Activation of Human Dendritic Cells by Recombinant Modified Vaccinia Virus Ankara Vectors Encoding Survivin and IL-2 Genes In Vitro

Abstract

Modified vaccinia virus Ankara (MVA) has attracted significant attention as a safe, promising vector for immunotherapy. However, the precise effects of MVA infection on immune responses in humans remain largely unknown. We constructed recombinant MVA (rMVA) encoding both a human tumor-associated antigen (survivin) and the proinflammatory cytokine interleukin (IL)-2 and investigated their effects on human monocyte-derived dendritic cells (DCs). The results showed that infection with rMVA slightly impaired the upregulation of CD83 and reduced the production of IL-10 in DCs after lipopolysaccharide stimulation. However, rMVA-infected DCs were still able to express high levels of target genes and the costimulatory molecules CD80 and CD86 and to produce significant amounts of the proinflammatory cytokine tumor necrosis factor alpha. Moreover, rMVA-infected DCs exhibited a greater capacity than uninfected cells to stimulate T-cell proliferation and to reverse MVA-induced apoptosis in syngeneic T cells. Coculture of lymphocytes with rMVA-infected DCs significantly increased cytotoxic potential and interferon gamma production by cytotoxic T cells. These findings suggest that rMVA encoding survivin and IL-2 can effectively stimulate the activation of human DCs and overcome defects such as impairment of DC maturation and apoptosis of lymphocytes that are caused by vector alone. Thus, this study may provide a rational basis for further optimization of MVA vector.

Introduction

M odified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, was derived from the vaccinia virus Ankara strain, which can be used under conditions of biosafety level 1 because of its avirulence and its inability to productively grow in human cells (Drexler et al., 2004; Shen and Nemunaitis, 2005). Several unique features make recombinant MVA (rMVA) an attractive candidate as a vector for use in vaccination or immunotherapy, namely, (i) large foreign gene capacity, (ii) high levels of recombinant protein expression in most human and mammalian cells, (iii) potent induction of humoral and cellular immune responses, and (iv) long-term stability in a frozen or lyophilized state (Moss, 1991; Pastoret and Vanderplasschen, 2003). Accordingly, significant progress has been made with respect to the development of rMVA vector technologies in recent years (Perez-Jimenez et al., 2006; Wang et al., 2008). Vaccine research based on rMVA expressing various tumor-associated antigens (TAAs) are currently being carried out in mice and humans with promising results (Nemeckova et al., 2003; Smith et al., 2005; Acres, 2007; Amato, 2007), and several rMVA vector vaccines have already been tested in phase I/II clinical trials (Harrer et al., 2005; Cebere et al., 2006).

One interesting vaccine strategy is based on the combination of dendritic cells (DCs) and rMVA for delivery of vaccine antigens (Chahroudi et al., 2006; Liu et al., 2008). DCs are the most potent antigen-presenting cells (APCs) in humans and play a critical role in the initiation of antitumor immune responses (Vulink et al., 2008). The premise on which association of the application of MVA vector vaccines with DCs is based is that more potent antigen-specific immune responses are likely to be induced in the context of DC-mediated immune signaling. This strategy could be advantageous for enhancing the immunogenicity of TAAs (Gasteiger et al., 2007).

Previous studies have shown that MVA infection of DCs resulted in early, but not late, viral gene expression (Bronte et al., 1997; Engelmayer et al., 1999; Jenne et al., 2000) and that viral DNA did not accumulate in infected cells (Drillien et al., 2000). Chahroudi also found that, unlike non-APCs that typically demonstrate cell lysis as a consequence of the viral cytopathic effect (Ramsey-Ewing and Moss, 1998), infection with MVA can block the differentiation of macrophages and DCs and even induce their apoptosis (Chahroudi et al., 2006), which may, in theory, impair the capacity of these APCs to elicit effective host immune responses. In the investigation of viral vaccines, the interaction between MVA and the host immune reaction has been shown to be multifactorial, and a strong immune response could serve either as a foe or as an ally of MVA-mediated cancer therapies. Therefore, the role of the immune system became one of the most important foci in research regarding immunotherapy mediated by rMVA vectors (Gomez et al., 2008).

Although several rMVA vector systems are under clinical evaluation for vaccine development against cancer, data on the influence of rMVA infection on DCs remain scarce. Therefore, the aim of this study was to further investigate the effect of rMVA vector on the differentiation, maturation, and functional activities of human DCs and to explore the possibility of overcoming the intrinsic defects induced by infection of these cells with rMVA vector by incorporating human survivin (Kato et al., 2001; Idenoue et al., 2005) and IL-2 genes. We found that DCs infected with rMVA slightly impair the expression of CD83 after lipopolysaccharide (LPS) stimulation. However, these infected DCs do express high levels of target genes, and they retain high expression of costimulatory molecules and Type 1 T helper cell (Th1) cytokines. Upon encountering syngeneic T cells, DCs infected by rMVA survivin/IL-2 can efficiently stimulate T-cell proliferation, with reduced apoptosis. Further, these DCs are more potent than rMVA-Sur-infected cells in stimulating the activation of syngeneic cytotoxic T cells. In general, our results provide evidence that rMVA vectors containing survivin and IL-2 genes may represent a promising strategy for future rational design of MVA vectors for clinical application.

