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Annals of Surgical Oncology 9:411-418 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Mature CD83+ Dendritic Cells Infected With Recombinant gp100 Vaccinia Virus Stimulate Potent Antimelanoma T Cells

Indira Prabakaran, MSc, Chandrakala Menon, PhD, Shuwen Xu, MD, Alicia Gómez-Yafal, PhD, Brian J. Czerniecki, MD, PhD and Douglas L. Fraker, MD

From the Department of Surgery, University of Pennsylvania, Philadelphia, Pennsylvania (IP, CM, SX, BJC, DLF); and Therion Biologics Corporation, Cambridge, Massachusetts (AG-Y).

Correspondence: Address correspondence and reprint requests to: Douglas Fraker, MD, Department of Surgery, 4th Floor, Silverstein Building, 3400 Spruce St., Philadelphia, PA 19104; Fax: 215-614-0765; E-mail: fraker{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Mature dendritic cells (DCs) are potent antigen-presenting cells that activate naive T lymphocytes and initiate cellular immune responses. The ability of CD83+ mature DCs infected with vaccinia virus encoding the gp100 melanoma transgene (rV-gp100) to stimulate an antimelanoma CD8+ T-cell response was investigated.

Methods: Monocyte-derived immature or CD83+ mature DCs were infected with rV-gp100. The activation state of the DCs and the expression of gp100 protein were evaluated by flow cytometry. The reactivity of antimelanoma CD8+ T cells was confirmed by measuring specific interferon {gamma} secretion by using enzyme-linked immunosorbent assay in a mixed-tumor lymphocyte culture.

Results: Both immature and CD83+ mature DCs expressed gp100 protein when the DCs were infected with rV-gp100. Calcium-signaling agents were required to induce maturation of both infected and noninfected immature DCs. Only rV-gp100-infected CD83+ DCs induced CD8+ T cells, after a single stimulation that recognized both peptide-pulsed target cells to multiple gp100 epitopes and a melanoma cell line that endogenously expressed gp100 antigen.

Conclusions: CD83+ DCs transduced with rV-gp100 are capable of generating a strong CD8+ T-cell response against melanoma tumor cells. Expression of melanoma antigens by mature DCs offers the potential advantage of presenting multiple endogenously processed T-cell epitopes and using multiple HLA restriction elements for antimelanoma vaccine therapy.

Key Words: Melanoma • Dendritic cells • Tumor-associated antigen • gp100 • Vaccinia virus • CD8+


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are highly efficient and specialized antigen-presenting cells capable of antigen capture and presentation to lymphocytes.1,2 DCs exist in an immature state in the peripheral tissues, where they sample the environment and, when stimulated by danger signals, begin to mature, express high levels of co-stimulatory molecules, and migrate to areas rich in lymphocytes to present antigens to T cells.3 Several groups have demonstrated, both in vivo and in vitro in human and murine studies, that DCs pulsed with antigenic peptides derived from tumor-associated antigens (TAAs), tumor lysates, RNA, microbial agents transduced with tumor-associated DNA, or DC-tumor cell fusions are capable of inducing specific antitumor responses.4–9 DC-based vaccines have entered clinical trials, such as in prostate cancer, melanoma, and lymphoma.10–13 Although a few of these trials have demonstrated clinical responses in patients with metastatic cancer, most trials have met with limited success.

One of the critical factors responsible for limited clinical success seems to be related to the maturation state of the DCs used in these trials, most of which have used immature DCs pulsed with peptides that bind major histocompatibility complex (MHC) class I molecules and therefore target only CD8+ T cells.14–17 Several studies now suggest that vaccination with immature DCs may even result in the development of suppressed T-cell responses.18

DC maturation requires one of several signals, including CD40 ligand, tumor necrosis factor-{alpha}, or calcium-signaling agents.19–21 Our group has previously documented that calcium-signaling agents induce maturation of DCs derived from peripheral blood monocytes21,22 and other myeloid cells.23 DCs activated with calcium-signaling agents, in the presence of cytokines in serum-free medium, rapidly express mature DC marker, CD83, and high levels of co-stimulatory molecules within 40 hours of culture.22 These activated DCs can efficiently sensitize T cells to tumor-derived peptide antigens that recognize tumor cells that endogenously express tumor antigen.

