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The Journal of Immunology, 2005, 175: 3431-3438.
Copyright © 2005 by The American Association of Immunologists

A Novel Viral System for Generating Antigen-Specific T Cells1

Timothy P. Moran*,{dagger}, Martha Collier*,{dagger}, Karen P. McKinnon*,{ddagger}, Nancy L. Davis*,{dagger}, Robert E. Johnston*,{dagger},{ddagger} and Jonathan S. Serody2,*,{ddagger},§

* Department of Microbiology and Immunology, {dagger} Carolina Vaccine Institute, {ddagger} Lineberger Comprehensive Cancer Center, and § Department of Medicine, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with ~50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-{alpha} treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-{alpha}, and IFN-{alpha}. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-{alpha}-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
There is significant interest in the use of dendritic cell (DC)3 vaccines as treatments for patients with malignancies and chronic infectious diseases (1, 2). Following activation by inflammatory cytokines or microbial products, DCs possess several characteristics that are necessary for efficient stimulation of tumor-specific T lymphocytes. These characteristics include enhanced homing to lymphoid tissues, high level expression of MHC class I and class II molecules in conjunction with costimulatory molecules, and secretion of immunostimulatory cytokines (3). The ability of DCs to prime tumor-specific T cell responses has been demonstrated in various animal models (4, 5, 6). These studies have led to several clinical trials evaluating the efficacy of DCs loaded ex vivo with tumor-associated Ags (TAAs) to initiate protective immune responses in cancer patients (7, 8, 9, 10, 11, 12, 13). Multiple techniques have been used for loading DCs with TAAs including pulsing with MHC class I- and/or class II-restricted peptides (14, 15, 16), incubation with tumor cell lysates (15), and electroporation with tumor cell RNA (17). Unfortunately, induction of measurable and durable antitumor T cell responses has been infrequent in most clinical trials, suggesting that the stimulatory capacity of current DC vaccines is inadequate (18). Therefore, alternative strategies for inducing optimal DC maturation and Ag presentation are warranted.

Viral vectors that encode TAAs may provide an alternative method for delivering Ags to DCs. Delivery of an entire TAA rather than TAA-derived peptides allows processing and presentation of multiple epitopes on both MHC class I and class II molecules, resulting in a broader CD8+ T cell response and incorporation of CD4+ T cell help (19, 20). In contrast to MHC-restricted peptide vaccines, viral vectors can be used to transduce DCs of all MHC haplotypes. Viral vectors can induce DC maturation through both TLR-dependent and -independent pathways, resulting in up-regulation of costimulatory molecules and secretion of Th1-inducing cytokines (21, 22). Additionally, viral vectors may provide stimuli that are required for overcoming tolerance against TAAs, specifically through the down-regulation of CD4+CD25+ regulatory T cell activity (23).

Several viral vectors have been used for transducing human DCs with TAAs (24, 25, 26, 27). Although some of these vectors have entered clinical trials (28), their widespread use is hampered by inefficient transduction efficiencies, interference with DC function, and induction of antivector responses due to pre-existing immunity (29). Because of these limitations, we have evaluated the potential use of vectors derived from Venezuelan equine encephalitis virus (VEE) for transduction of human DCs. Nonpropagating VEE replicon particles (VRPs) possess intriguing characteristics including 1) significant expression of the inserted gene in infected cells (30), 2) induction of both cell-mediated and humoral immunity (31), 3) potential for repeated immunizations without significant induction of antivector immune responses (30), and 4) potential tropism for DCs. Although our group has shown that VRPs can infect murine DCs in vivo (32), the capacity of VRP to transduce human DCs is unknown. In this report, we demonstrate that VRPs can infect human immature monocyte-derived DCs. VRP-transduced DCs can efficiently process and present VRP-encoded Ags, leading to robust proliferation of Ag-specific T cells and acquisition of effector function. Thus, vaccines consisting of VRP-transduced DCs may prove highly effective for the induction of tumor-specific CD8+ T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

PE-conjugated mAbs specific for human CD8 (SK2), CD11c (B-LY6), CD14 (M5E2), CD40 (5C3), CD80 (L307.4), CD83 (HB15e), and HLA-DR (G46–6) were purchased from BD Pharmingen. Anti-human CD86-PE (HA5.2B7) was purchased from Beckman Coulter. Mouse anti-influenza A matrix protein (FMP) mAb was purchased from Serotec. All isotype control Abs were purchased from BD Pharmingen. Recombinant human GM-CSF, IL-4, IL-2, IL-7, and TNF-{alpha} were purchased from PeproTech. Human AB serum was purchased from Gemini Bio-Products.