Materials and Methods

Cells and reagents

Syrian hamster kidney cell line BHK-21 was obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and was maintained in complete medium composed of RPMI-1640 supplemented with 10% fetal bovine serum. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were obtained from R&D Systems (Minneapolis, MN). Fluorescence-conjugated antibodies (Abs) against human CD3, CD8, CD80, CD83, CD86, survivin, CD107a, perforin, and interferon gamma (IFN-γ), along with relevant isotype controls, were obtained from BD PharMingen, R&D Systems, or eBioscience (San Diego, CA). Tissue culture plates were purchased from Corning-Costar (Corning, NY). Cell isolation and other tissue culture reagents were purchased from Invitrogen (Grand Island, NY). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated in the text.

Generation of rMVA

Full-length cDNA template of survivin or IL-2 was obtained by RT-PCR. Total RNAs were isolated from human peripheral blood lymphocytes using Trizol reagent (Invitrogen). Reverse transcription was performed using oligo-(dT) primer and Reverse Transcriptase System (no. A3500; Promega, CA, USA) according to the manufacturer's protocol. One recombinant cDNA survivin was amplified by PCR using the primers F-sur-5′-TTTGTCGACGAATTGGATCCGCCACCATGGGT-3′ and R-sur-5′-TTTAGATCTATTCTCAATCCATGGCAGCCAGC-3′ and was cloned into the SmaI and SalI sites of the vaccinia pSC65 plasmid under control of the synthetic vaccinia early/late promoter (pE/L) (Chakrabarti et al., 1997). The plasmid also contained the selectable marker LacZ under the control of the vaccinia p7.5 promoter. KOD-plus-DNA polymerase (Toyobo, Osaka, Japan) was used in all PCR reactions to produce PCR products with blunt ends suitable for cloning into the SmaI site. In addition, the overlapping PCR method (Heckman and Pease, 2007) was used to splice the recombinant fragment survivin-pE/L-IL-2 (Fig. 1A) containing a survivin gene, the pE/L promoter, and the IL-2 gene to express each of these genes from an independent pE/L promoter placed in head-to-tail orientation to avoid undesired recombinant events in mammalian cells (He et al., 1998). The primers used for overlapping the PCR primer were as follows:

F-sur-5′-TTTGTCGACGAATTGGATCCGCCACCATGGGT-3′ (P1);

R2-sur-5′-CGATAAAAATTAATTAATTAGAACTTCAATCCATGGCAGCCAGCT-3′ (P2);

F1-IL2-5′-ATGGTCGACTAATCAATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTG-3′ (P3);

F2-IL2-5′-GAATATAAATAAGCTCGTAATAAAGATCTATGTACAGGATGCAACTCCTGTCTTG-3′ (P4);

F3-IL2-5′-TGGTACCAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAGCTCGT-3′ (P5);

F4-IL2-5′-AGTTCTAATTAATTAATTTTTATCGATCTAAGCTTGGTACCAAAAATTGAAATT-3′ (P6);

R-IL2-5′-GGGTTAATTATCAAGTCAGTGTTGAGATGATGCTTTGACAAAAGGTAATCC-3′ (P7).

Overlapping PCR was designed as shown in Fig. 1B. The primers P3–P5 are the overlapping primers used to ligate the sequence of pE/L promoter into IL-2 cDNA sequence. To construct plasmid pSC65-Sur-IL-2, the recombinant fragment survivin-pE/L-IL-2 was also cloned into the pSC65 plasmid digested by SalI and SmaI. The correct recombinant plasmids were confirmed by PCR using survivin and IL-2-specific primers and sequencing. To construct rMVA, vSur (rMVA encoding survivin), and vD10 (rMVA encoding survivin and IL-2), the recombinant plasmids were transfected into wild type MVA-infected BHK-21 cells using lipofectamine (Gibco BRL, CA, USA) according to standard homologous recombination protocols (pSC65 and wild type MVA were provided by Linda Wyatt and Bernard Moss Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases [NIAID], National Institutes of Health) (Broder and Earl, 1997). An empty pSC65 plasmid was similarly transfected to construct recombinant vaccinia virus expressing only LacZ (v65) as a negative control. All the rMVA were purified after 8–10 rounds of plaque screening in the presence of 40 ng/ml X-gal (Takara, Shiga, Japan).