Besides the activation state of DCs, the delivery of antigen may also affect the outcome of an antitumor T-cell response. Specific peptide-pulsed DCs have been shown to be somewhat effective in activating antitumor T cells.6,9–12 Such peptide-pulsed DCs may not always result in T cells that recognize tumor cells expressing the antigen. Specific peptides designed to bind particular MHC-binding domains are hampered by the need to identify the relevant peptide-binding domains; this leads to the exclusion of a significant number of patients from such DC vaccine trials.

The availability of cloned genes that encode melanoma TAAs offers a strategy for using TAA gene-modified DCs as tumor vaccines. Several groups have documented that microbial agents transduced with genes expressing TAA can be used to infect DCs and to generate antitumor T cells that can recognize tumor targets.24–28 It has also been shown that such DCs can be used to treat animals with established tumors.29 Infection of mature DCs with an entire TAA gene could allow DCs to process and present TAA in a physiological manner, thus generating cytotoxic T lymphocytes (CTLs) capable of recognizing naturally processed antigenic epitopes presented on a tumor surface. Another potential advantage of the expression of the TAA in an endogenous antigen-presenting system is the prolonged and constitutive expression of the antigen to T lymphocytes as compared with the short-term effect of exogenous peptide loading. This approach is appealing because multiple epitopes of a particular antigen can be presented to T cells,25 and the process is not limited by the need to identify MHC restriction elements. In addition, these DCs may also present MHC class II-restricted epitopes to CD4+ T cells, which are important for long-term maintenance of activated CTLs and the induction of memory T cells.30,31 Most of the studies demonstrating that viruses transduced with TAA induce antitumor CTLs were performed with immature DCs.24,25 It is not known whether mature DCs infected with virus-transduced TAA are more efficient in inducing an antitumor T-cell response.

In this study, we demonstrate that CD83+ mature DCs infected with recombinant gp10032 vaccina virus (rV-gp100) express the gp100 protein. CD83+ DCs are more potent than immature DCs infected with rV-gp100 in stimulating antitumor CD8+ T cells. Such DCs induce peptide-specific HLA-restricted CD8+ T cells and CD8+ T cells capable of recognizing naturally expressed multiple antigenic epitopes on an HLA-A2+ cell line. An immunotherapeutic approach that promotes the induction of tumor-specific CTL and HLA restrictions could theoretically provide an enhanced immune response with a greater potential to either slow down or eliminate tumor growth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Human Monocytes
Human monocytes were prepared as previously described.21 Briefly, 5 to 7 L of whole blood from human donors was leukapheresed after informed consent was obtained. Cells were washed and resuspended in Ca2+/Mg2+-free, pyrogen-free Hanks’ balanced salt solution (BioWhittaker, Walkerswille, MD) and then elutriated with a model J-6MTM centrifuge equipped with a JE5.0TM elutriation rotor operating at 1725 x g at 20°C (Beckman Instruments, Palo Alto, CA). Monocyte-rich and lymphocyte-rich fractions were collected on ice in Life CellTM tissue culture vessels (Baxter HealthCare, Deerfield, IL) to inhibit cellular adherence. Lymphocyte-rich fractions were further purified through a Ficoll-Hypaque gradient (Biowhittaker) to remove red blood cells. These elutriated fractions were then cryopreserved at 5 x 107/ml in 90% human inactivated AB serum and 10% dimethyl sulfoxide (Sigma Chemical Co, St. Louis, MO) for future use.