Generation of human monocyte-derived DCs

Peripheral blood was obtained from volunteer donors by venipuncture and diluted 1/2 with PBS. PBMCs were isolated by centrifugation over lymphocyte separation medium (ICN Biomedicals), washed twice with PBS, and resuspended in serum-free AIM-V medium (Invitrogen Life Technologies). Monocytes were enriched by culturing 107 PBMC/well in six-well tissue culture plates for 2 h. Nonadherent PBMCs were removed and cryopreserved in 90% FBS/10% DMSO. In experiments evaluating cytokine secretion, highly purified monocytes (>90% CD14+) were obtained by immunodepletion of nonmonocytic cells using the Monocyte Isolation kit II (Miltenyi Biotec) according to the manufacturer’s instructions. Monocytes isolated by either method were cultured at 37°C/5% CO2 in complete AIM-V/10% human AB serum supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml). Fresh cytokine was added on days 3 and 6 of culture. The cells were harvested on day 6 as immature DCs, or further matured for 24–48 h with LPS (0.1–1 µg/ml) or for 48 h with recombinant human TNF-{alpha} (20 ng/ml) added daily. All of the clinical reagents were generated under protocols approved by the Committee for the Protection of the Rights of Human Subjects at the University of North Carolina School of Medicine (Chapel Hill, NC).

Generation of recombinant VRPs

The production of VRPs that encode GFP (GFP-VRP) has been previously described (32). The absence of propagating recombinant virus was confirmed by passage in baby hamster kidney (BHK) cells. VRPs were concentrated from supernatants by centrifugation through a 20% sucrose cushion and resuspended in PBS. Titration of GFP-VRPs was determined by infecting BHK monolayers with 10-fold dilutions of VRPs for 16–18 h at 37°C/5% CO2. The infected cells were fixed with 4% paraformaldehyde and GFP-expressing cells were directly visualized by fluorescent microscopy. VRPs that encode FMP (FMP-VRP) were generated by directionally cloning the FMP cDNA, kindly provided by P. Palese (Mount Sinai School of Medicine, New York, NY), immediately downstream of the 26 S mRNA promoter of the pVR21 replicon plasmid; proper orientation was confirmed by DNA sequencing. The FMP replicon plasmid was used to generate FMP-VRPs. For titration, BHK monolayers were infected with 10-fold dilutions of FMP-VRPs for 16–18 h at 37°C/5% CO2. Infected cells were fixed with ice-cold methanol and sequentially stained with mouse anti-FMP mAb, biotinylated anti-mouse IgG, and FITC-conjugated streptavidin. FITC-positive cells were directly enumerated by fluorescent microscopy.

Infection of human DCs with VRPs

Immature or mature DCs were resuspended in serum-free AIM-V at 0.5–1.0 x 106 cells/ml and seeded at 1–2 x 105 DCs per well in 24-well ultra low attachment plates (Corning). For infectivity experiments, 1–2 x 105 DCs were infected with VRPs at different multiplicity of infection (MOI) values over specific time intervals as indicated in the figures. Infections were performed in serum-free conditions at 37°C/5% CO2. After 1–2 h, DCs were washed with AIM-V/10% human AB serum, resuspended in media supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml), and cultured in 24-well ultra low attachment plates at 37°C/5% CO2.