FIG. 1.

A schematic map of construction of recombinant plasmids pSur and pD10 encoding survivin or both survivin and IL-2 target genes. (A) The coding gene sequence of survivin or the double gene cassette was placed under the transcriptional control of pE/L promoter and cloned into the SmaI and SalI sites of the recombinant vaccinia pSC65 plasmid. (B) The flow diagram of construction of the double gene cassette using overlapping PCR. The sequences of forward and reverse primers shown in the diagram are given in the Materials and Methods section. E, early; IL-2, interleukin-2; L, late; P, primers; pE/L or p7.5, promoter.

Isolation and preparation of DCs and CD3⁺ T cells

Monocyte-derived DCs were prepared as previously described (Kuang et al., 2007, 2008). Briefly, peripheral blood mononuclear cells (PBMCs) from buffy coats of normal healthy donors were separated by Ficoll-hypaque density gradient centrifugation and purified using anti-CD14 magnetic beads in a MACS column purification system (Miltenyi Biotec, Bergisch Gladbach, Germany). The resulting monocytes were cultured for 6–7 days in RPMI-1640 supplemented with 10% fetal bovine serum, 40 ng/ml GM-CSF, and 40 ng/ml IL-4. On days 3 and 5, half of the medium was replaced with fresh medium and immature DC (iDC) (CD1a⁺ HLA-DR⁺ CD80^low CD83^low CD86^low cells) were collected after 6–7 days. The purity of the iDC obtained was >85%. In some experiments, syngeneic CD3⁺ T cells were isolated from PBMCs by negative selection using magnetic microbeads in the MACS column purification system according to the manufacturer's instructions and were used as responder cells in the later mixed leukocyte reaction (MLR). The purity of the T cells obtained, as determined by CD3 staining, ranged from 88% to 95%.

Western blotting for detection of survivin protein

Cells were lysed in buffer containing 1% Igepal, 50 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) (pH 7.5), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 10% glycerin, 1 mM phenylmethylsulfonyl fluoride, and 2 μg/ml aprotinin. Total protein was separated on 15% polyacrylamide gels, blotted onto nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), and probed with a survivin-specific Ab (rabbit mAb, no. 2808; Cell Signaling, Boston, USA). Bands were visualized by electrogenerated chemiluminescence staining (Amersham Pharmacia, Freiburg, Germany).

Fluorescence-activated cell sorting analysis of DC phenotype and expression of exogenous survivin

Expression of molecular markers and exogenous survivin on DC surfaces was determined by flow cytometry. Briefly, DCs (infected or uninfected) were washed and incubated with fluorochrome-conjugated mAb (anti-human survivin, CD80, CD83, CD86, or relevant isotype-matched controls) according to the manufacturer's instructions. The cells were gated according to their size and granularity using a BD FACSVantage flow cytometer (BD, San Diego, CA). At least 10,000 cells were acquired for each sample. Expression was analyzed by the CellQuest software. To examine survivin intracellular expression by infected DCs, cells underwent fixation using 4% paraformaldehyde for 10 min and permeabilization for 15 min at 20°C using SAP buffer (a sterile solution containing 0.1% [w/v] saponin and 0.05% [w/v] NaN₃ in Hanks' balanced salt solution). The samples were then washed twice, stained using survivin mAb according to the manufacturer's instructions, and analyzed by fluorescence-activated cell sorting (FACS).

ELISA for cytokine detection

Immature DCs were treated as indicated and incubated for 24 hr. The supernatants were centrifuged to remove particulate debris and stored at −80°C until use. The cytokines IL-2, tumor necrosis factor alpha (TNF-α), IL-10, IL-12p70, and IFN-γ in the culture supernatants were measured by specific ELISA kits (eBioscience) according to the manufacturer's protocols.

MLR and proliferation assay of T cells

DCs were infected with rMVA v65, vSur, or vD10 for 1 hr at 37°C as described, with uninfected DCs in parallel experiments serving as negative control. After washing, 4 × 10⁴ DCs were cultured with 4 × 10⁵ purified syngeneic T cells at a ratio of 1:10 in 96-well round-bottom plates. To detect T-cell proliferation, triplicate cultures were maintained for 20 or 40 hr at 37°C in a 5% CO₂ incubator, followed by a 6-hr period of pulsing with 5-bromo-2′-deoxyuridine (BrdU) (10 μM) to label the proliferating cells. The BrdU assay procedure was carried out according to the manufacturer's original protocol (Cell Proliferation ELISA BrdU, colorimetric; Roche Diagnostics, Mannheim, Germany). The reaction was quantified by measuring the optical density at a wavelength of 450 nm and at a reference wavelength of 570 nm.

