Experimental and Molecular Pathology 91 (2011) 325–330

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Experimental and Molecular Pathology

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Dendritic cells treated with HPV16mE7 in a three-dimensional model promote thesecretion of IL-12p70 and IFN-γ

Wang Ya Ting, Li WenSheng⁎, Liu QinShe, Guan XiaoYing, Hu JunShaanxi Provincial People's Hospital, Third Affiliated Hospital of the School of Medicine, Xi'an Jiaotong University, Xi'an, 710068, China

⁎ Corresponding author. Fax: +86 2985236987.E-mail address: liwensheng263@sohu.com (W. Li).

0014-4800/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.yexmp.2011.03.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 February 2011and in revised form 22 March 2011Available online 2 April 2011

Keywords:Dendritic cellThree-dimensionalHPVE7 vaccineImmune

Although the human papillomavirus (HPV) DNA therapeutic vaccine represents a promising approach to theprevention and treatment of cervical cancer, the mechanism of the HPV DNA vaccine is poorly understood.Moreover, current strategies have met with only limited success in preclinical and dendritic cell-based (DC-based) clinical research. In addition, two-dimensional (2-D) DC monolayers poorly mimic the physiologyfunction in vivo. We used a three-dimensional (3-D) DC culture model in vitro to explore the immunemechanism of the HPV DNA vaccine. DCs were generated from peripheral bloodmonocytes with interleukin-4(IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells, growing in 3-D collagengel, were treated with pcDNA3.1-HPV16mE7 in vitro for 48 h. Compared to DCs treated with E7 in a 2-Dculture model, the expression of co-stimulatory molecules CD80 and CD40 were significantly increased in the3-D model (pb0.05), and a remarkable increase of IL-12 p70 was observed. However, we did not detect anyobvious change in IL-10 in 3-D culture. In addition, we found that IFN-γ expression increased whenHPV16mE7-DC cells were co-cultured with T-cells for 96 h in the 3-D model, and HPV16mE7-DCs stimulatedthe proliferation of T lymphocytes more efficiently in the 3-D model than in the 2-D model (pb0.05). Theseresults suggest that DCs in 3-D culture model have a notable effect on the enhancement of the HPV16 DNAvaccine's immune reaction and indicate that the DC-based 3-D model is a novel approach to study the HPVvaccine.

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Introduction

High-risk human papillomavirus (HPV) is the major initiator ofhuman cervical cancer, and the recognition of a strong etiologicalrelationship between infections of high-risk HPV and cervical cancerhas prompted the development of prophylactic and therapeuticvaccines against such agents. In 2006, the U.S. Food and DrugAdministration (FDA) approved the first prophylactic quadrivalentHPV types 6/11/16/18 L1 virus-like particle (VLP) vaccine (Olssonet al., 2007). However, the VLP vaccine is expensive and unaffordablefor many people, especially those in developing countries. Moreover,this preventive vaccine does not have therapeutic effects on asignificant number of people who already have an HPV infectionand/orHPV-associated lesions. Compared to theHPVVLP vaccine, DNAvaccination has become a promising approach for antigen-specificimmunotherapy due to its safety, stability and ease of preparation(Monie et al., 2009; Hung et al., 2006). Many of the studies on DNAvaccines have been performed on preclinical models. Encouragingresults from impressive preclinical studies have led to several clinicaltrials, but current strategies have met with only limited success in

preclinical and dendritic cell-based (DC-based) clinical research(Nieto et al., 2010; Radulovic et al., 2009). Dendritic cells are themost efficient professional antigen presenting cells (APCs) in vivo,as they capture and process antigens and display large amounts ofMHC-peptide complexes on their surface (Palucka et al., 2011). Toimprove DNA vaccine immune potency, it is important to increase thenumber of antigen-expressing/antigen-loaded DCs, improve antigenprocessing and presentation in DCs, and enhance the interactionbetween DCs and T cells.

Moreover, compared to the immune mechanism of the VLPvaccine, the role of the HPV DNA vaccine in the initiation of adaptiveimmune responses is poorly understood. However, a few studies havedemonstrated that DCs treated with the HPV DNA vaccine can inducea specific CTL immune reaction (Santin et al., 1999, Davidson et al.,2001).

Three-dimensional (3-D) collagen models have been widely usedfor in vivo-like culture of various types of cells, including DCs, becauseDC monolayer cell culture models neglect the interactions betweendendritic cells and the impact of the stromamicroenvironment. DC2-Dmonolayers are believed to poorly mimic the physiological functionof DCs in vivo. (Tasaki et al., 2004).

