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. 2010 Dec 14;60(3):381–388. doi: 10.1007/s00262-010-0948-4

Adenoviral-transduced dendritic cells are susceptible to suppression by T regulatory cells and promote interleukin 17 production

Adele Y Wang 1, Sarah Q Crome 1, Kristina M Jenkins 2, Jeffrey A Medin 3, Jonathan L Bramson 2, Megan K Levings 1,✉
PMCID: PMC11028621  PMID: 21153637

Abstract

Dendritic cell (DC) vaccines offer a robust platform for the development of cancer vaccines, but their effectiveness is thought to be limited by T regulatory cells (Tregs). Recombinant adenoviruses (RAdV) have been used successfully to engineer tumor antigen expression in DCs, but the impact of virus transduction on susceptibility to suppression by Tregs is unknown. We investigated the functional consequences of exposure to adenovirus on interactions between human monocyte-derived DCs and Tregs. Since the development of Tregs is linked to that of pro-inflammatory Th17 cells, the role of Th17 cells and IL-17-producing Tregs in the context of DC-based immunotherapies was also investigated. We found that Tregs potently suppressed the co-stimulatory capacity of RAdV-transduced DCs, regardless of whether the DCs were maturated by inflammatory cytokines or by exposure to Th1 or Th17 cells. Furthermore, exposure of Tregs to RAdV-exposed DCs increased IL-17 production and suppressive capacity, and correlated with enhanced secretion of IL-1β and IL-6 by DCs. The findings that DCs exposed to RAdV are suppressed by Tregs, promote Treg plasticity, and enhance Treg suppression indicates that strategies to limit Tregs will be required to enhance the efficacy of such DC-based immunotherapies.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-010-0948-4) contains supplementary material, which is available to authorized users.

Keywords: Adenoviral vector, T regulatory cells, T helper 17 cells, Dendritic cells, Cancer immunotherapy

Introduction

Extensive pre-clinical work has established that recombinant adenoviral (RAdV) vectors are efficient delivery platforms for gene therapies and vaccine applications [1]. Because RAdV infects various cell types, including antigen-presenting cells (APCs) and incorporates and expresses large transgenes at high levels [2], there is much interest in using them as tools to express tumor-associated antigens (TAAs) in APCs and thus boost tumor immunity in cancer patients. Indeed virally transduced dendritic cells (DCs) are superior to DCs transfected by other methods [3], and compared to peptide-pulsed DCs, RAdV-transduced DCs have improved migratory capacity [4]. Although RAdV-based genetic vaccinations have proven both clinically safe and feasible in melanoma patients [1], RAdV-transduced DC-based regimens have yet to result in consistent tumor regression [5, 6].

It is hypothesized that a major obstacle preventing the efficacy of DC-based cancer vaccines is the presence of numerous immunosuppressive mechanisms in the tumor microenvironment [7]. For example, many tumors evade immunity by promoting the development and expansion of T regulatory cells (Tregs), by secreting immunosuppressive cytokines such as IL-10 and TGF-β, and by inhibiting antigen (Ag) presentation [8]. Indeed Treg-mediated suppression of immune responses to tumor Ags represents a significant hurdle to successful cancer immunotherapy [7–10]. In addition to T cells, Tregs also modulate the maturation and function of APCs, including DCs and monocytes [11]. For example, Tregs regulate the stimulatory capacity of both human and murine DCs by reducing expression of CD80 and CD86, two co-stimulatory molecules known to be critical for optimal T cell priming [12, 13]. Additionally, the binding of lymphocyte activation gene 3 on Tregs to MHC class II molecules expressed on DCs delivers an inhibitory signal that interferes with DC maturation and decreases Ag presentation [12, 14]. Moreover, Tregs induce programmed death ligand-1 expression on DCs hence reducing the ability of DCs to stimulate T conventional (Tconv) cell responses [15]. Whether or not any of these Treg-mediated suppressive mechanisms impact the success of immunotherapy based on RAdV-transduced DCs remains an important outstanding question.

