activity. Separate groups of mice were also treated with vehicle as a negative control or with the Ad2/hugp100v1 vector itself for comparison. The vector was delivered under conditions previously determined to be optimal for immunization (3 × 109 i.u., intradermally (i.d.)).

After in vitro restimulation with syngeneic fibroblasts transduced with Ad2/hugp100v1, effector splenocytes were tested for cytolytic activity against 51Cr-labeled target fibroblasts that were either untransduced or transduced with Ad2/hugp100v1 or wt E3-deleted Ad (Ad2Δ2.9). The CTLs were also tested against B16 tumor cells, a cell line originally derived from a spontaneously arising melanoma in C57BL/6 mice that expresses the murine equivalent of hugp100.

As expected, mice treated with vehicle failed to develop any significant CTL activity against any of the targets (Fig. 3,A). Mice immunized with transduced DCs developed high levels of CTL activity against target fibroblasts infected with the Ad2/hugp100v1 vector. Interestingly, the bulk of the CTL response appeared to be directed against the hugp100 transgene product rather than adenoviral protein(s) since there was very little lysis of fibroblasts infected with E3-deleted wt Ad (Fig. 3 B). A similar bias in the specificity of the CTL response toward the transgene product was also observed by Wan et al. (20) and Gong et al. (18), following immunization of mice with DCs transduced with Ad vector encoding the polyoma middle T Ag or the DF3/MUC1 tumor-associated Ag, respectively.

FIGURE 3.

Induction of CTL activity following immunization with Ad2/hugp100v1 vector or Ad2/hugp100v1-transduced DCs. Spleens from groups of three animals were collected 15 days after i.v. administration of vehicle (A), Ad2/hugp100v1-transduced DCs (B), or i.d. delivery of Ad2/hugp100v1 vector (C). Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Targets consisted of B16 cells and SVB6KHA fibroblasts untransduced or transduced with Ad2/hugp100v1 or wt Ad2 deleted for E3 (SVB6KHA-Ad2Δ2.9). SD for mean percent lysis values was below 15%. Similar results were obtained in three separate studies.

FIGURE 3.

Induction of CTL activity following immunization with Ad2/hugp100v1 vector or Ad2/hugp100v1-transduced DCs. Spleens from groups of three animals were collected 15 days after i.v. administration of vehicle (A), Ad2/hugp100v1-transduced DCs (B), or i.d. delivery of Ad2/hugp100v1 vector (C). Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Targets consisted of B16 cells and SVB6KHA fibroblasts untransduced or transduced with Ad2/hugp100v1 or wt Ad2 deleted for E3 (SVB6KHA-Ad2Δ2.9). SD for mean percent lysis values was below 15%. Similar results were obtained in three separate studies.

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Mice immunized i.d. with the Ad2/hugp100v1 vector itself, developed robust but comparatively lower levels of CTL activity against Ad2/hugp100v1-transduced fibroblasts. Furthermore, in contrast to the response obtained with transduced DCs, a significant proportion of the CTL response appeared to be specific for Ad Ag, as indicated by the greater level of lysis of fibroblasts infected with E3-deleted wt Ad (Fig. 3,C). Importantly, CTLs from mice immunized with transduced DCs and, to a lesser extent, with Ad vector were both able to lyse B16 tumor cells, suggesting that the CTLs induced by immunization with Ad2/hugp100 also recognized the endogenous mouse gp100 expressed by the tumor cells (Fig. 3, B and C). A similar cross-reactivity between the human and murine Ag was observed by Overwijk et al. following immunization of C57BL/6 mice with recombinant vaccinia virus encoding hugp100 (32).

An ELISPOT assay was used to confirm the presence of CTLs specific for gp100 since CTLs against culture medium components could potentially be present that may participate in the in vitro lysis of B16 cells. In these studies, mice were immunized with DCs transduced with Ad2/hugp100v2, or Ad2/EV as a negative control, and spleen cells were stimulated in vitro with peptide corresponding to either a known H-2b-binding CTL epitope from hugp100 (32), the homologous sequence from mgp100 (32), or a known H-2b-binding CTL epitope from OVA as a negative control (17). The number of class I-restricted CTLs that produced IFN-γ upon specific peptide recognition was measured after 48 h. Results shown in Fig. 4 confirm the presence of CTLs specific for hugp100 peptide in mice immunized with Ad2/hugp100v2-transduced DCs and demonstrate the extensive cross-reactivity between the human and murine epitope. As expected, spleen cells from mice that received DCs transduced with Ad2/EV show little or no reactivity against gp100 peptides, and neither group of mice shows any significant reactivity against the negative control OVA peptide.