Flow cytometry analysis

For quantification of VRP transduction efficiency, GFP-VRP- or mock-infected DCs were harvested at 6, 12, or 24 h postinfection (p.i.) and washed once with cold FACS buffer (PBS/0.5% human serum albumin). DCs were fixed with PBS/1% formaldehyde before FACS analysis. In some experiments, DC viability was determined using the Fixation and Dead Cell Discrimination kit (Miltenyi Biotec) according to the manufacturer’s instructions. For phenotypic analysis, 5 x 104 DCs were incubated with 200 µg/ml mouse IgG (Sigma-Aldrich) at 4°C for 20 min. Following blocking, the DCs were stained with 2 µl of PE-conjugated specific or isotype control Abs for 30 min at 4°C, washed once with FACS buffer, and fixed with PBS/1% formaldehyde. FACS data were acquired using a FACScan flow cytometer (BD Biosciences), and analyzed using FlowJo software (TreeStar).

Cytokine assays

For evaluation of cytokine secretion by DCs, immature DCs were either mock-infected or infected with GFP-VRPs (MOI = 20) for 2 h at 37°C/5% CO2. Fully mature DCs were generated by treatment for either 24 h with LPS (100 ng/ml) or 48 h with TNF-{alpha} (20 ng/ml). Mock-infected immature DCs, VRP-infected immature DCs, or fully mature DCs were washed and seeded into 96-well flat-bottom tissue culture plates at 105 DCs per well. Supernatants were harvested at 12, 24, 36, or 48 h posttreatment and stored at –80°C. Quantification of IL-6, IL-8, IL-10, IL-12p70, and TNF-{alpha} in the supernatants was performed using the cytometric bead array according to the manufacturer’s instructions (BD Pharmingen). Measurement of IFN-{alpha} was determined by ELISA (BioSource International) according to the manufacturer’s instructions.

Allogeneic MLR

Mock- or GFP-VRP-infected (MOI = 10) DC cultures were harvested after 1 h of infection, washed with media, and resuspended in AIM-V/10% human AB serum. Decreasing numbers of DCs were added in triplicate to 1 x 105 nonadherent allogeneic PBMCs per well in 96-well round-bottom plates, and T cell proliferation assays were performed as previously described (33).

In vitro expansion of FMP-specific T cells

Immature DCs from HLA-A*0201-positive donors were infected for 2 h with either GFP-VRPs or FMP-VRPs (MOI = 10) and washed with AIM-V/10% human AB serum media. DCs (0.2–2 x 105) were added to 2 x 106 autologous nonadherent PBMCs per well in 24-well tissue culture plates. For comparative stimulation of T cells with peptide-pulsed DCs, TNF-{alpha}-matured, or LPS-matured DCs from the same donors were incubated with 10 µg/ml FMP peptide in AIM-V/10% human AB serum for 2 h. FMP peptide-pulsed DCs were washed with media and added to autologous nonadherent PBMCs as earlier described. PBMCs were incubated for 7 days in AIMV/10% human AB serum supplemented with IL-2 (20 U/ml) and IL-7 (10 ng/ml). Fresh cytokine was added on days 3 and 6 of culture, and cell density was maintained at <2 x 106 cells/ml during the entire assay. On day 7, the responders were harvested and evaluated for either Ag-specific expansion by tetramer staining or for specific lysis of peptide-pulsed T2 cells by a conventional 51Cr release assay (34). The percentage of specific lysis was determined using the following formula: Percentage of specific lysis = 100 x [(sample CPM – spontaneous CPM)/(total CPM – spontaneous CPM)].

For tetramer staining, 1 x 106 responders were stained for 30 min with 20 µl of anti-human CD8-FITC (BD Pharmingen) and 10 µl of either PE-conjugated HLA-A*0201/influenza M1 peptide tetramer or HLA-A2*0201/negative tetramer (Beckman Coulter). Cells were washed with PBS, fixed with PBS/0.5% formaldehyde, and analyzed by FACS within 6 h.

Statistical analysis

Statistical differences were calculated using a Student t test when sample data distribution was parametric. Sample data that exhibited nonparametric distribution were evaluated using a Mann-Whitney rank sum test. Differences in costimulatory molecule expression between mock- and VRP-infected DCs from several donors were analyzed using a Wilcoxon signed-rank test. Values of p ≤ 0.05 were considered significant. All statistical analyses were performed with SigmaStat 3.0 software (Systat Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
VRPs can efficiently transduce human immature DCs

Our group has previously demonstrated that VRPs can infect mouse DCs in vivo following footpad injection (32). To determine whether human DCs could be infected with VRPs in vitro, immature monocyte-derived DCs were generated from normal donors. By day 6 of culture, DCs exhibited a typical immature phenotype (CD11c+, HLA-DR+, CD86+, CD14–, CD40–, CD80–) when evaluated by flow cytometry (data not shown). Immature DCs were infected with GFP-VRPs at an MOI of 10. GFP expression in DCs was first detectable at around 4 h p.i. and reached a maximum value between 6 and 12 h p.i. (Fig. 1A).