In addition to classical T helper 1 (Th1) and Th2-mediated responses, increasing evidence suggests that the activity of pro-inflammatory Th17 cell responses also has a critical role in determining the outcome of anti-tumor immunity. In some cases Th17 cells appear to bolster anti-tumor responses by enhancing cytotoxic T cell activity [16], but on the other hand they may promote angiogenesis and tumor growth [17]. In mice, the development of Tregs is linked to that of Th17 cells, with the local cytokine milieu influencing lineage commitment [18]. Furthermore, human IL-17+ Tregs can be isolated ex vivo or converted from IL-17− Tregs in vitro [19, 20]. Thus, knowledge of how genetically modified DCs influence the cytokine phenotype and/or suppression function of Tregs is key to understanding how DC-based immunotherapies could impact the relative balance between suppression and inflammation.

Here we investigated how interactions between ex vivo Tregs, Th1 and/or Th17 cells affect the phenotype and function of DCs exposed to RAdV. We found that despite their mature phenotype, RAdV-transduced DCs remained susceptible to Treg-mediated suppression. Surprisingly, RAdV-exposed DCs promoted Treg plasticity, stimulated them to produce IL-17 and increased their suppressive function. These data support the hypothesis that the efficacy of RAdV-transduced DCs in cancer immunotherapy is limited by pre-existing Tregs and indicate that strategies to block stimulation of Tregs should be incorporated in future RAdV-transduced DC- based vaccine strategies.

Results

RAdV-transduced DCs remain susceptible to suppression by Tregs

In addition to their well-known ability to suppress T cells, Tregs also interact with DCs and reduce their capacity to stimulate T cells via a variety of mechanisms [21, 22]. We first investigated whether transduction with RAdV alters the ability of Tregs to suppress DCs. DCs were differentiated from CD14+ monocytes in the presence of IL-4 and GM-CSF for 5 days, then cultured for an additional 24 h in the absence (for iDC) or presence of a maturation cocktail (IL-1β, IL-6, TNF-α and PGE-2) (for mDC), with or without the addition of RAdV-GFP. Expression of CD80 and CD86 on RAdV-transduced DCs was determined on gated CD11c+GFP+ cells. Exposure to RAdV alone at an MOI of 60 did not mature the DC (data not shown), consistent with previous reports [23–25]. As expected, maturation stimulated high expression of CD80 and CD86 compared to iDCs (data not shown), and RAdV transduction did not alter this phenotype (Fig. 1a). After maturation, in the absence or presence of RAdV, mDCs were co-cultured with allogeneic Tregs in order to provide the required TCR-dependent activation signal [13] and the phenotype of CD11c+ DCs was determined. After 96 h of exposure of mDCs to Tregs, there was a significant reduction of CD80 and CD86 expression (Fig. 1b) [12, 13]. In comparison, mDCs co-cultured with Tconv cells maintained their state of maturation (Fig. S1). Moreover, mDCs isolated from co-cultures with Tregs had a significantly reduced ability to stimulate the proliferation of CD4+ T cells compared with mDCs isolated from co-cultures with Tconv cells (Fig. S2). Notably, mDCs that were transduced with RAdV also remained fully susceptible to Treg-mediated suppression of CD80 and CD86 expression.

Fig. 1.

Fig. 1

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Treg-mediated down-regulation of CD80 and CD86 on mature untransduced and RAdV-transduced DCs. After 5 days of differentiation in IL-4 and GM-CSF, DCs were matured for 24 h by addition of IL-1β, IL-6, TNF-α and PGE-2 in the absence or presence of RAdV. DCs were then cultured alone or co-cultured with Tregs at a 1:5 ratio (DCs to T cells) for 96 h and expression of CD80 and CD86 was determined on gated CD11c+ cells. RAdV-transduced DCs were further gated as GFP+ cells. a A representative experiment with percentages of positive cells, set according to fluorescent minus one (FMO) controls (not shown), are displayed in each quadrant. Mean fluorescence intensities (MFI) is shown below the percentages. b Average fold change in MFI in four independent experiments. *p < 0.05 compared to DCs not exposed to Tregs