FIGURE 4.

Specificity and cross-reactivity of effector cells induced by Ad2/hugp100v2-transduced DCs. Spleen cells from mice immunized with DCs transduced with Ad2/hugp100v2 or Ad2/EV were tested in an ELISPOT assay. The number of IFN-γ-producing cells was counted after 48 h of stimulation with an MHC class I-restricted CTL peptide epitope from hugp100 (open bars), the homologous epitope from mgp100 (filled bars), or an irrelevant H-2b-binding CTL epitope from OVA (slashed bars). Results shown are the mean number of IFN-γ-producing spleen cells ± SEM of triplicate wells after subtracting the background values obtained with spleen cells incubated alone. ∗, p < 0.005, and ∗∗, p < 0.025, compared with OVA-stimulated spleen cells by Student’s t test.

FIGURE 4.

Specificity and cross-reactivity of effector cells induced by Ad2/hugp100v2-transduced DCs. Spleen cells from mice immunized with DCs transduced with Ad2/hugp100v2 or Ad2/EV were tested in an ELISPOT assay. The number of IFN-γ-producing cells was counted after 48 h of stimulation with an MHC class I-restricted CTL peptide epitope from hugp100 (open bars), the homologous epitope from mgp100 (filled bars), or an irrelevant H-2b-binding CTL epitope from OVA (slashed bars). Results shown are the mean number of IFN-γ-producing spleen cells ± SEM of triplicate wells after subtracting the background values obtained with spleen cells incubated alone. ∗, p < 0.005, and ∗∗, p < 0.025, compared with OVA-stimulated spleen cells by Student’s t test.

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Further investigation was conducted regarding the route of DC administration. As shown in Fig. 5, mice immunized with Ad2/hugp100v1-transduced DCs delivered s.c. or i.v developed similar levels of CTL activity against B16 melanoma cells with comparatively little lysis of syngeneic gp100-negative EL4 lymphoma cells. Significant lysis of the YAC NK cell target was also observed. However, NK cells could not account entirely for the lysis of B16 tumor cells since cold target inhibition with an excess of unlabeled YAC cells successfully prevented lysis of labeled YAC cells without significantly affecting specific lysis of B16 cells. These results indicate that the s.c. and i.v. routes of immunization with transduced DCs elicit equivalent levels of CTL activity against B16 tumor cells.

FIGURE 5.

Induction of CTL activity by transduced DCs delivered via the s.c. or i.v. route. Spleens from groups of three mice were collected 16 days after the s.c. (A and B) or i.v. (C and D) delivery of Ad2/hugp100v1-transduced DCs. Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Target cells consisted of 51Cr-labeled YAC cells (NK cell target), C57BL/6-derived EL4 lymphoma cells, and B16 melanoma cells incubated with effector CTLs in the presence (B and D) or absence (A and C) of excess unlabeled “cold” YAC cells. SD for mean percent lysis values was below 15%.

FIGURE 5.

Induction of CTL activity by transduced DCs delivered via the s.c. or i.v. route. Spleens from groups of three mice were collected 16 days after the s.c. (A and B) or i.v. (C and D) delivery of Ad2/hugp100v1-transduced DCs. Pooled spleen cells from each group were restimulated in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2/hugp100v1 and were tested for cytolytic activity after 6 days of culture. Target cells consisted of 51Cr-labeled YAC cells (NK cell target), C57BL/6-derived EL4 lymphoma cells, and B16 melanoma cells incubated with effector CTLs in the presence (B and D) or absence (A and C) of excess unlabeled “cold” YAC cells. SD for mean percent lysis values was below 15%.