Apoptosis assay of T cells

To detect CD3⁺ T-cell apoptosis induced by infected or uninfected DCs, cells were labeled with anti-human CD3-PE (CD3-phycoerythrin) and annexin V after coculture for 4 days. Optimal compensation and gain setting were determined for each experiment based on unstained and single-stained samples. In all cases, gates based on forward light scatter and side light scatter were set to exclude cell debris. The apoptosis ratio was calculated using the following formula: Apoptosis ratio = annexin V⁺ CD3⁺ cells/total CD3⁺ cells.

FACS assay on CD107a, perforin, and IFN-γ expression of T cells

Syngeneic T cells were incubated with infected DCs in a 10:1 ratio for 40 hr at 37°C in a 5% CO₂ incubator, followed by an additional 5-hr incubation during which 0.2 μg/ml each of soluble anti-CD3 and anti-CD28 mAbs (eBioscience) were added in the presence of brefeldin A (10 μg/ml). Unstimulated or mock DC-stimulated cells were included as controls. For intracellular staining, the cells were first stained with anti-CD8-PEcy5 and anti-CD107a-FITC (fluorescein isothiocyanate) and then fixed and permeabilized with 4% paraformaldehyde and SAP buffer. Thereafter, the cells were incubated with anti-IFNγ-PE (or anti-perforin-PE) Abs for 30 min and then washed and fixed in 0.5% paraformaldehyde. Multicolor flow cytometry data were acquired on a FACSVantage SE and analyzed with CellQuest software, first gating on the lymphocyte population identified by forward and side scatter and subsequently by analysis of IFN-γ, CD107a, or perforin-positive populations within the CD8^+/− gate.

Statistical analysis

The data in the study represent mean ± standard error of the mean. Statistical significance was determined by Student's t-test; p < 0.05 was considered statistically significant.

Results

Dose-dependent expression of exogenous protein by rMVA-infected iDCs

To examine the expression of recombinant proteins induced by rMVA virus, BHK-21 cells were infected by rMVA v65, vSur, or vD10 for 1 hr at a multiplicity of infection (MOI) of 0.05. After 48 hr, the cells were collected for western blot. HepG2 cells were used as the positive control for survivin expression (Qiao et al., 2008). The results showed that BHK-21 cells infected with vSur or vD10 expressed significant amounts of survivin protein (Fig. 2A) and that no detectable survivin was found in uninfected or v65-infected cells. Unlike BHK-21, DCs were nonpermissive for MVA replication. Studies have shown that MVA infection in nonpermissive cells proceeds through early and late gene expression, but is blocked at later times at a step of virion morphogenesis (Sutter and Moss, 1992). To measure the expression of exogenous protein in rMVA-infected DCs, immature DCs were generated as reported previously (Kuang et al., 2007, 2008) and were infected with the vSur vector at four different MOIs. As shown in Fig. 2B, the total expression of survivin in infected DCs after 24 hr exhibited a dose-dependent pattern. However, higher MOI (>2) of rMVA in some cases led to programmed cell death of DCs (data not shown). Further, when infected with vD10 instead of vSur, the production of IL-2 in the culture supernatant was not affected by the presence of LPS (Fig. 2C and D).

FIG. 2.

Expression analysis of target genes and antigen presentation of survivin. (A) Western blot analysis of BHK-21 cell lysates, as described in the Results section. (B) Western blot analysis of iDCs lysates prepared at 24 hr after exposure to v65 or vSur at indicated MOIs for 1 hr. (C) ELISA analysis of IL-2 expression by infected BHK-21 cells or DCs (D). (C) BHK-21 cells were infected as previously described; supernatants were collected at 24 hr postinfection and tested using IL-2 ELISA. (D) DCs were infected on day 6 at an MOI of 1 by vSur and vD10 for 2 hr; supernatants and cells were collected and tested after 24 hr culture. Some groups underwent a 24-hr, 40 ng/ml LPS treatment after infection, as indicated by the gray columns. (E) FACS analysis of survivin intracellular expression in vSur-infected DCs at different MOIs indicates the infection efficiency, as described in the Materials and Methods section. (F) FACS analysis of survivin presentation on the surface of DCs, as described in the Materials and Methods section. DC, dentritic cell; FACS, fluorescence-activated cell sorting; iDC, immature DC; LPS, lipopolysaccharide; MOI, multiplicity of infection.

To examine antigen presentation by DCs infected with vSur or vD10, we used flow cytometry to detect total and surface levels of survivin proteins. The results demonstrated that these recombinant proteins were not only distributed in the cytoplasm of infected cells but were also present on the surface of DCs at 24 hr postinfection (Fig. 2E and F). This result suggests that an rMVA-based DC vaccine might allow effective presentation of specific tumor antigens.