Th17-induced maturation of RAdV-transduced DCs is suppressed by Tregs

Since RAdV-transduced DCs remained susceptible to suppression by Tregs, we next asked whether this also held true in the presence of polarized Th1 and/or Th17 cells. Ex vivo Th1 cells were sorted as CXCR3+ cells and Th17 cells were sorted as CXCR3−CCR4+CCR6+ cells; ELISAs for IL-17 and IFN-γ confirmed the expected polarized cytokine phenotype of these cells (Fig. S3) [26]. Exposure of iDCs (in the absence of cytokine maturation) to Th1 or Th17 cells resulted in significant up-regulation of CD80 and CD86 expression (Figs. 2 and S4), consistent with previous reports [27, 28]. Addition of Tregs at a 1:1 ratio to co-cultures with Th1 or Th17 cells reduced T cell-induced maturation of iDCs (Figs. 2 and S4). When Th1 or Th17 cells were co-cultured with mDCs, which were either untransduced or transduced with RAdV (gated on CD11c+GFP+ cells), there was a negligible change in the expression of CD80 and CD86, likely because these DCs were already maximally matured (Figs. 2 and S4). Similar to the effect on iDCs, when Tregs were added to co-cultures with mDCs, they significantly decreased expression of CD80 and CD86 on both untransduced and transduced cells. Therefore, the presence of inflammatory Tconv cells, in this case Th1 or Th17 cells, unable to protect RAdV-transduced DCs from Treg-mediated suppression.

Fig. 2.

Fig. 2

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Treg-mediated suppression of Th17-induced maturation of untransduced and RAdV-transduced DCs. The indicated type of DC was cultured alone, or co-cultured with ex vivo Th17 (CCR4+CCR6+CXCR3−) cells at a 1:5 ratio, or co-cultured with Th17 cells in the presence of a 1:1 ratio of Tregs for 96 h. Expression of CD80 and CD86 on DCs, gated as CD11c+ cells, was analyzed by flow cytometry. RAdV-transduced DCs were further gated as GFP+ cells. Left panels depict representative experiments, and right panels depict average fold change of MFI from three independent experiments. *p < 0.05

RAdV-exposed DCs induce IL-17 production and up-regulate IL-23R expression on Tregs

Generation of potent immunity involves a bidirectional feedback between DCs and T cells. Therefore, we examined the phenotype of CD4+ T cell subsets stimulated with RAdV-exposed DCs. After 96 h of co-culture, intracellular cytokine staining of T cells revealed that a significant percentage of Tregs stimulated with RAdV-exposed mDCs, expressed IL-17 (Fig. 3a) (mean % IL-17+ for Tregs cultured with RAdV-exposed mDCs was 9.6 ± 2.2% vs. 2.3 ± 0.4% for Tregs cultured with untransduced mDCs). Similarly, stimulation of Th1 or Th17 cells with RAdV-exposed mDCs also caused up-regulation of IL-17 (Fig. 3a). In contrast, the proportion of IFN-γ produced by all three CD4+ T cell subsets remained consistent upon stimulation with RAdV-exposed DCs (Fig. 3a). A characteristic phenotype of Th17 cells is expression of the IL-23 receptor (IL-23R) [29, 30], which confers responsiveness to the Th17-stabilizing properties of IL-23 [31]. In addition to IL-17, we also found that RAdV-exposed DCs caused a significant up-regulation of IL-23R on Treg, Th17 and Th1 cells (Fig. 3b). Taken together, these data indicate that exposure of DCs to RAdV induces a Th17-polarizing program, and support the notion that the cytokine profile of polarized Th cell subsets can be re-directed by APCs.

Fig. 3.