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The ability of Ad2/hugp100v1-transduced DCs to induce effector CTLs capable of lysing B16 tumor cells in vitro suggested that DC immunization may also provide antitumor protection in vivo. This was first tested in a pretreatment model whereby mice were immunized with an i.v. injection 5 × 105 Ad2/hugp100v1-transduced DCs and challenged 15 days later with a lethal s.c. injection of 2 × 104 B16 tumor cells. Animals in negative control groups that were pretreated with vehicle or untransduced DCs developed rapidly growing tumors leading to death of the animals within 30 days (Fig. 6). In contrast, mice preimmunized with transduced DCs showed significant resistance to tumor growth, and only one of five animals developed a tumor with delayed kinetics. To test for the presence of immunological memory, the remaining tumor-free mice were given a second B16 challenge, 50 days after the first B16 cell injection. Three of the four mice preimmunized with transduced DCs remained tumor-free upon rechallenge, indicating that a single administration of DCs was sufficient to induce long-term antitumor immunity.

FIGURE 6.

Induction of long-term antitumor protection by Ad2/hugp100v1-transduced DCs. Groups of five C57BL/6 mice were injected i.v. with vehicle (A), 5 × 105 untransduced DCs (B), or 5 × 105 Ad2/hugp100v1-transduced DCs (C). The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. Results shown depict tumor growth in individual animals over time. All animals that were still tumor-free 50 days after B16 challenge received a second injection of B16 cells to test for immunological memory. Results are representative of six separate studies using five to eight mice per group.

FIGURE 6.

Induction of long-term antitumor protection by Ad2/hugp100v1-transduced DCs. Groups of five C57BL/6 mice were injected i.v. with vehicle (A), 5 × 105 untransduced DCs (B), or 5 × 105 Ad2/hugp100v1-transduced DCs (C). The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. Results shown depict tumor growth in individual animals over time. All animals that were still tumor-free 50 days after B16 challenge received a second injection of B16 cells to test for immunological memory. Results are representative of six separate studies using five to eight mice per group.

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Nature of the Ag.

The Ad2/hugp100v1 vector expresses the hugp100 Ag, which, upon presentation by DCs, was found to elicit a protective immune response against murine B16 melanoma cells. To determine whether immunization against a homologous murine MAA would be as effective in inducing protective antitumor immunity, mice were preimmunized i.v. with DCs transduced with Ad vectors encoding the murine MAAs gp100 (Ad2/mgp100) or tyrosinase-related protein 2 (Ad2/mTRP-2). Protection from B16 tumor cell challenge administered 15 days later was compared with that obtained following immunization with Ad2/hugp100v1-transduced DCs, or untransduced DCs as a negative control. As observed previously, mice pretreated with untransduced DCs developed rapidly growing tumors whereas mice preimmunized with Ad2/hugp100v1-transduced DCs showed resistance to tumor challenge so that only two of five mice developed tumors (Fig. 7,A). In contrast, four of five mice treated with DCs transduced with Ad vector encoding the murine homologue of gp100 showed progressive tumor growth, indicating that protective immunity failed to develop in these animals. This finding is in agreement with the results of Overwijk et al., who reported that recombinant vaccinia virus encoding murine gp100 was nonimmunogenic in C57BL/6 mice (32). The failure to induce antitumor protection may have been attributed to the difficulty in breaking immunological tolerance against a self Ag as opposed to the heterologous human protein. However, mice immunized against mTRP-2 with Ad2/mTRP-2-transduced DCs did develop a protective immune response against B16 cells, and five of five mice remained tumor-free (Fig. 7 A). This result indicates that it is in fact possible to generate an effective immune response against a tumor self Ag, but that not all tumor-associated self Ags can be expected to be equally potent.

FIGURE 7.

Factors involved in the effectiveness of immunization with Ad vector-transduced DCs. A, Nature of the Ag: groups of five C57BL/6 mice were injected i.v. with 5 × 105 DCs that were either untransduced or transduced with Ad2/hugp100v1, Ad2/mgp100, or Ad2/mTRP-2 vector. The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. B, Involvement of CD4+ T cells: groups of eight wt or 10 CD4 KO C57BL/6 mice were immunized s.c. with 5 × 105 DCs transduced with Ad2/mTRP-2, or Ad2/EV as a negative control. The animals were challenged 14 days later with a s.c. injection of 2 × 104 B16 melanoma cells. C, Dose dependence: groups of eight C57BL/6 mice were immunized s.c. with increasing doses (5 × 103-5 × 106) of Ad2/mTRP-2-transduced DCs. One group received vehicle as a negative control. Animals were challenged with 2 × 104 B16 cells s.c. 15 and 64 days later. All results are shown as the percentage of tumor-free mice in each group over time.