Effect of MVA or rMVA on DC phenotype

To investigate the phenotype of human monocyte-derived DCs after infection by rMVA, iDCs were infected with rMVA at an MOI of 1 for 1 hr on day 6 and were then incubated for 24 hr to determine CD80, CD83, and CD86 expressions. As shown in Fig. 3, rMVA promoted iDC differentiation, evidenced by increased CD80, CD83, and CD86 levels on these cells. To further elucidate the functional state of infected DCs, cells that responded to LPS were examined in the following experiments. Addition of LPS resulted in maturation of control DCs with significant upregulation of CD80, CD86, and CD83 molecules (Fig. 3). The pattern of upregulation of CD80, CD86, and CD83 was not obviously influenced by rMVA infection except for a slight decrease in CD83 upregulation after stimulation with 40 ng/ml LPS for 24 hr. These results indicate that rMVA-infected iDCs demonstrate enhanced expression of surface markers and that their capacity to respond to LPS is not strongly influenced by the infection.

FIG. 3.

DC activation by rMVA infection. DCs were mock-infected with PBS (Con), or infected with v65, vSur, or vD10 on day 6 at an MOI of 1 for 2 hr. The remaining viruses were removed by washing three times with RPMI-1640 and the cells were then cultured for another 24 hr. Some groups underwent a 40 ng/ml LPS treatment during this period of time. After 24 hr, the cells were collected, labeled with mouse anti-CD80 (top), anti-CD83 (middle), or anti-CD86 (bottom) antibody, and analyzed by flow cytometry; the histograms of MFI were plotted. The numbers in the figure represent the MFI values in one representative experiment. Dashed histograms are shown for immature DCs; solid histograms are shown for LPS-treated DCs. The MFI of each is given in the bottom histogram. These results are representative of four consecutive experiments with DCs from different donors. The purity range of the in vitro differentiated DCs was 86%, as shown in dotted histogram. Statistical difference between groups was calculated using Student's t-test. *p < 0.05, compared with controls (con or con-LPS group). MFI, mean fluorescence intensity; rMVA, recombinant modified vaccinia virus Ankara.

Cytokines secreted by DCs are important for their functions and determine the Th1/Th2 polarization of the immune response. The expressions of IL-10, IL-12, and TNF-α are closely related to the ability of DCs to stimulate and regulate the activation of naive T cells (Steinbrink et al., 1999). Therefore, we next determined whether rMVA infection altered the production of these cytokines by DCs. Among immature DCs, rMVA-infected cells secreted TNF-α (0.5–2 ng/ml) in a dose-dependent manner, whereas control cells did not. At the highest MOI tested (MOI = 2), the levels of TNF-α failed to increase to the expected level because of the impaired viability of infected DCs. After LPS stimulation, the levels of TNF-α produced by infected DCs were comparable to those of the control group (Fig. 4A and B). In addition, both control and infected cells released little IL-10, and rMVA markedly reduced IL-10 production after treatment with LPS (Fig. 4C and D). We failed to detect any IL-12p70 (the bioactive form of IL-12) in either infected or uninfected iDCs prior to LPS stimulation; however, on LPS stimulation, the ability of infected DCs to produce IL-12p70 was restored to about 80% (Fig. 4E). Moreover, various exogenous proteins introduced by rMVA did not alter the levels of these cytokines secreted by infected DCs, as shown in Fig. 4A and C.

FIG. 4.

The profile of cytokines secreted by rMVA-infected iDCs and mDCs. DCs were infected as described in Fig. 3; some groups underwent a 40 ng/ml LPS treatment as indicated by the black columns in (B), (D), and (E). After 24 hr, supernatants were collected and tested by TNF-α, IL-10, and IL-12p70 ELISA. The gray, white, or shadowed columns in (A) and (C) represent v65, vSur, or uninfected iDC groups, respectively. (A and B) TNF-α was secreted by infected DCs in a dose-dependent manner. (C and D) IL-10 production was impaired by MVA infection after LPS treatment. (E) The production of IL-12p70 could be restored after LPS treatment. The data shown represent mean ± SEM of results from three separate experiments; *p < 0.05, compared with mDC group. mDC, mature DC; SEM, standard error of the mean; TNF-α, tumor necrosis factor alpha.