Preparation of DCs
Monocyte-rich fractions were thawed in serum-free medium and supplemented with .1 mM of minimum essential medium nonessential amino acids, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM of L-glutamine (Gibco BRL, Gaithersburg, MD). Cells were cultured for 24 hours with granulocyte-macrophage colony-stimulating factor (Peprotech, Rocky Hill, NJ) at 100 IU/ml, interleukin (IL)-2 (Chiron, Emeryville, CA) at 300 IU/ml, and IL-12 (R&D Systems, Minneapolis, MN) at 80 IU/ml and cultured an additional 24 hours (immature DCs). To obtain mature DCs, immature DCs were treated with calcium ionophore (CI; Sigma) at a concentration of 150 ng/ml for 16 hours. The cells were collected at 40 hours and analyzed for DC surface markers and co-stimulatory molecules by fluorescence-activated cell sorter (FACS) analysis or co-cultured with autologous CD8+ T cells for sensitization assays.

Preparation of Vaccinia-Infected Mature DCs
Vaccinia viruses encoding the LacZ transgene (rV-LacZ) and the gp100 transgene (rV-gp100) were obtained from Dr. Alicia Yafal (Therion Biologicals, Cambridge, MA). Virus stocks were prepared in RK13 cells (American Type Culture Collection No. CCL-37).32 After 24 hours in culture, immature DCs were infected at various multiplicities of infection (MOIs) to determine optimal expression of LacZ or gp100. In studies using mature DCs, CI was added 1 hour after viral infection of immature DCs. To determine the viability of vaccinia-infected DCs, cells were collected at 1, 2, 4, 8, 16, and 24 hours after infection, and trypan blue exclusion was used to determine live/dead cells. Staining for LacZ expression at different time points in rV-LacZ-infected DCs was performed on chamber slides (Nunc, Roskilde, Denmark). A total of 1 x 105 infected DCs were plated on the chamber slide and allowed to adhere for 2 hours at 37°C. DCs were then stained for LacZ expression. To determine whether there was prolonged gene expression in the vaccinia-infected DCs, rV-LacZ/rV-gp100-infected cells were collected 1, 2, and 3 days after infection for FACS analysis and stained for LacZ and gp100 expression. Also, immature and mature DCs were collected 16 hours after infection, and co-stimulatory molecules and CD83 expression were determined.

Melanoma Cell Lines
624A2+, 624A2-, and MW115 cell lines33 were a generous gift from Dr. Steven Rosenberg (National Cancer Institute, Bethesda, MD). The 624A2+ cell line, which was transduced with the HLA-A2+ gene, was originally derived from a metastatic lesion of a patient and expressed high levels of gp100. The 624A2- cell line is HLA-A2-/gp100+, whereas MW115 is HLA-A2+/gp100-. The cell lines were cultured in RPMI 1640 medium containing 10% fetal calf serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 2 mM of L-glutamine.

T-2 Cell Line
The T-2 human cell line that was used in cytokine release assays is an HLA-A2.1+ T-cell hybrid that is peptide transporter-associated protein deficient and defective in endogenous processing, thereby enhancing the effectiveness of exogenous peptide loading.34 This cell line was used to prepare gp100(gp154–162), gp100(gp209–217), gp100(gp280–288),35 and Her-2/neu(46–56) peptide-pulsed stimulator cells for CD8+ T-cell sensitization assays. The peptides were synthesized at the University of Pennsylvania Cancer Center Protein Chemistry Laboratory, as previously described.36

Preparation of CD8+ T Lymphocytes
Cryopreserved lymphocyte-rich fractions were thawed on the day of culture in Iscoves Modified Dulbecco’s Medium with 5% human AB serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 2 mM of L-glutamine, and 1 mM of nonessential amino acids. Autologous CD8+ cells were prepared by using a negative immunoselection column (R&D Systems) that allowed for enrichment of the CD8+ T-cell subset. Purity of >95% was obtained with this method.