Fig. 3

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Tregs exposed to RAdV convert into IL-17 producing and IL-23R expressing cells. Tregs, Th17 (CCR4+CCR6+CXCR3−) or Th1 (CXCR3+) cells were cultured with either untransduced or RAdV-exposed DCs at a 5:1 ratio (T cells to DCs). a After 96 h of co-culture, cells were stimulated with PMA and ionomycin for 6 h and expression of IL-17 and IFN-γ gated on CD4+ cells was analyzed by intracellular staining. Top panel depicts representative data and the bottom panel depicts the average % of IL-17+ cells from three independent experiments. b IL-23R expression on CD4+ T cells was determined by flow cytometric analysis after 96 h of co-culture. Top panel depicts representative data and the bottom panel depicts the average % of IL23R+ cells from three independent experiments. *p < 0.05

Tregs exposed to RAdV-transduced DCs have enhanced suppressive capacity

We next investigated whether exposure to RAdV-transduced DCs alters the suppressive capacity of Tregs. Tregs were co-cultured with either mDCs or RAdV-exposed mDCs for 7 days. At the end of the co-culturing period, Tregs were re-isolated from the DC–T cell mixture by positive selection of CD4+ T cells. Various amounts of Tregs (from mDC co-cultures) or RAdV Tregs (from RAdV mDC co-cultures) were incubated with autologous CFSE-labeled PBMCs for 72 h. Suppression was assessed by analyzing the amount of CFSE dilution in gated CD8+ T cells. Interestingly, RAdV Tregs displayed a significantly enhanced suppressive capacity compared to Tregs (Fig. 4). Hence RAdV-exposed mDCs are not only susceptible to Treg-mediated suppression themselves, they further promote suppression by increasing the potency of Tregs.

Fig. 4.

Fig. 4

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Exposure of Tregs to RAdV-transduced DCs enhances their suppressive capacity. Tregs were cultured with either mDCs or RAdV mDCs for 7 days and then purified as CD4+ T cells by positive selection. a Autologous CFSE-labeled PBMCs were stimulated with α-CD3 and cultured with Tregs (from mDC co-cultures; closed circle) or RAdV Tregs (from RAdV mDC co-cultures; open circle) at a 1:8 ratio (Tregs to PBMCs) for 72 h. The percentage of CD8+CFSE+ cells was determined by flow cytometry. b Averaged data from three independent experiments are expressed as percentage suppression, calculated by the formula: 1 (proliferation in the presence of Tregs/proliferation of CD8+ cells alone) × 100. *p < 0.05

RAdV exposure stimulates mature DCs to secrete Th17-promoting cytokines

In order to better understand the mechanistic basis for why RAdV promotes Th17-polarizing DCs, we examined how RAdV infection impacts cytokine production from DCs. Although the nature of Th17-polarizing cytokines in humans is a subject of much debate, substantial evidence suggests that the combination of IL-1β and IL-6 is required for induction of IL-17 [32]. Other cytokines that could also be involved in Th17 development include: IL-21, IL-23 and TGF-β [30]. In order to test the cytokine profile of DCs exposed to RAdV, supernatants were collected from mDCs and RAdV mDCs 24 h after transduction. Consistent with their ability to induce IL-17 production, exposure to RAdV significantly enhanced production of IL-1β and IL-6, but not IL-10, IL-12, or IL-23 (Fig. 5). Similarly, exposure of DCs matured by CD40 stimulation to RAdV also resulted in a specific increase in IL-1β and IL-6 production (data not shown). Hence, RAdV confers DCs with the capacity to polarize T cells towards Th17 cells by modulating their cytokine production profile.

Fig. 5.

Fig. 5

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Mature RAdV-exposed DCs produce Th17-polarizing cytokines. After 5 days of differentiation, DCs were matured overnight with the maturation cocktail with or without the addition of RAdV. The next day, mDCs and RAdV mDC (500,000 cells/mL) were collected and washed extensively and stimulated for 24 h with IL-4 (50 ng/mL) and GM-CSF (50 ng/mL). Supernatants were collected and analyzed for IL-1β, IL-6, IL-10, IL-12, IL-23 and TNF-α. Data represent averages from three independent experiments. *p < 0.05

Discussion

Successful cancer immunotherapy must simultaneously stimulate effector immunity and break tumor-induced tolerance. A great deal of work has focused on RAdV-transduced DCs to stimulate Tconv cell immunity, but little is known about how such cells impact Tregs. Here we show that DCs transduced with RAdV remain susceptible to suppression by Tregs and develop a Th17-polarizing cytokine profile. Although RAdV-exposed DCs stimulate IL-17 production in both regulatory and effector cells, these Tregs remain suppressive and are capable of blocking Th1- and Th17-induced maturation of DCs. Together these data indicate that to effectively stimulate T cell immunity, delivery of Ag by RAdV-transduced DCs must be coupled with strategies to remove or prevent Treg-mediated suppression.