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  		FIGURE 1. VRPs can efficiently transduce immature human DCs. A, Human immature monocyte-derived DCs were infected with GFP-VRPs for 1 h (MOI = 10). Cells were analyzed for GFP expression at 12 h p.i. by fluorescent microscopy. The white arrow indicates possible apoptosis of infected DCs. B, Infectivity of immature or LPS-matured DCs (n = 3 donors) was quantified by FACS. DCs were infected for 1 h (MOI = 10) and GFP expression was evaluated at 6–24 h p.i. as indicated. Symbols represent mean percentage of GFP-positive DCs ± SEM. C, DCs were infected with GFP-VRP as described, harvested at 24 h p.i., stained with PE-conjugated Abs specific for CD11c, HLA-DR, or CD14, and analyzed by two-color FACS.

 
To quantify VRP transduction efficiency, mock- or GFP-VRP-infected (MOI = 10) immature DCs were harvested at 6, 12, or 24 h p.i. and analyzed for GFP expression by flow cytometry. As shown in Fig. 1B, VRPs could infect human immature DCs at an MOI of 10, with a mean of 10.8% expressing GFP by 6 h p.i. The mean percentage of GFP-positive immature DCs peaked at 18.4% at 12 h p.i. and then decreased to 15.5% by 24 h p.i. To determine whether the maturation status of the DCs affected the transduction efficiency, immature DCs were stimulated with LPS for 2 days, resulting in marked up-regulation of CD40, CD80, CD83, and CD86 (data not shown). LPS-matured DCs were minimally transduced by GFP-VRPs (Fig. 1B). DCs matured with TNF-{alpha} for 2 days were also less susceptible to VRP infection (mean 6.2% GFP-positive at 12 h p.i.), although not to the same degree as found using LPS-matured DCs. Thus, VRP transduction efficiency was inversely related to the degree of DC maturation.

To verify that the GFP-positive cells exhibited a DC phenotype, we performed two-color FACS analysis on infected DC cultures. GFP-VRP-infected DCs were harvested at 24 h p.i. and stained with PE-conjugated Abs specific for CD11c, HLA-DR, and CD14. GFP-positive cells expressed high levels of CD11c and HLA-DR, and did not express the monocyte-marker CD14 (Fig. 1C). To demonstrate that VRPs specifically infected immature DCs, the ability of VRPs to transduce peripheral blood T cells, B cells, and monocytes was determined. We did not observe infection of CD3+ T cells and CD19+ B cells with GFP-VRPs (MOI = 10), and only minimal (~2%) transduction of CD14+ monocytes (data not shown). Thus, VRPs specifically infected immature DCs.

We next evaluated approaches that could enhance the efficiency of VRP transduction of immature DCs. Increasing the MOI improved the transduction of immature DCs by GFP-VRPs. At an MOI of 100, ~50% of DCs expressed GFP (Fig. 2A). The percentage of GFP-positive DCs began to plateau between an MOI of 50 and 100, suggesting that transduction efficiency was near maximum. In an effort to maximize transduction efficiency at a lower MOI, we increased the duration of infection and the DC concentration during infection. By doubling both the time of infection and the DC concentration during infection at an MOI of 20, the transduction efficiency increased from a mean of 22.5% to 37.0% (n = 3, p = 0.002) (Fig. 2B). Thus, immature DCs can be efficiently transduced with relatively small quantities of VRPs.