Flow Cytometric Analysis of Cell Populations
Monocytes/DCs were collected and resuspended in cold FACS buffer (phosphate-buffered saline with 1% fetal calf serum and .1% sodium azide). Cells were immunostained by using a cocktail containing fluorescein isothiocyanate-conjugated anti-CD3, anti-CD14, anti-CD20, and anti-CD56 antibodies (Becton Dickinson, Mountain View, CA). The cells were simultaneously immunostained with phycoerythrin (PE)-conjugated antibodies to CD80, CD83, CD86, CD40, CD54, and MHC class I or II antigens to identify populations that lacked monocytic (CD14) and lymphocytic (CD3, CD20) markers while expressing DC markers (CD83, CD86, and CD 80) and their co-stimulatory molecules (CD40 and CD54). PE-conjugated mouse anti-human CD80, CD86, CD54, MHC class I and II, and corresponding fluorescein isothiocyanate and PE immunoglobulin G isotype control antibodies were purchased from Becton Dickinson (San Diego, CA). PE-conjugated mouse anti-human CD40 was purchased from Serotec (Oxford, UK), and PE-conjugated CD83 was obtained from Immunotech (Westbrook, ME). A total of 1 x 106 cells were incubated for 30 minutes at 4°C with antibodies, washed once with cold FACS buffer, and fixed in 2% paraformaldehyde (Sigma); two color analyses were performed on a FACSscanTM (Becton Dickinson). An intracellular staining method was used for the detection of gp100 protein in rV-gp100-infected DCs. Mature and immature DCs were fixed by incubation in 2% paraformaldehyde. Cell membranes were permeabilized in .1% saponin (Sigma) and then incubated with Cy5-conjugated HMB50 antibody against gp100 (Neomarkers, Fremont, CA) for 30 minutes at 4°C. The cells were then washed once in FACS buffer, resuspended, and phenotyped on a FACScaliburTM (Becton Dickinson).

In Vitro Sensitization of Autologous CD8+ T Cells
Mature and immature DCs (1 x 105 cells) that had been infected with rV-gp100 for 16 hours or pulsed with gp100 peptide for 2 hours were collected and co-cultured with 1 x 106 autologous CD8+ lymphocytes in 48-well flat-bottomed plates. IL-1{alpha} at .4 ng/ml and IL-6 at 4 ng/ml were added to cells at the time of co-culture. IL-2 at 30 IU/ml was added to all wells 24 hours later and 2 to 3 days thereafter. After 1 week, CD8+ T cells were collected and tested for specificity by using T-2 cells that had been pulsed with specific gp100(gp154–162), gp100(gp209–217), gp100(gp280–288), or irrelevant peptide; Her-2/neu(46–56); and the melanoma cell lines MW115, 624A2+, and 624A2-. A total of 1 x 105 CD8+ T cells were co-incubated with 1 x 105 target cells in a 96-microwell plate in 200 µl of culture medium for 24 hours at 37°C. After 24 hours, the supernatant was collected, and the amount of interferon (IFN)-{gamma} released was determined by enzyme-linked immunosorbent assay (Pharmingen, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Vaccinia LacZ Expression in Immature DCs to Determine Viability and Optimal MOI
Immature DCs were infected with rV-LacZ at different MOIs. Twenty-four hours after infection, cells were collected, viability was determined by trypan blue exclusion, and the cells were stained for LacZ expression (Table 1). The viability of the cells infected at the various MOIs at the time of collection was similar (90%–96%). However, at the lowest MOI of .01, only 15% to 20% of the cells stained positive for LacZ. At an MOI of .1, the percentage of cells staining positive for LacZ increased to 45% to 60%, whereas at an MOI of 1, 90% to 95% of cells stained positive for LacZ. At the highest MOIs of 10 and 100, all the cells stained positive for the LacZ gene. However, when the viability was followed out for another 24 hours, a feature essential for the sustained stimulation of CD8+ T cells, a majority of the DCs were not viable because of lysis by the vaccinia virus (data not shown). On the basis of these data, an MOI of 1 was chosen for further experiments.