FIGURE 7.

Factors involved in the effectiveness of immunization with Ad vector-transduced DCs. A, Nature of the Ag: groups of five C57BL/6 mice were injected i.v. with 5 × 105 DCs that were either untransduced or transduced with Ad2/hugp100v1, Ad2/mgp100, or Ad2/mTRP-2 vector. The animals were challenged 15 days later with a s.c. injection of 2 × 104 B16 melanoma cells. B, Involvement of CD4+ T cells: groups of eight wt or 10 CD4 KO C57BL/6 mice were immunized s.c. with 5 × 105 DCs transduced with Ad2/mTRP-2, or Ad2/EV as a negative control. The animals were challenged 14 days later with a s.c. injection of 2 × 104 B16 melanoma cells. C, Dose dependence: groups of eight C57BL/6 mice were immunized s.c. with increasing doses (5 × 103-5 × 106) of Ad2/mTRP-2-transduced DCs. One group received vehicle as a negative control. Animals were challenged with 2 × 104 B16 cells s.c. 15 and 64 days later. All results are shown as the percentage of tumor-free mice in each group over time.

Close modal

Involvement of CD4+ cells.

Overwijk et al. attributed the nonimmunogenicity of mgp100 to the low affinity of a mapped H-2Db-restricted CTL epitope in the murine compared with the human Ag (32). However, whereas much attention has been focused on the induction of tumor-specific CD8+ CTLs, results shown in Fig. 7,B underscore the additional importance of CD4+ T cells, and consequently class II-restricted epitopes, in the development of an optimal antitumor response. In the experiment shown, wt and CD4 KO C57BL/6 mice were immunized in parallel with Ad2/mTRP-2-transduced DCs and challenged with B16 cells 14 days later. As observed above, preimmunization of wt mice with Ad2/mTRP-2-transduced DCs offered 100% protection from B16 challenge (eight of eight mice tumor-free). In contrast, only three of ten CD4 KO mice were able to inhibit tumor growth, indicating that, despite the presence of CD8+ cells, the development of antitumor immunity was severely impaired in these animals (Fig. 7 B).

Dose dependence.

The level of antitumor protection achieved by preimmunization with Ad-transduced DCs was also found to be dependent on the dose of DCs administered. As shown in Fig. 7 C, maximal 100% protection from B16 challenge was achieved with s.c. administration of 5 × 105 Ad2/mTRP-2-infected DCs, with an observed decrease in levels of antitumor protection as the dose was reduced to 5 × 104 and 5 × 103 transduced DCs. Increasing the dose to 5 × 106 DCs did not provide any additional benefit but also failed to induce any discernible toxicity.

Most individuals in the general population have been preexposed to wt Ad and are expected to possess some level of preexisting immunity against Ad. To mimic the expected clinical situation and evaluate the impact of Ad immunity on the activity of Ad-transduced DCs, mice were preimmunized intranasally with wt virus until they developed high titers of Ad-specific Abs (Fig. 8) and, as documented previously, virus-specific CTLs (34). Ad-immune and naive mice were then immunized s.c. with 5 × 105 Ad2/mTRP-2-transduced DCs and were challenged 15 days later with B16 tumor cells. As shown in Fig. 8, naive and Ad-immune mice developed comparable levels of tumor protection with twelve of twelve and ten of twelve tumor-free mice, respectively. Similar results were also obtained with Ad2/hugp100v1-transduced DCs delivered via the i.v. route of immunization (not shown). As expected, negative control animals, which received DCs transduced with Ad2/EV lacking a transgene, developed tumors whether the mice were naive (zero of eight tumor-free) or preimmunized against Ad (one of eight tumor-free). These results suggest that immunization with Ad vector-transduced DCs is unlikely to be impaired significantly in individuals previously exposed to Ad.

FIGURE 8.

Immunization with Ad vector-transduced DCs in animals with preexisting immunity against Ad. Groups of 12 naive or 12 Ad-immune mice were immunized s.c. with 5 × 105 Ad2/mTRP-2-transduced DCs. In parallel, groups of 8 naive or 8 Ad-immune mice received the same number of DCs transduced with Ad2/EV as a negative control. The ELISA titers of Ad-specific Abs present in immune serum collected the day before DC immunization ranged from 12,800 to 51,200. All mice were challenged s.c. with 2 × 104 B16 cells 15 days after administration of DCs. The kinetics of tumor growth are depicted for each individual animal.