DCs infected by vD10 effectively maintain the viability of syngeneic T cells through IL-2-mediated proliferation and overcome the intrinsic defect of MVA

Previous investigations have shown that high doses of MVA can elicit the death of DCs (Trevor et al., 2001; Smith et al., 2005). Studies were therefore conducted to determine whether rMVA-infected DCs could influence the viability of T cells. To address this issue, the MLR assays were performed as described in the Materials and Methods section. DCs were exposed to rMVA for 1 hr and subsequently cocultured with purified syngeneic T cells at a ratio of 1:10 for 4–5 days. Apoptosis was measured as annexin V binding by the gated proportion of CD3⁺ lymphocytes. As illustrated in Fig. 5A, T cells cocultured with v65-infected DCs (control group) underwent apoptosis measured by annexin V positivity ranging up to 50%, but apoptosis in T cells cocultured with vD10-infected DCs was comparable to the uninfected control. These results led to the question of whether rMVA per se are able to influence the viability of T cells. Further experiments revealed increased apoptosis in T cells infected with control rMVA v65 or with vSur; however, normal levels of apoptosis, similar to those of uninfected T cells, were found in vD10-infected T cells (data not shown). The fact that MVA per se or rMVA-infected DCs could induce T-cell apoptosis shows that MVA possesses an intrinsic defect as a candidate for vaccine development. However, rMVA encoding IL-2 shows potential for use in overcoming this defect.

FIG. 5.

The viability of syngeneic T cells after coculture with rMVA-infected DCs. (A) The apoptosis of syngeneic CD3⁺ T cells in mixed lymphocyte reaction. The MLR was performed as described in the Materials and Methods section. After coculture for 4 days, apoptosis was measured by FACS analysis as annexin V binding by the gated proportion of CD3⁺ lymphocytes. (B) The proliferation of syngeneic CD3⁺ T cells in MLR assay. T cells were cocultured with syngeneic rMVA-infected DCs for 14 hr (white column) or 34 hr (gray column) and then cultured for additional 6 hr in the presence of BrdU. Proliferation was measured as described in the Materials and Methods section. (C and D) Release of IFN-γ and IL-2 into the supernatants of coculture systems. The supernatants were collected at day 4 for ELISA assay. The levels of IFN-γ and IL-2 were elevated in rMVA-infected groups and further increased in the vD10-infected group. All data shown represent the mean ± SEM of results from three to five separate experiments; **p < 0.01, compared with v65-infected group. Uninfected DCs were included in parallel experiments as negative controls (con). IFN-γ, interferon gamma; MLR, mixed leukocyte reaction.

We hypothesized that maintenance of the viability of syngeneic T cells by DCs infected with vD10, which we demonstrated, might be due to exogenous IL-2 secreted by vD10-infected DCs. In theory, both active DCs and IL-2 cytokine could facilitate the proliferation of T cells. The following experiments utilized an ELISA BrdU assay to examine the proliferation of syngeneic T cells stimulated by control or rMVA-infected DCs. As shown in Fig. 5B, all infected DCs stimulated the proliferation of T cells when compared with uninfected controls; however, infection of DCs with vD10 significantly increased this stimulation after 40 hr of coculture.

IFN-γ and IL-2 are the main cytokines of Th1 produced by active T cells. To determine whether these cytokines exist in the coculture systems used in our experiments, supernatants from such systems were used in ELISA assays. The results showed that vD10-infected cells had a markedly increased ability to produce IFN-γ; IFN-γ was not detected in control supernatants (Fig. 5C). Similarly, the level of IL-2 produced by the vD10 group was much higher than that produced by the v65 or vSur groups (Fig. 5D), in accord with our expectation.

Activation of cytotoxic T lymphocytes by coculture with rMVA-infected DCs

To further investigate the activation of T-cell subsets, we employed FACS analysis to detect the expression of IFN-γ in CD8⁺ and CD4⁺ cells in the MLR assay. In these experiments, low-dose anti-CD3 and anti-CD28 (≤0.2 μg/ml) were used as stimuli to provoke terminal maturation of DCs and mimic the microenvironment in vivo (Harris et al., 2008). The results indicated that, in comparison to coculture with v65- or vSur-infected DCs, coculture with vD10-infected DCs resulted in an approximately two- to threefold increase in the production of IFN-γ by both CD8⁺ and CD4⁺ T cells (Fig. 6).

FIG. 6.

Activation of cytotoxic T lymphocytes by coculture with rMVA-infected DCs. CD3⁺ T cells were stimulated by coculture with infected DCs for 40 hr, followed by an additional 5-hr stimulation in the presence of stimulus. FACS analysis was employed to detect the expression of CD107a, perforin, and IFN-γ in the CD8^+/− T-cell subset of MLR groups. Uninfected DCs were included in parallel experiments as negative controls (co-con); the co-sti group was included as another uninfected control that was only treated with stimulus. The numbers in the figure represent the percentage of positive cells. Similar results were obtained in three separate experiments.