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  		FIGURE 2. VRP infection of immature DCs is dependent upon the MOI, length of infection and cell density during infection. A, Immature DCs (n = 3 donors) were infected for 1 h with GFP-VRPs at increasing MOI. The percentage of GFP-positive cells was determined at 12 h p.i. by FACS. B, Immature DCs (n = 3 donors) were infected with GFP-VRPs (MOI = 20) for 1 h at 0.5 x 106 DC/ml or for 2 h at 1 x 106 DC/ml. The percentage of GFP-positive cells was determined at 12 h p.i. by FACS. Graphs represent the mean percentage of GFP-positive cells ± SEM. *, p = 0.002, Student’s t test.

 
The percentage of GFP-expressing DCs began to decline between 12 and 24 h p.i. (Fig. 1B), suggesting that VRP infection may be cytopathic to human DCs. Alphaviruses and alphaviral vectors induce apoptosis in cultured cells (35), although their ability to similarly induce cell death in human DCs is unknown. We therefore compared the viability of VRP-infected (GFP-positive) DCs to uninfected (GFP-negative) DCs in the culture by exclusion of a vital dye. VRP-infected DCs exhibited >90% viability between 6 and 12 h p.i., and remained ~75% viable at 24 h p.i. (Fig. 3). However, by 48 h p.i. only 26% of the DCs remained viable compared with 66% of the uninfected DCs. This loss in viability was associated with increased expression of annexin V by VRP-infected DCs (data not shown), suggesting that VRP-induced apoptosis was likely responsible for the death of human DCs. In summary, the viability of VRP-transduced DCs remained high for 24 h following infection, but steadily decreased between 24 and 72 h p.i.



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  		FIGURE 3. VRP-infected DCs remain predominantly viable during the first 24 h following infection. Immature DCs were infected for 2 h with GFP-VRP (MOI = 20). At various times p.i., viability of infected (GFP-positive) DCs and uninfected (GFP-negative) DCs was determined as described in Materials and Methods. Graphs represent the mean percentage of viable cells from two experiments.

 
VRP infection induces DC maturation and secretion of proinflammatory cytokines

The studies we described indicated that immature DCs could be easily transduced with VRPs. However, immature DCs are poor stimulators of Ag-specific T cells and have been shown to induce tolerance (36, 37). Thus, we wanted to determine whether VRP-infection induced maturation of immature DCs by evaluating expression of costimulatory and maturation surface markers (Fig. 4). At 12 h p.i., the expression of various costimulatory/maturation markers in VRP-infected DC cultures was similar to DCs that were mock-infected or treated with TNF-{alpha}. In contrast, DCs treated with a strong maturation stimulus (100 ng/ml LPS) had up-regulated CD80 and CD86 expression at this time. By 24 h p.i., however, the expression of CD40, CD80, and CD86 was significantly elevated in VRP-infected DC cultures when compared with mock-infected or TNF-{alpha}-treated DCs. CD86 expression in VRP-infected DC cultures at 24 h p.i. was comparable to that seen with LPS treatment, whereas LPS induced higher levels of CD80 and CD83. Interestingly, VRP-infection induced higher levels of CD40 expression when compared with LPS treatment, a trend that was consistent in four different experiments. We next determined whether VRP infection induced maturation of both infected and uninfected bystander DCs by analyzing costimulatory/maturation marker expression on GFP-positive and -negative DCs in the culture (Table I). The expression of costimulatory/maturation molecules was increased on both GFP-positive and GFP-negative DCs, although the latter exhibited the highest expression levels at 24 h p.i. These observations indicate that VRP infection resulted in phenotypic maturation of both infected and uninfected immature DCs within the same culture.



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  		FIGURE 4. VRP infection induces maturation of immature DCs. Immature DCs were either mock-infected (gray filled histogram), infected for 2 h with GFP-VRPs at an MOI of 20 (thick line histogram), treated with TNF-{alpha} at 20 ng/ml (thin line histogram), or treated with LPS at 100 ng/ml (dashed line histogram). DCs were harvested at 12 or 24 h p.i. and stained with the indicated PE-conjugated specific Abs. Staining with isotype control Abs was negative. The numbers indicate the median PE fluorescence intensity. The median costimulatory/maturation marker expression in VRP-infected DC cultures includes both GFP-positive and GFP-negative cells. Data are representative of four experiments.