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  		TABLE 1. LacZ expression in rV-LacZ-infected monocytes
 
Time Course of Vaccinia LacZ Expression in DCs to Determine Time of Maximal Expression/Viability
Immature and mature DCs were then infected with rV-LacZ at an MOI of 1. Cells were collected at 1, 2, 4, 8, 16, and 24 h and stained for LacZ expression, and viability was determined (Fig. 1). LacZ expression was seen as early as 1 hour in both immature and mature DCs, with mature DCs showing a slightly lower percentage of positively stained cells. This trend in LacZ expression continued up to 8 hours after infection. At 16 and 24 hours after infection, 90% to 95% of cells stained positive in both immature and mature DCs. Whereas the viability of mature DCs was 88% at 16 hours after infection, it decreased to 80% at 24 hours after infection (data not shown). LacZ expression was sustained up to 3 days after infection in both mature and immature DCs. On the basis of these data, the 16-hour postinfection time point was used in experiments.



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  		FIG. 1. Time course of infection with vaccinia viruses encoding the LacZ transgene (rV-LacZ) in immature/mature dendritic cells (DCs). Immature DCs (granulocyte-macrophage colony-stimulating factor/interleukin [IL]-2/IL-12) ({blacksquare}) or mature DCs (calcium ionophore) (•) were infected with rV-LacZ at a multiplicity of infection of 1. At 16 hours after infection (the optimal time for conversion from immature DCs to mature DCs), approximately 90% of mature DCs were positive for LacZ expression. Data shown are representative of 3 independent experiments that produced similar results.

 
Effect of rV-gp100 Vaccinia Virus on DC Maturation
To establish that mature DCs infected with rV-gp100 expressed co-stimulatory molecules, mature and immature DCs infected and uninfected with rV-gp100 were analyzed for cell-surface expression of monocytic marker (CD14), T-cell marker (CD3), B-cell marker (CD20), natural-killer cell marker (CD56), mature DC marker (CD83), co-stimulatory molecules (CD80, CD86, CD40, and CD54), and MHC class I and class II molecules. Immature DCs expressed CD86 and MHC class I and class II molecules and little or no CD40, CD83, and CD80 (Table 2, Fig. 2), as expected. These cells also had diminished CD14 surface expression compared with monocytes (not shown). Infection of immature DCs with rV-gp100 did not result in increased expression of CD83 or CD80 and CD86, suggesting that virus infection alone did not result in DC maturation (Table 2, Fig. 2). These cells also continued to express diminished levels of the monocytic marker CD14. In contrast, mature DCs activated with CI demonstrated an increased percentage of cells expressing CD83, as previously described.22 In addition, mature DCs also showed a marked upregulation of CD80, CD83, CD86, CD40, CD54, and MHC class I and II molecules. These cells also demonstrated a further downregulation of the monocytic marker CD14. Viral infection of mature DCs did not result in significant increases or decreases in CD83, CD80, or CD86 expression, suggesting that the virus had little effect on DC maturation (Table 2).


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  		TABLE 2. Expression of DC markers and co-stimulatory molecules on immature/mature and uninfected/infected DCsa
 


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  		FIG. 2. Fluorescence-activated cell sorter histograms showing expression of dendritic cells (DCs) and co-stimulatory markers in mature versus immature DCs with or without recombinant gp100 vaccina virus (rV-gp100) infection. Mature uninfected DCs (top panel) converted to a phenotype of activated DCs demonstrated by high levels of CD83 expression and showed upregulation of CD40, CD54, major histocompatibility complex (MHC) class I and II and co-stimulatory molecules, CD80, and CD86. Mature infected DCs (upper middle panel) showed similar CD83 expression and upregulation of co-stimulatory and MHC class I and II molecules. Immature uninfected DCs (lower middle panel) and immature infected DCs (bottom panel) showed very little upregulation of CD80, CD40, and CD83 markers compared with mature infected DCs. There was low but evident expression of CD86, CD54, and MHC class II; relevant mean channel fluorescence intensities are shown as an inset in each histogram. A lighter histogram represents the distribution of control antibodies. Data shown are representative of 3 independent experiments that produced similar results.