RAdV are commonly used gene-delivery vectors in immunotherapy as they efficiently transduce DCs and induce T cell and antibody responses against the delivered transgene products [2]. Previous reports have shown that exposure of DCs to Tregs suppresses the expression of co-stimulatory molecules [12–14]; here we found that despite their mature phenotype RAdV-transduced DC are also susceptible to these effects. In addition to stimulation with inflammatory cytokines or pathogen-associated molecular patterns, DCs can also be matured by interaction with T cells, at least partly via CD40-dependent signals [27, 33]. Notably, our data represent the first demonstration that human Th17 cells stimulate DC maturation and thus likely enhance their ability to prime Tconv cells. We found that Tregs suppress DC maturation stimulated by ex vivo Th1 or Th17 cells, indicating that even in the presence of fully polarized T cells, Tregs manifest their inhibitory effects on DCs. Recent findings in mice have suggested that Tregs suppress DCs by outcompeting naive T cells in the formation of LFA-1-dependent aggregates around DCs in vitro [13]. Further investigation will be required to define whether a similar mechanism underlies the ability of Tregs to suppress Th1 and Th17-driven maturation. Overall these data indicate the existence of Tregs in the tumor microenvironment could limit the effectiveness of RAdV-transduced DCs to present tumor Ags even if inflammatory T cells are present.

Accumulating evidence suggests that Tregs are plastic and can differentiate into IL-17-producing cells in the presence of IL-1β, IL-2, IL-21, and IL-23 [20, 34]. In support of Treg plasticity, we found that stimulation of Tregs to RAdV-exposed mDCs resulted in elevated proportions of IL-17 but not IFN-γ secreting cells. Moreover, an increased proportion of IL-17-producing cells was also observed in both Th1 and Th17 cells. We found that DCs exposed to RAdV produced high amounts of IL-1β and IL-6, cytokines involved in the differentiation of human Th17 cells [30, 32]. Induction of IL-1β and IL-6 by RAdV is consistent with previous findings [35], and provides at least part of the mechanistic basis for why mDCs exposed to RAdV stimulate CD4+ T cells to produce IL-17. Elevated IL-17 correlated with increased expression of the IL-23R in Tregs, Th1 and Th17 cells, a molecule that is known to be regulated by RORC2 [26] and is required for IL-23-dependent stabilization of the Th17 lineage [36].

Circulating IL-17+ Tregs have been identified in humans and shown to be equally suppressive as IL-17-Tregs in vitro [20, 34]. In contrast, we found that Tregs isolated from co-cultures with RAdV mDCs displayed enhanced suppressive capacity compared to Tregs isolated from co-cultures with untransduced mDCs. Further investigation into the molecular basis for how RAdV-transduced DCs potentiate Treg suppression and whether it involves regulation of IL-17 production will be of considerable interest. It will also be of interest to determine whether similar mechanisms may be operational in the context of natural viral infections.

Although more potent Tregs would be predicted to be detrimental to anti-tumor immunity, the possibility that they may have a beneficial effect cannot be ruled out. Indeed the concept that inflammation precedes cancer is gaining increased support [37] and depending on the context IL-17-producing Tregs could act to control inflammation and therefore reduce cancer progression.