As a candidate for cancer vaccine vectors, the ability of rMVA-infected DCs to activate cytotoxic T lymphocytes is a crucial issue. Accordingly, to analyze the effective cytotoxic response of CD8⁺ T cells, a CD107a mobilization assay was used to measure the degranulation of CD8⁺ T cells (Betts et al., 2003); perforin levels (Voskoboinik et al., 2006) were also measured. As shown in Fig. 6, more than 70% of the CD8⁺ T cells in our study expressed perforin protein after coculture with infected DCs. In comparison with stimulated control cells, the expressions of CD107a on the cell membrane and of intercellular IFN-γ were also increased. Further, vD10 infection caused an approximately twofold increase in both CD107a and IFN-γ over v65- or vSur-infected cells, suggesting that the vD10-infected DCs could effectively activate cytotoxic T cells.

Discussion

MVA is now considered to be a suitable platform for the next generation of safer smallpox vaccines and recombinant poxvirus vectors (McCurdy et al., 2004). Vaccines based on MVA strains have already been tested in clinical trials for safety and effectiveness as vaccines against various infectious diseases and as cancer therapeutics. Compared with adenovirus system, MVA vector has several unique features, including larger foreign gene capacity, better biosafety level, and broader infection host spectrum which is CAR (coxsackie/adenovirus receptor) independent. Future advances in the exploitation of MVA will most likely focus on modification of the immune responses of the infected host to specifically optimize presentation of key immunogenic epitopes.

DCs are the most potent “professional” APCs in the body and are responsible for integrating a variety of incoming signals and for orchestrating a potent innate and adaptive immune response. Bidirectional interactions between DCs and T cells initiate either an immunogenic or a tolerogenic pathway, each of which plays a crucial role in immunotherapy (Lanzavecchia and Sallusto, 2004; Kuang et al., 2008). In the case of viruses that infect DCs, it is crucial to investigate the consequences of infection in order to obtain a better understanding of the mechanisms by which immunity is generated and to provide methods for modification of rMVA vaccines for optimal immunogenicity. Previous studies have shown that MVA replication in DCs is abortive and is characterized by early, but not late, viral gene expression and absence of viral DNA accumulation (Chahroudi et al., 2006). In view of the limited replication capacity of MVA, it is necessary for clinical trails to design more efficient strategies that further enhance the immune responses of MVA vector, which in turn might confer a higher protection against tumors when MVA is used as a vaccine. Consequently, we constructed survivin- or survivin/IL-2-producing rMVA to further analyze the effect of rMVA infection on the phenotype, maturation, and antigen presentation capacity of human DCs. In this study, we confirmed that immature DCs can be activated by rMVA infection, as demonstrated by the upregulation of CD80, CD83, and CD86 which we observed. This result is coincident with that of Drillien's study showing a moderate maturation of MVA-infected human DCs without further maturation stimulus (Drillien et al., 2004). Moreover, we extended our finding by additionally treating rMVA-infected DCs with LPS. Our data revealed that rMVA-infected mDCs exhibited slightly impaired CD83 and unimpaired costimulatory molecular expression compared with mock mDCs, properties which might contribute to prolonging the period of antigen presentation and promoting immune evasion of virus vectors.

Many TAAs have been reported to have the capacity of efficient presentation on the DC surface after rMVA infection, including MUC1, Her2, CEA, and 5T4 (Trevor et al., 2001; Hodge et al., 2003; Kastenmuller et al., 2006; Amato, 2007). In this context, we found that rMVA-infected DCs could also successfully express and present the intracellular protein survivin. Survivin is a member of inhibitor of apoptosis gene family. Increased survivin expression has been found in a variety of broad spectrum epithelial and hematologic malignancies, including renal carcinomas, breast cancer, colon cancer, multiple myeloma, and leukemias, and thus was recognized as a universal tumor antigen (Idenoue et al., 2005; Pennati et al., 2007). Despite the restriction to early viral gene expression, infected DCs maintained the expression of recombinant survivin at very high levels.

Cytokines secreted by DC are critical determinants for the induction of Th1 and Th2 responses and the expression of proinflammatory cytokines has been suggested to contribute to immune activation. After infection with rMVA, DCs extinguish their synthesis of IL-10 but release significant amounts of TNF-α. This altered cytokine profile, together with increased expression of costimulatory molecules, may trigger responding T cells toward a Th1 response.