FIGURE 8.

Immunization with Ad vector-transduced DCs in animals with preexisting immunity against Ad. Groups of 12 naive or 12 Ad-immune mice were immunized s.c. with 5 × 105 Ad2/mTRP-2-transduced DCs. In parallel, groups of 8 naive or 8 Ad-immune mice received the same number of DCs transduced with Ad2/EV as a negative control. The ELISA titers of Ad-specific Abs present in immune serum collected the day before DC immunization ranged from 12,800 to 51,200. All mice were challenged s.c. with 2 × 104 B16 cells 15 days after administration of DCs. The kinetics of tumor growth are depicted for each individual animal.

Close modal

Up to 100% protection against a lethal challenge of B16 tumor cells was achieved by preimmunization with DCs transduced with Ad vector-expressing MAAs. To extend these findings to a more clinically relevant model, Ad-transduced DCs were also tested in an active treatment setting. In the study shown in Fig. 9 A, mice received a lethal s.c injection of B16 tumor cells, which were allowed to establish themselves for 4 days before treatment with Ad-transduced DCs. As expected, negative control animals that were untreated or treated with Ad2/EV-transduced DCs were unable to control tumor growth. Mice treated with Ad2/hugp100v1-transduced DCs, which provided significant antitumor protection in a preimmunization setting, showed little or no protection from tumor growth (one of five tumor-free) in an active treatment setting. Treatment with Ad2/mTRP-2-transduced DCs, which provided 100% protection in a pretreatment setting, gave rise to partial antitumor protection with three of five mice remaining tumor-free. Therefore, an overall reduction in efficacy was seen in the more stringent active treatment model, which requires rapid mobilization of an immune response against aggressive tumor cell growth.

FIGURE 9.

A, Active treatment of established B16 tumor cells with Ad vector-transduced DCs. Twenty C57BL/6 mice were injected s.c. with 1.5 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into groups of five and were treated s.c. with 5 × 105 DCs that were untransduced or transduced with Ad2/EV, Ad2/hugp100v1, or Ad2/mTRP-2 vector. Results shown represent the percentage of tumor-free mice in each group over time. Note that several animals had already developed tumors when tumor measurement was initiated on day 16. Results are representative of six separate studies using five to eight mice per group. B, Active treatment of established B16 tumor cells using transduced DCs presenting two vs one MAA. Forty-six C57BL/6 mice were injected s.c. with 2 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into six groups, which were treated with a s.c. injection of 5 × 105 DCs transduced with either Ad2/EV (•), Ad2/hugp100v2 (▪) or Ad2/mTRP-2 (▴). Two groups received a mixture of DCs transduced separately with Ad2/hugp100v2 or Ad2/mTRP-2 in the amount of 2.5 × 105 (♦) or 5 × 105 (○) of each DC population. An additional group was treated with vehicle (□) as a negative control. All groups contained eight animals, except for the vehicle control group, which was limited to six mice. Results are presented as the mean tumor size over time. Similar results were obtained in two separate studies.

FIGURE 9.

A, Active treatment of established B16 tumor cells with Ad vector-transduced DCs. Twenty C57BL/6 mice were injected s.c. with 1.5 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into groups of five and were treated s.c. with 5 × 105 DCs that were untransduced or transduced with Ad2/EV, Ad2/hugp100v1, or Ad2/mTRP-2 vector. Results shown represent the percentage of tumor-free mice in each group over time. Note that several animals had already developed tumors when tumor measurement was initiated on day 16. Results are representative of six separate studies using five to eight mice per group. B, Active treatment of established B16 tumor cells using transduced DCs presenting two vs one MAA. Forty-six C57BL/6 mice were injected s.c. with 2 × 104 B16 cells on day 0. Four days later, the animals were divided randomly into six groups, which were treated with a s.c. injection of 5 × 105 DCs transduced with either Ad2/EV (•), Ad2/hugp100v2 (▪) or Ad2/mTRP-2 (▴). Two groups received a mixture of DCs transduced separately with Ad2/hugp100v2 or Ad2/mTRP-2 in the amount of 2.5 × 105 (♦) or 5 × 105 (○) of each DC population. An additional group was treated with vehicle (□) as a negative control. All groups contained eight animals, except for the vehicle control group, which was limited to six mice. Results are presented as the mean tumor size over time. Similar results were obtained in two separate studies.