In conclusion, we found that Tregs block the ability of RAdV-transduced DCs to stimulate immune responses by down-regulating the expression of co-stimulatory molecules, CD80 and CD86. These data indicate that pre-existing Tregs may limit the effectiveness of DC-based vaccines in vivo. Indeed, depletion of Tregs enhanced the efficacy of DC immunotherapy and protected vaccinated mice against glioma [38]. Evidence that RAdV-exposed DCs promote IL-17 production from multiple T cell subsets indicates that further investigation into how IL-17 impacts immunity will be crucial to understanding and optimizing the therapeutic efficacy of this approach. Moreover, our data clearly indicate that for DC-based immunotherapies to be maximally effective, a combination therapy where RAdV-transduced DC-based vaccines are administered in parallel with Treg inhibitory agents may be required.

Materials and methods

Differentiation, maturation and transduction of DC

Peripheral blood was obtained from healthy volunteers following approval by the University of British Columbia Clinical Research Ethics Board and after obtaining written informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll. CD14+ monocytes were purified from PBMCs by positive selection (StemCell Technologies) and immature DCs (iDCs) were generated by culturing monocytes for 5 days in DC medium [RPMI 1640 containing 10% fetal calf serum (FCS) (Invitrogen), penicillin/streptomycin (Invitrogen), non-essential amino acids (0.1 mM, StemCell Technologies), HEPES (10 mM, StemCell Technologies), sodium pyruvate (1 mM, Invitrogen) and 2 mercaptoethanol (50 μM, Bio-Rad)]. DC medium along with rh-IL-4 (50 ng/mL, kind gift from Ulf Korthaeuer, Novartis) and rhGM-CSF (50 ng/mL, StemCell Technologies) was replenished every 2 days. Immature DCs were CD11c+HLA-DRintCD80loCD86loCD83− (data not shown). To mature DCs, an inflammatory cytokine cocktail (IL-1β, 10 ng/mL, Sigma–Aldrich; TNF-α, 10 ng/mL, eBiosciences; IL-6, 5 ng/mL, eBiosciences; and prostaglandin-2 [PGE-2], 1 μg/mL, Sigma–Aldrich) was added to the iDCs for 24 h at day 5. Mature DCs (mDCs) were CD11c+HLA-DRhiCD80hiCD86hiCD83+ (data not shown).

For DC transduction, cells were infected with RAdV, at a multiplicity of infection (MOI) of 60, either alone (for iDC) or with the inflammatory cytokine cocktail (for mDC) for 24 h. The RAdV type 5 vectors contained deletions of E1 and E3 regions [39]. A GFP expression cassette was inserted into the E1 region under the control of the murine CMV promoter and the SV40 polyadenylation sequence. The virus was propagated using 293 T cells and purified using CsCl gradient centrifugation as described previously [40]. The transduction efficiency was ~50% based on expression of GFP (data not shown). On day 6, DCs were washed extensively before co-culture with T cells.

Isolation of T cell subsets and co-culture with DC

CD4+ T cells were enriched from PBMCs by negative selection (StemCell Technologies, Vancouver, Canada). Tregs were enriched from CD4+ T cells by positive selection for CD25 expression (Miltenyi Biotec, Auburn, CA, USA) over two columns to ensure 81–90% purity based on expression of CD25 (BD Pharmingen) and FOXP3 (eBiosciences). To isolate Th17 (CD4+CXCR3−CCR4+CCR6+) and Th1 (CD4+CXCR3−) cells, CD4+CD25− were purified by incubation with CD25 beads (Miltenyi Biotec) and passed over a LS depletion column. The resultant CD4+CD25− T cells were labeled with antibodies to CD4 (eBiosciences), CXCR3 (BD Pharmingen), CCR4 (BD Pharmingen) and CCR6 (eBiosciences), and sorted using FACS Aria (BD Biosciences) to a purity of >96% as previously described [24]. The cytokine-polarized phenotype of the Th1 and Th17 cells was confirmed by measuring levels of secreted IFN-γ and IL-17 (Fig. S3). For T cell co-cultures, allogeneic DCs were incubated at a ratio of 5:1 (T cells to DCs) with Tregs, Th1, and/or Th17 cells as indicated. When DCs were co-cultured with Th1 or Th17 cells and Tregs, the T cell subsets were at a 1:1 ratio. Cells were cultured for 96 h in DC medium with recombinant human IL-2 (rh-IL-2) (50 U/mL, Chiron).