High doses of vaccinia virus may elicit apoptosis or necrosis of DCs, as described in previous reports (Trevor et al., 2001; Behboudi et al., 2004; Smith et al., 2005), but so far little information exists as to the influence of rMVA-infected DCs on the viability of T cells. Yates et al. (2008) recently reported T-cell apoptosis following respiratory infection with vaccinia virus (WR strain) in C57BL/6 mice. By comparison, our results suggest that coculturing with MVA-infected DCs can trigger apoptosis of syngeneic T cells in 4–5 days; however, DCs producing exogenous IL-2 transferred by rMVA vector exhibited different effects. IL-2 appears to be one of the key molecules conferring unique T-cell stimulatory capacity on DCs (Granucci et al., 2002; Liu et al., 2004). In our studies, we chose IL-2 as an immune stimulus and constructed double-gene rMVA (vD10) to increase the therapeutic potential of this virus and overcome its intrinsic defect. Previous evidence has demonstrated that DCs infected with vaccinia virus IL-2 vector secrete bioactive IL-2, as confirmed by the stimulation of allogeneic T cells or PBMCs in studies with other VV strains or in mouse models (Trevor et al., 2001; Mukherjee et al., 2003). In our context, we showed that the combination of IL-2 coexpression in DCs maintained the viability of syngeneic T cells at a similar level as in uninfected controls. Further investigation of the ability of rMVA-infected DCs to stimulate syngeneic T cells showed that rMVA-infected DCs could induce the proliferation of T cells in 20 hr; in the case of vD10, this ability increased markedly after 40 hr. Further, high levels of IFN-γ and IL-2 from T cells after coculture were found in the vD10 coculture system when compared with other infected systems. Taken together, these findings indicate that vD10-infected DCs possess enhanced ability to stimulate syngeneic T cells and reverse the limitations of the MVA vector. Although Behboudi et al. (2004) reported the controversial opinion that MVA-infected DCs retain immunogenicity in vivo despite in vitro dysfunction, as characterized by the impairment of allogeneic T-cell stimulatory capacity after infection with MVA in vitro (Behboudi et al., 2004), this impairment in iDCs was presumably due to DC death elicited by high doses of MVA, as described by Engelmayer et al. (1999).

Cytotoxic T lymphocytes are important executants in adaptive immune responses, and therefore, it is absolutely necessary to investigate the activation of cytotoxic T lymphocytes induced by rMVA-infected DCs. The upregulation of CD107a, perforin, and IFN-γ in CD8⁺ T lymphocytes cocultured with rMVA-infected DCs indicated that these T cells had been activated by infected DCs and were ready to release degradation particles to exert cytotoxic effects. The expression of perforin did not change as significantly as that of CD107a in vD10 group, which might be due to the activation of cytotoxic T lymphocytes by Sur and MVA antigens, resulting in the rapid exocytosis of perforin and expression of CD107a on their surface. Generally, the CD8⁺ T lymphocytes stimulated with vD10-infected DCs also exhibited the most efficient activation in comparison with v65- or vSur-infected cells, suggesting that coexpression of Sur and IL-2 in rMVA-infected DCs may have promising application in further vaccine research in vivo.

In conclusion, in this study, we investigated the maturation status and functional capacity of DCs infected by rMVA encoding survivin/IL-2 and the ability of these DCs to activate syngeneic cytotoxic T cells in vitro. Further studies are necessary to quantify Sur-specific cytotoxic T lymphocyte cells stimulated by vD10-infected DCs in vitro and in vivo. Despite its preliminary character, this study provides further evidence that rMVA-infected DCs display an activated phenotype as confirmed by high levels of costimulatory molecules and increased proinflammatory cytokine versus reduced immunosuppressive cytokine expression, as well as the capacity to stimulate syngeneic T-cell proliferation and cytotoxic potential. Further, DCs infected with rMVA encoding antigen and IL-2 could effectively maintain the viability of syngeneic T cells and overcome intrinsic defects, such as impairment of DC maturation and apoptosis of lymphocytes, caused by vector alone. These findings allow us to better define the requirements for MVA-mediated antigen delivery to DC and provide valuable help in deriving optimized vectors for this advanced therapy strategy.

Footnotes

Acknowledgments

This work was supported by the Outstanding Young Scientist Fund and by project grants from the National Natural Science Foundation of China (30425025 and 30672388), the “973” program (2004CB518801 and 2005CB724600), and the Natural Science Foundation of Guangdong (05200303).

Author Disclosure Statement

The authors declare that no conflicts of interest exist.

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Activation of Human Dendritic Cells by Recombinant Modified Vaccinia Virus Ankara Vectors Encoding Survivin and IL-2 Genes In Vitro

Abstract

Introduction

Materials and Methods

Cells and reagents

Generation of rMVA

Isolation and preparation of DCs and CD3+ T cells

Western blotting for detection of survivin protein

Fluorescence-activated cell sorting analysis of DC phenotype and expression of exogenous survivin

ELISA for cytokine detection

MLR and proliferation assay of T cells

Apoptosis assay of T cells

FACS assay on CD107a, perforin, and IFN-γ expression of T cells

Statistical analysis

Results

Dose-dependent expression of exogenous protein by rMVA-infected iDCs

Effect of MVA or rMVA on DC phenotype

DCs infected by vD10 effectively maintain the viability of syngeneic T cells through IL-2-mediated proliferation and overcome the intrinsic defect of MVA

Activation of cytotoxic T lymphocytes by coculture with rMVA-infected DCs

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Isolation and preparation of DCs and CD3⁺ T cells