 
Tumor Antigen Expression by DCs Infected With rV-gp100
Cytoplasmic expression of gp100 protein was confirmed by FACS analysis. Intracellular FACS with HMB50 antibody was used to determine the expression of gp100 protein in rV-gp100-infected DCs, 16 hours after infection, by using rV-LacZ-infected or -noninfected DCs and isotype antibody as negative controls. By FACS, gp100 expression was greater in the rV-gp100-infected DCs than in rV-LacZ-infected DCs or in noninfected DCs, whereas the normal isotype immunoglobulin G control was negative for gp100 expression (Fig. 3), as expected. In addition, immature and mature DCs infected with rV-gp100 demonstrated similar expression of gp100, suggesting that both cell types were equally infected and that protein was similarly transcribed (not shown).



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  		FIG. 3. Fluorescence-activated cell sorter (FACS) histogram showing increased expression of gp100 in recombinant gp100 vaccina virus (rV-gp100)-infected dendritic cells (DCs). Immature DCs cultured for 16 hours with granulocyte-macrophage colony-stimulating factor IL-2, IL-12, and calcium ionophore and simultaneously infected with rV-gp100 were immunofluorescently stained for gp100 antigen by using HMB50 antibody and were analyzed by FACS. Mature DCs with or without infection with vaccinia viruses encoding the LacZ transgene and that were immunofluorescently stained for gp100 were used as control. Mature DCs infected with rV-gp100 were immunofluorescently stained with immunoglobulin G1 (IgG1) as an isotype control.

 
Sensitization of Autologous CD8+ T Cells by DCs Infected With rV-gp100
Autologous antimelanoma CD8+ T cells were generated by a single stimulation with immature or mature DCs infected with rV-gp100. CD8+ T cells sensitized with immature DCs resulted in little evidence of antimelanoma T-cell reactivity, because very little IFN-{gamma} secretion was seen with gp100 peptide-pulsed target cells or melanoma cell lines that endogenously express gp100 (Fig. 4A and B). In contrast, CD8+ T cells that were sensitized by co-culturing mature DCs infected with rV-gp100 recognized multiple peptide epitopes of gp100, as assessed by 2- to 3-fold increased IFN-{gamma} secretion to peptide-pulsed target cells (Fig. 4A). In addition, these T cells recognized a melanoma cell line that expressed endogenous gp100 and caused secretion of high levels of IFN-{gamma} (Fig. 4B).



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  		FIG. 4. Sensitization of autologous CD8+ T cells by mature dendritic cells (DCs) infected with recombinant gp100 vaccina virus (rV-gp100) as assayed by interferon (IFN)-{gamma} release assay. Mature DCs infected with rV-gp100 for 16 hours were collected and co-cultured with autologous CD8+ T cells for 7 days. Sensitized CD8+ T cells were co-incubated with target T-2 cells pulsed with specific gp100 peptide or irrelevant peptide (A) and gp100+ (624A2+), gp100- (MW115), or HLA-A2- (624A2-) cells (B). Supernatants were collected 24 hours later and were assayed for IFN-{gamma} release. Data show that mature infected DCs could sensitize CD8+ T cells not only to multiple epitopes of gp100, but also to naturally processed melanoma antigens on a gp100-expressing cell line. Data shown are representative of 3 independent experiments that produced similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have documented that calcium-signaling agents induce maturation and expression of CD83+ on human DCs.21,22 These DCs, pulsed with tumor-derived peptides, result in enhanced antitumor T-cell responses.22 In this study, we establish that mature DCs activated with calcium-signaling agents and cytokines lead to a superior CD8+ antitumor T-cell activation when the tumor antigen is delivered by vaccinia virus-expressing gp100. The CD8+ T cells not only recognized tumor cells endogenously expressing gp100, but also recognized multiple peptide epitopes within gp100, as demonstrated after only a single stimulation with mature rV-gp100-infected DCs.