To test the stimulatory capacity of DCs exposed to Tregs, DCs were incubated with either CD4+CD25− Tconv cells or CD4+CD25+ Tregs for 72 h at a 1:1 ratio (DCs to T cells). T cells were depleted using positive selection for CD3+ cells (StemCell Technologies), and the remaining DCs (10,000 cells/well) were tested for their stimulatory capacity in an MLR with 50,000 allogeneic CD4+ T cells/well. Proliferation of T cells was assessed after 96 h by [3H]thymidine incorporation (1 μCi/well, Amersham Biosciences), added for the final 16 h of culture.

Flow cytometric analyses

DCs were monitored for cell surface expression of CD11c (eBiosciences), CD80 and CD86 (both BD Pharmingen). T cells were stained for CD4 (eBiosciences) and IL-23R (R&D Systems). For analysis of intracellular cytokine production, T cells were activated with 10 ng/mL PMA and 500 ng/mL Ca2+ ionophore (both Sigma–Aldrich) for 6 h, with brefeldin A (10 μg/mL, Sigma–Aldrich) added half-way through activation. Following surface staining, cells were fixed in 2% formaldehyde and permeabilized with 0.5% saponin. Intracellular cytokine staining was performed with antibodies against IL-17 (eBiosciences) and IFN-γ (BD Pharmingen). A minimum of 20,000 live cell events were acquired on a BD FACSCanto and analyzed with FCS Express Pro Software Version 3 (De Novo Software, Thornhill, Canada).

Determination of cytokine concentration

Supernatants from untransduced and RAdV-transduced DCs (500,000 cells/mL/well) were collected after 24 h and frozen at −80°C until analysis. IL-1β, IL-6, IL-10, IL-12p70 and tumor necrosis factor-alpha (TNF-α) were measured by Human Inflammatory Cytokines cytometric bead arrays (BD Biosciences). IL-23 was measured by ELISA (eBiosciences).

Suppression assays

Tregs (125,000 cells/well) were co-cultured with mDCs or RAdV mDCs (25,000 cells/well; ratio of 5:1 T cells to DCs) in RPMI complete medium for 7 days. Tregs were then re-isolated from co-cultures by positive selection for CD4 (StemCell Technologies). To test for suppressive capacity, autologous PBMCs, which were frozen down when the Tregs were obtained, were labeled with 2.5 mM 5- (6-)CFSE (Molecular Probes) and stimulated at 100,000 cells/well with α-CD3 (1 μg/mL) in the presence or absence of various numbers of Tregs corresponding to the indicated ratios. As a control, various numbers of CD4+CD25− T cells were titrated in, and as expected had no detectable suppressive capacity (data not shown). Suppression was assessed after 72 h by staining the samples for CD8 expression (BD Pharmingen) and analysis of CFSE dilutions in the CD8+ T cells using flow cytometry.

Statistical analysis

All analyses for statistically significant differences were performed with 1-tailed paired Student’s t test. P values of <0.05 were considered significant. All error bars represent mean ± standard errors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 485 kb) (485.1KB, pdf)
Supplementary material 2 (TIFF 958 kb) (958.5KB, tiff)
Supplementary material 3 (TIFF 710 kb) (710.3KB, tiff)
Supplementary material 4 (TIFF 1082 kb) (1.1MB, tiff)

Acknowledgments

This work was supported by a Terry Fox Foundation program project grant in cancer immunotherapy awarded to MKL, JAM and JLB. MKL holds a Canada Research Chair in Transplantation. AYW holds a CIHR Canada Graduate Award and a CIHR/SRTC Strategic Training Program in Transplantation award. SQC holds a MSFHR Senior Graduate Studentship award and CIHR/SRTC Strategic Training Programs in Skin and Transplantation awards. Core support for flow cytometry sorting provided by Lixin Xu and was funded by the Immunity and Infection Research Centre MSFHR Research Unit.

Conflict of interest

The authors declare no competing financial interests.

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