In this study, we investigated the immune function of DCs treatedwith the HPV16E7 DNA vaccine using a 3-D culture model. We showthat DCs treated with the HPV16E7 DNA vaccine in the 3-D model

326 Y.T. Wang et al. / Experimental and Molecular Pathology 91 (2011) 325–330

strongly express IL-12 and the co-stimulatory molecules CD48 andCD40. In addition, IFN-γ expression is increased when HPV16mE7-DCcells were co-cultured with T-cells. In turn, T cell proliferation isstimulated in co-culture. Our study not only serves to elucidate theimmune mechanism of the HPV DNA vaccine, but it also suggests thata DC-based HPV16E7 vaccine in a 3-D model is a new and promisingapproach.

Methods

Cloning of the HPV16E7 gene

HPV16E7 was amplified from the plasmid pHPV16 by two PCRprimers (5′-GGGAATTC ATGCATGGAGATACACCTAC-3′ and 5′-GGCAAGCTTATGGTTTCTGAGAACAGATGG-3′), and the resulting frag-ment was inserted into T-Vector (Takara). The HPV16E7 gene wasconfirmed by sequencing.

Site-directed mutagenesis of the HPV16E7 gene

To obtain the HPV16E7 mutants, we performed site directedmutagenesis using a QuickChange™ Site-Directed Mutagenesis Kitwith the primers listed below (the mutated nucleotides areunderlined):

M1 5′-GAG ACA ACT GAT CTC TAC GGT TAT GAG CAA TTA AAT G-3′M1 5′-C ATT TAA TTG CTC ATA ACC GTA GAG ATC AGT TGT CTC-3′M2 5′-C AAT ATT GTA ACC TTT GGT TGC AAG TGT GAC TCT AC-3′M2 5′-GT AGA GTC ACA CTT GCA ACC AAA GGT TAC AAT ATT G-3′

The mutations were confirmed by sequencing.

Construction of pcDNA3.1-HPV16mE7

HPV16mE7was subcloned into the expression vector pcDNA3.1 byEcoRI and HindIII (Takara) from PMDTM19-T-HPV16mE7. The recom-binant plasmid pcDNA3.1-HPV16mE7 was confirmed by doublerestriction enzyme digestion and by sequencing.

Generation of bone marrow-derived DCs

Bone marrow-derived dendritic cells (BMDCs) were generatedfrom the bone marrow of normal BALB/c mice. Briefly, bone marrowcells were prepared from the femurs and tibias of sacrificed mice, anderythrocytes were depleted with 0.14 M NH4Cl buffer (pH 7.2). Aftercentrifugation at 500 g for 5 min, the cells collected from the interfacewere cultured in RPMI1640 (Gibco, Carlsbad, CA) containing 10% FBS,500 U/ml GM-CSF (PeproTech, USA) and 500 U/ml IL-4 (PeproTech,USA). Non-adherent granulocytes, T and B cells were gently removedby lymphocyte separation medium, and fresh media was added. Twodays later, the loosely adherent proliferating DC aggregates weredislodged and replated. Media was changed every 2 days by gentlyswirling the plates. At day 7, the non-adherent, relatively mature DCswere harvested and used for in vitro assays. DCs generated in thismanner displayed the typical morphological features of DCs that havesignificant expression of MHC II antigens and co-stimulatorymolecules (CD40, CD80) (data not shown).

DC culture in three-dimensional model

Rat Tail Collagen HC (BD, USA) is used as a three dimensional gel.First, the following was placed on ice: Rat Tail Collagen, P/N354249,sterile 10×phosphate buffered saline (10×PBS), sterile dH2O andsterile 1 N NaOH. Collagen type I solution was prepared according tothe methods recommended by the manufacturer. A sterile tube ofsufficient capacity to contain the final volume of collagen was placed

on ice, and the following steps were performed using aseptictechnique in a Class 100 Hood. Briefly, 10×PBS, sterile ice cold 1 NNaOH and sterile ice cold dH2O were added to the tube, the contentsof tube were mixed while still on ice, the calculated volume ofcollagenwas added and the solutionwasmixed and stored on ice untilready for use. The cell suspension was added to the collagen solutionbefore the gel solidified. The collagen mixture was dropped into 96-well plates at a 3 mg/ml final concentration, and then the plate wasincubated at 37 °C under 5% CO2 for 15 min. After the gel was formed,200 μl of full-DC culture media was added to each well, and DCs weregrown for 48 h.

Transfection of BMDCs with pcDNA3.1-HPV16mE7

DCs were transfected with pcDNA3.1-HPV16mE7 using Lipofecta-mine 2000 (Molecular Probes/Invitrogen). Transfection was per-formed as described in manufacturer's instructions. Briefly, DCs weretransfected with 10 μl of Lipofectamine 2000 in 240 μl RMPI1640(GIBCO) containing 2 μg of pcDNA3.1-HPV16mE7. Empty pcDNA3.1was used as a transfection control.