Close modal

As a first strategy to improve the efficacy of active treatment with Ad-transduced DCs, mice were immunized against two MAAs simultaneously in an attempt to potentiate the immune response and/or minimize escape of tumor cell variants expressing low or nonexistent levels of a given target Ag. As shown in Fig. 9 B, combination therapy with a mixture of two DC populations transduced with Ad2/hugp100v2 or Ad2/mTRP-2 did in fact result in enhanced inhibition of tumor growth, compared with administration of either DC population alone.

Results from this study support the concept of cancer immunotherapy using DCs transduced with Ad vectors encoding TAAs. Bone marrow-derived DCs transduced with Ad vector were found to retain their phenotype as determined by FACS analysis (Table I). Transduced DCs were also functionally active in vitro as assessed in a standard mixed lymphocyte reaction and as demonstrated by their ability to induce primary Ag-specific proliferation of syngeneic T lymphocytes (Fig. 2). In vivo testing of Ad2/hugp100v1-transduced DCs demonstrated their ability to induce a specific CTL response following i.v or s.c. delivery. The appearance of CTLs specific for gp100 peptide and capable of lysing B16 tumor cells in vitro correlated with the development of protective immunity against B16 tumor challenge in vivo.

Immunization with DCs transduced with Ad vectors encoding murine MAAs (mgp100, mTRP-2), as opposed to a heterologous human Ag (hugp100), indicated that it was possible to break immunological tolerance and induce effective immunity against a melanoma self Ag using a DC-based approach. However, the two murine Ags tested differed markedly in their ability to induce antitumor immunity. Ad2/mTRP-2-transduced DCs typically provided complete protection from B16 tumor challenge whereas Ad2/mgp100-transduced DCs gave rise to little or no protective activity. It is likely that multiple factors underlie such differences in activity. For example, the number and potency of CTL (32) and/or Th epitopes within a particular protein, as well as the density of target epitopes expressed by the tumor cells, are two variables that could influence the development of an effective antitumor response. In fact, whereas much emphasis has been placed on the identification of MHC class I-restricted CTL epitopes for induction of antitumor immunity, our results indicate that epitopes recognized by CD4+ T cells also play an important role. Although the murine TRP-2 Ag is known to contain an H-2Kb-restricted CTL epitope (22), the impaired ability of CD4 KO mice to develop protective immunity following immunization with Ad2/mTRP-2-transduced DCs (Fig. 7 B) underscores the involvement of CD4+ T cells and suggests that presentation of MHC class II-restricted epitopes, either by transduced DCs or through secondary processing of the expressed TRP-2 protein, is essential to the development of an effective antitumor response. The exact contribution of CD4+ T lymphocytes in the afferent or efferent phase of the immune response remains to be determined, but the lack of class II expression by B16 cells, even after treatment with IFN-γ (not shown), suggests that CD4+ T lymphocytes are more likely to function as helper cells in the induction of antitumor responses rather than as effector cells against the largely class II-negative tumor cells. In any case, the results suggest that optimal immunization protocols should incorporate the inclusion of class II-restricted epitopes in addition to class I-restricted CTL epitopes, a criterion likely fulfilled by delivery of a complete TAA gene to DCs.

The induction of antitumor immunity by Ad vector-transduced DCs was found to be dose dependent. A single dose of 5 × 103 Ad2/mTRP-2-transduced DCs was sufficient to provide a benefit compared with vehicle-treated animals, and complete protection from tumor challenge was attained with 5 × 105 transduced DCs (Fig. 7 C). These results suggest that, in a therapeutic setting, a clinical benefit may be achievable even with relatively low doses of transduced DCs.