 

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  		Table I. Median fluorescence intensity of costimulatory/maturation proteins following VRP infection of DCsa

 
We next evaluated secretion of proinflammatory cytokines by GFP-VRP-infected DC cultures (Fig. 5). Because DC cultures generated from adherent PBMCs contained a small but significant population of contaminating lymphocytes (15–45%), we generated highly purified DCs from monocytes that had been isolated by negative selection using immunomagnetic beads. DC cultures generated by this method were >95% CD11c+, and were similar to adherent monocyte-derived DCs in both surface marker phenotype and susceptibility to VRP infection (data not shown). These DCs were mock- or VRP-infected and supernatants were collected and assayed for proinflammatory cytokines at various time points p.i. In contrast to mock-infected DCs, VRP-infected immature DCs secreted significant amounts of TNF-{alpha}, IL-6, and IFN-{alpha} at 24–48 h following infection. Low but statistically significant levels of IL-12p70 were detected at later time points (36–48 h p.i.). IL-10 was also barely detectable in the supernatants from VRP-infected DCs, but the levels were not significantly higher than in mock-infected DC supernatants. By comparison, DCs that had been matured by either 24 h with LPS or 48 h with TNF-{alpha} did not secrete significant amounts of TNF-{alpha}, IL-6 and IFN-{alpha}. LPS- and TNF-{alpha}-matured DCs secreted IL-8 (Fig. 5) and displayed increased costimulatory molecule expression (Fig. 4), demonstrating that these cells had been activated. Additionally, incubating mature DCs with an MHC class I-restricted peptide (FMP peptide 58–66) did not affect cytokine secretion (data not shown). In summary, VRP infection of immature human DCs induced maturation and proinflammatory cytokine secretion.



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  		FIGURE 5. VRP-infected DCs, but not fully matured DCs, secrete high levels of proinflammatory cytokines. Supernatants from immature DCs that were either mock-infected (Mock-DC) or GFP-VRP-infected (VRP-DC) at an MOI of 20 were harvested and analyzed for specific cytokines by cytometric bead array (TNF-{alpha}, IL-6, IL-12p70, or IL-8) or ELISA (IFN-{alpha}). Supernatants from DCs that had been previously matured by 24 h of LPS treatment (LPS-DC) or 48 h of TNF-{alpha} treatment (TNF-DC) were also analyzed. The mean cytokine concentration ± SEM from three donors is shown. Data are representative of two experiments. *, p < 0.05 (Student’s t test) when compared with mock-infected DC, TNF-{alpha} treated DC, or LPS-treated DC.

 
VRP-infected human DCs can stimulate allogeneic and Ag-specific T cells

To initially evaluate the functionality of VRP-infected DC cultures, we performed a standard allospecific T cell stimulation assay. DCs infected with GFP-VRP stimulated substantial proliferation of allogeneic T cells, indicating that VRP infection did not have a detrimental effect on DC function (data not shown). More importantly, we determined whether VRP-transduced DCs could stimulate expansion of autologous T cells specific for a VRP-encoded Ag. For this set of experiments, we used recombinant VRPs expressing FMP. When autologous PBMCs were stimulated with an irrelevant VRP expressing GFP, there was no significant increase in the percentage of FMP-specific CD8+ T cells (Fig. 6, A and B). However, stimulation of PBMCs with FMP-VRP-transduced DCs led to a significant increase in the percentage of FMP-specific CD8+ T cells (Fig. 6, A and B). VRP-transduced DCs were highly efficient at expanding FMP-specific CD8+ T cells at even low DC numbers (Fig. 6C). Furthermore, the expanded FMP-specific CD8+ T cells were functional as they could lyse T2 cells pulsed with the FMP peptide (Fig. 6D). When we compared FMP-VRP-transduced DCs to TNF-{alpha}-matured DCs pulsed with FMP peptide, we found that FMP-VRPs were significantly more effective at inducing expansion of FMP-specific CD8+ T cells compared with FMP-pulsed DCs (Fig. 6, A and B). However, peptide-pulsed DCs matured with a more potent stimulus (100 ng/ml LPS) induced comparable expansion of FMP-specific CD8+ T cells when compared with FMP-VRP-infected DCs (42 and 38% tetramer-positive cells, respectively, responder to stimulator ratio is 10:1). VRP-infected DC can thus process and present vector-encoded Ags to reactive T cells, resulting in significant T cell expansion and acquisition of effector function.