CTLs generated by using peptides that are derived from a given TAA and selected on the basis of the binding motif for the possible HLA class I restriction elements are not always able to recognize tumor cells; this suggests that such epitopes may not always be naturally processed.37 In contrast, however, introducing the entire TAA transgene into DCs may allow them to process and present the antigen in a natural and immunologically effective form. Thus, CD8+ T cells generated by mature DCs expressing TAA were able to recognize multiple gp100 epitopes that may be naturally processed by tumor cells and therefore may account for the enhanced reactivity seen by the anti-gp100 CD8+ T cells generated by the mature DCs in this study.

Despite the facts that both immature and mature DCs were able to be equally infected with rV-gp100 and that protein expression seemed similar, immature DCs were unable to stimulate significant antimelanoma CD8+ T cells. This may explain the requirement for multiple re-stimulations with immature DCs infected with virus containing TAA to generate antitumor T cells.24,25 Clearly, in this study, CD83+ DCs stimulated more potent antimelanoma T cells than did immature DCs. Whether other agents, such as CD40 ligands that signal DC maturation, can result in enhanced antimelanoma T-cell activity needs to be studied. We have demonstrated that DCs activated with IFN-{gamma} and CD40 ligand result in mature DCs that secrete high levels of IL-12 and induce greater high IFN-{gamma} secreting T-cell antitumor activity compared with calcium-activated DCs.38 Whether a similar response would be seen in DCs infected with rV-gp100 is under investigation.

Vaccinia virus infection of immature DCs did not result in significant maturation, as evidenced by the lack of CD83 expression. However, not all TAA transduced viruses used to infect DCs are inert. Adenovirus has been shown to induce maturation of human DCs derived from monocytes.39 Thus, the effect of transduced viruses on DC maturation must be considered in vaccine preparation because the subsequent T cell-stimulating capacity of DCs may differ.

The advantage of the approach of TAA gene expression, as compared with peptide immunizations, is the ability to introduce the gene for whole antigens without identification of the specific epitope or the necessary restriction element. Most of the melanoma antigens in the HLA*A0201 system have been identified, but thus far the accessibility of peptide-based vaccines is limited to patients who are of a limited HLA phenotype. An autologous endogenous system of antigen presentation, in which the specific epitope or the restriction element of the antigen is not known, could circumvent the previously mentioned limitations. In addition, this autologous endogenous system could be useful in identifying new restriction epitopes and their restriction elements.

Another advantage of DCs infected with virus transduced with TAA is that such DCs can stimulate antitumor CD4+ T cells in addition to CD8+ T cells.29,30 CD4+ T cells may be required to maintain the activity of antitumor CD8+ T cells.25 CD4+ T cells also induce regression of metastatic tumors through an indirect mechanism involving cytokine secretion.40,41 The effect of DC maturation on CD4+ T-cell sensitization using DCs infected with virus transduced with TAA needs to be evaluated.

One of the major barriers to effective vaccination with viruses such as vaccinia and adenovirus is the inhibition of vaccine "take" by preexisting neutralizing antibodies.26 These antibodies could be the result of previous exposure to cross-reacting viruses or previous immunizations (vaccinia). Ultimately, realization of the full clinical value of recombinant viral vaccines will require the development of methods to transiently eliminate neutralizing antibodies, to design viral vectors to which most individuals have not been exposed, or both of these.

In summary, CD83+ DCs infected with rV-gp100 stimulated potent antimelanoma CD8+ T cells that recognized multiple gp100 epitopes. This study suggests that use of mature DCs infected with viruses transduced with TAA would result in greater antitumor activity and has important implications for developing anticancer DC vaccines.


   Acknowledgments
 
The authors thank Andy Gelman of the Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, for his technical advice. Supported by the Georgene S. Harmelin Fund.

Received for publication October 9, 2001. Accepted for publication January 23, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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