An important issue to consider in the context of a clinical setting is that of preexisting immunity against Ad since most individuals in the general population have been exposed to the wt virus. However, as shown in Fig. 8, immunity to Ad appears unlikely to interfere with DC-based immunization since mice preimmunized with wt Ad2 were not significantly impaired in their ability to develop antitumor immunity following injection of Ad2/mTRP-2-transduced DCs. This finding is in agreement with the results of Brossart et al., who reported that DCs transduced with an Ad vector encoding OVA were able to induce an OVA-specific CTL response in mice that had been previously immunized with two injections of Ad vector and had developed Ad-neutralizing Abs (17). Virus neutralizing Abs were not expected to interfere with DC-based immunization, but Ad-specific CTLs, which are also present in Ad-immune mice (34), have the potential to destroy Ad-transduced DCs. Even though the “half-life” of transduced DCs administered to Ad-immune mice was not determined in this study, the observed development of antitumor protection in these animals indicates that the transduced DCs were present long enough to induce effective immunity against tumor cells. The observation that Ad vector-transduced DCs appear to induce a CTL response largely directed against the transgene product rather than Ad proteins (Refs. 18 and 20 ; Fig. 3) raises the possibility that limited presentation of Ad Ags by Ad/TAA-transduced DCs may provide some level of protection from lysis by Ad-specific CTLs.

The efficacy of Ad vector-transduced DCs was also tested in a therapeutic setting against established B16 s.c. tumor cells. The level of antitumor protection achieved was reduced, compared with that obtained in a preimmunization model. Nevertheless, a single injection of Ad2/mTRP-2-transduced DCs resulted in complete inhibition of tumor growth in an average of three of five mice (Fig. 9,A). Several approaches are being considered to improve this outcome further. For example, simultaneous immunization against two MAAs, as opposed to a single Ag, was tested as a means to potentiate the immune response and prevent the escape of tumor variants that may express insufficient levels of a target Ag for recognition by CTLs. The results obtained support the validity of this type of approach since administration of a mixture of DCs transduced separately with Ad vector encoding hugp100 or murine TRP-2 resulted in greater levels of tumor growth inhibition, compared with either DC population alone (Fig. 9 B). In addition, preliminary results suggest that the therapeutic efficacy of Ad vector-transduced DCs can also be enhanced by multiple administrations of DCs (40% increase in day 40 survival with three doses compared with a single dose) or by coadministration of low dose IL-2 (not shown). In agreement with the latter observation, Shimizu et al. (35) have reported recently that low dose IL-2 dramatically enhanced the antitumor response elicited by DCs pulsed with tumor lysate in a murine sarcoma model.

Overall, the data obtained in this study provide supporting evidence for the concept of melanoma immunotherapy based on the administration of DCs transduced with Ad vectors encoding MAAs. This type of approach is considered feasible in humans since protocols have been established that allow for the expansion of large numbers of DCs from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 (36, 37). The DCs obtained can be effectively transduced with Ad vector and can induce a primary CTL response against the transgene product in vitro (G. Linette, S. Shankara, R. Doll, L. Eaton, B. Roberts, and C. Nicolette; manuscript in preparation). These observations, in conjunction with the protective antitumor activity elicited by transduced DCs in the B16 melanoma model, suggest that immunization of melanoma patients with autologous DCs transduced with Ad vector expressing human MAAs may provide a therapeutic benefit.

We thank Drs. Richard Gregory, Michael Perricone, and Charles Nicolette for helpful advice throughout the study, as well as Karen Smith, Kristin Hubley, and Samantha Rudginsky for technical help. We are also grateful to the Genzyme Virus Production and Animal Care Units, the laboratory of Dr. Donald Kufe (Harvard Medical School, Boston, MA) for providing information on the preparation of murine DCs, and Drs. Willem Overwijk and Jim Yang (NCI Surgery Branch, Bethesda, MD) for providing the mgp100 and mTRP-2 cDNAs used in the construction of Ad vectors.

2

Abbreviations used in this paper: TAA, tumor-associated Ag; DC, dendritic cell; β-gal, β-galactosidase; EGFP, enhanced green fluorescent protein; wt, wild type; TRP, tyrosinase-related protein; mTRP, murine TRP; EV, empty vector; hugp100, human gp100 melanoma Ag; mgp100, murine gp100 melanoma Ag; KO, knockout; Ad, adenovirus; MAA, melanoma-associated Ag; MOI, multiplicity of infection; ELISPOT, enzyme-linked immunospot; i.u., infectious unit; i.d., intradermal.

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