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  		FIGURE 6. VRP-transduced DCs stimulate greater expansion of Ag-specific CD8+ CTL compared with TNF-{alpha}-matured DCs pulsed with peptide. Immature DCs were transduced with either FMP-VRPs or irrelevant GFP-VRPs for 2 h (MOI = 20). DCs were washed and cocultured with autologous nonadherent PBMCs at various responder to stimulator ratios in the presence of IL-2 and IL-7 for 7 days. A, Expansion of FMP-specific CD8+ T cells was determined by tetramer analysis. Baseline indicates the percentage of FMP-specific T cells before stimulation. The stimulatory capacity of VRP-infected DCs was compared with TNF-{alpha}-matured DCs (TNF-DC) that had been pulsed with FMP peptide (10 µg/ml) for 2 h or left untreated (responder to stimulator ratio = 20:1). Numbers represent the percentage of FMP-specific cells of total CD8+ T cells. B, Mean percentage of FMP-specific CD8+ T cells on day 7 of stimulation from three experiments. *, p < 0.05 (Student’s t test). C, Percentage of FMP-specific CD8+ T cells on day 7 of stimulation at various responder to stimulator ratios. One of two similar experiments is shown. D, PBMCs that had been stimulated with FMP-VRP-infected DCs for 7 days were assayed for effector function in a standard 51Cr release assay. Labeled FMP peptide-pulsed ({circ}) or unpulsed ({blacktriangleup}) T2 cells were incubated with effector cells for 4 h and specific lysis was calculated as described in Materials and Methods. Lysis of the NK-sensitive cell line K562 ({blacksquare}) was similar to that found using unpulsed T2 cells indicating that the enhanced lytic activity using FMP-pulsed T2 cells was not due to NK-mediated lysis. The graphs represent the mean of triplicate wells ± SEM. One of two similar experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Consistent generation of tumor-specific T cell responses in patients treated with DC-based vaccines has remained elusive. The shortcomings of current vaccines are due in part to inefficient Ag loading of DC, as well as unsatisfactory induction of DC maturation and proinflammatory cytokine secretion (38). To address these issues, we evaluated the use of vectors derived from VEE. We have demonstrated that VRPs can efficiently transduce human immature DCs in vitro, leading to DC maturation and secretion of proinflammatory cytokines. Furthermore, transduced DCs processed and presented a VRP-encoded Ag and stimulated significantly greater expansion of Ag-specific T cells in comparison to peptide-pulsed DCs matured with TNF-{alpha}.

Several vectors have been used for transducing human DCs with TAAs (24, 25, 26, 27). However, the potential clinical use of these vectors is hindered by poor transduction efficiencies, inhibition of DC maturation, questionable safety, and induction of detrimental antivector immune responses. The use of VRPs for DC transduction successfully addresses many of these concerns. We have shown that VRP transduction efficiency is appreciable at an MOI of 20; efficiency can be further enhanced at higher MOIs. VRPs have an outstanding safety record in thousands of animal experiments including both rodents and primates (30, 39). Because VEE is only endemic to specific subtropical regions, pre-existing immunity to VRPs is unlikely to be present in the majority of patients. Finally, our group and others have shown that VRPs can induce cell-mediated and humoral immune responses (31, 40). Recent work suggests that induction of both a humoral and cellular antitumor response may increase the effectiveness of tumor vaccines (41).

One concern from our data was the cytopathic effect of VRPs on human DCs. After terminal maturation, DCs remain viable in vitro for ~96 h (42). However, we found that VRP-infected DCs began to lose viability within 48 h following infection. The shortened lifespan of VRP-transduced DCs could limit their effectiveness in tumor immunotherapy. However, studies have shown that DCs injected intradermally migrate rapidly (within 12–24 h) to draining lymph nodes (43). Additionally, several groups have found that DC-T cell interactions in vivo occur during the first 24 h following immunization (44, 45). As shown, the majority of VRP-infected DCs are viable during the first 24 h following infection, which should allow sufficient time for infected DCs to migrate to regional lymph nodes and interact with T cells. Furthermore, the induction of apoptosis in transduced DCs may actually be advantageous because cross-presentation of Ag from apoptotic DCs can effectively induce Ag-specific CD8+ T cells (46, 47, 48). Indeed, apoptosis was necessary for the enhanced efficacy of alphaviral replicase-based DNA vaccines in an in vivo tumor challenge model (47). Our group has preliminary evidence that VRP-infected DCs can generate protective immunity in a tolerant breast cancer animal model (T. Moran and J. Serody, unpublished observations). Thus, we do not believe that the shortened lifespan of VRP-infected DCs should provide a significant impediment to their use in vivo.

VRP infection not only resulted in production of TAAs within the cytoplasm of human DCs, but also induced maturation of DCs and proinflammatory cytokine secretion. VRP infection resulted in up-regulation of the costimulatory molecules CD40, CD80, and CD86, and the maturation marker CD83. This is consistent with previous observations that replicons derived from Sindbis virus, a related alphavirus, induced maturation of human DCs (49). Interestingly, costimulatory/maturation marker expression was induced on both infected and uninfected DCs within the same cultures. VRP-induced maturation of bystander DCs is potentially advantageous, as this could enhance cross-presentation of Ag from infected DCs undergoing apoptosis. In addition to phenotypic maturation, VRP infection resulted in secretion of proinflammatory cytokines including IFN-{alpha}, TNF-{alpha}, IL-6, and IL-12p70. IFN-{alpha}, TNF-{alpha}, and IL-6 are important for activation of APCs, and are likely responsible for maturation of uninfected bystander DCs (50, 51). IFN-{alpha} also enhances the efficiency of cross-presentation of Ag by DCs (52), a mechanism that is important for in vivo priming of tumor-specific CD8+ CTL (53). Furthermore, recent studies suggest that IL-6 secretion by DCs is important for inhibiting CD4+CD25+ regulatory T cell activity (54, 55). In contrast to VRP-infected DCs, DCs that had been terminally matured with TNF-{alpha} or LPS did not secrete significant levels of IFN-{alpha}, TNF-{alpha}, IL-6, or IL-12p70. This observation is in line with other publications describing the inability of fully matured DCs to secrete several proinflammatory cytokines (56, 57). In summary, VRPs represent a novel strategy to deliver TAAs and a strong maturation signal simultaneously to human DCs.

In the current study, we have shown that VRP-infected DCs could efficiently stimulate Ag-specific T cell responses against a VRP-encoded Ag. Furthermore, VRP-infected DCs were more efficient at expanding Ag specific CD8+ T cells when compared with TNF-{alpha}-matured DCs pulsed with an MHC class I-restricted peptide. Increased costimulatory molecule expression and secretion of proinflammatory cytokines are likely responsible for the enhanced immunostimulatory capacity of VRP-infected DCs. It is also possible that VRP-transduced DCs are presenting MHC class II-restricted epitopes to CD4+ Th cells in the PBMC cultures, which would augment activation and expansion of CTL (58). The ability of VRP-transduced DCs to stimulate CD4+ T cells is under current investigation.

In conclusion, we have found that immature DCs can be readily transduced with VRPs and these DCs can induce potent Ag-specific T cell expansion. Our group is currently pursuing this strategy for vaccination of patients with cancer.


   Acknowledgments
 
We thank members of the laboratories of Drs. Serody and Kirby for donation of PBMC, Kathryn A. Chwastiak for technical assistance, and Arlene Mendoza-Moran for critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
N. L. Davis and R. E. Johnston own stock in AlphaVax, a company formed to develop the VEE vectors.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by Grant P50 CA58223 from the National Institutes of Health (to J.S.S.). Back

2 Address correspondence and reprint requests to Dr. Jonathan S. Serody, Lineberger Comprehensive Cancer Center, Campus Box 7295, University of North Carolina, Chapel Hill, NC 27599-7295. E-mail address: Jonathan_Serody{at}med.unc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; TAA, tumor-associated Ag; VEE, Venezuelan equine encephalitis virus; VRP, VEE replicon particle; p.i., postinfection; FMP, influenza matrix protein; MOI, multiplicity of infection. Back

Received for publication November 12, 2004. Accepted for publication June 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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