Immunocytochemistry analysis

DCs transfected with HPV16mE7 were grown in 3-D and 2-Dmodels for 48 h, and then DCs were harvested. This was followed byimmunostaining with an anti-HPV16E7 monoclonal antibody (Neo-Marker clone SP1, 1:100 dilution). EnVision+ horseradish peroxidase(HRP)-labeled detection system (DAKO) was used as the detectionsystem.

Flow cytometric analysis

DCs transfected with HPV16mE7 were embedded in collagen gelfor the 3-D culture model. Forty-eight hours later, DCs were collectedfrom collagen gels by collagenase digestion, and the expression ofantigen presentation-related molecules such as MHCII, CD80, andCD40 was examined by FACS analysis. Harvested DCs were washedtwice with PBS supplemented with 1% FBS. Fc receptors were blockedwith excess human IgG (Sigma) on ice for 10 min. Then, DCs wereincubated with fluorescence labeled-antibodies against CD11c, MHCII,CD80 and CD40 for 30 min at 4 °C. FITC-CD11c, PE-MHCII, PE-CD80and PE-CD40 were from Ebioscience. Relevant isotype controls werealways used. At least 5000 mononuclear cells were gated using acombination of forward-angle and side scatter to exclude dead cellsand debris. Data were analyzed with FACSD via software.

ELISA analysis

DCs cultured in the 3-D model for 48 h were collected fromcollagen gels by collagenase digestion. DCs were then seeded in 96well plates with 1×105 cells/well for 48 h, and the expression of IL-12p70 and IL-10 were analyzed by an ELISA kit (Ebiosciences, USA).The test was performed in triplicate three separate times.

Preparation of T lymphocytes

Spleen were extracted from Sterile sacrificed BABLIc mice. Spleencells were isolated from spleen by Ficoll–Hypaque density-gradientcentrifugation. The spleen cells were suspended in RPMI1640supplemented with 10% FBS. After incubation for 2 h at 37 °C in 5%CO2, the nonadherent cells were harvested. Sterile nylon–woolisolation column (Wako, Japan) was soaked in complete RPMI1640supplemented with 10% FBS, 100 u/ml penicillin and 100 μg/mlstreptomycin for 1 h at 37 °C. Then, the nonadherent cells isolatedfrom spleen lymphocytes were applied to the column and cultured for

Fig. 1. Morphological characterization of BMDCs in 3-D collagen culture. BMDCs werecultured in a collagen matrix for 2 days and were observed under phase-contrastmicroscopy. (A) Some DCs had a round shape right after cultivation in the collagenmatrix. (10×40). (B) Some DCs extended long “dendritic” processes. (10×40).

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additional 1 h. T lymphocytes were eluted from the column with10 ml RPMI1640.

Lymphocyte proliferation assays

Lymphocyte proliferation assays were performed with fourdifferent kinds of DCs (3-DT, HPV16E7-DCs in the 3-D model; 3-D,untransfected DCs in the 3-D model; 2-DT, HPV16E7-DCs in the 2-Dmodel; 2-D, untransfected DCs in the 2-D model) as stimulator cellsand T lymphocytes as responder cells. Stimulator cells were incubatedwith Mitomycin C (MMC) at 25 μg/ml at 37 °C for 30 min, and thenwashed with PBS twice. T lymphocytes isolated from the spleen cellswere plated in 96-well tissue culture plates (Costar, USA) at 4×105

cells per well. Then, stimulators were added and co-cultured withresponders at ratios of 1:50, 1:25, 1:10 for 96 h at 37 °C in 5%CO2. Tcells incubated in media alone served as a control. The cells were thenincubated with 5 mg/ml metrizamide (MTT, Sigma) at 20 μl per wellfor 4 h. The supernatant was removed and 150 μl of dimethylsulfoxide (DMSO, Amresco, USA) was added to each well and agitatedfor 10 min to fully dissolve the crystals. Absorbance was measured at570 nm on an automatic multiwell spectrophotometer (Bio-TekInstruments Inc, USA). All determinations were carried out intriplicate three separate times. Stimulation index (SI) was calculatedas follows: SI=(experimental-blank)/(control-blank).

IFN-γ secretion assays by ELISA

T lymphocytes isolated from spleen cells were plated in 96-welltissue culture plates (Costar, USA) at 4×105 cells per well. Thenstimulators were added and co-cultured with responders at ratios of1:10 for 96 h at 37 °C in 5% CO2. T cells incubated in medium aloneserved as a control. The supernatants were collected, and IFN-γexpression was analyzed by ELISA. The assay was performed induplicate. Using a standard curve provided in the kit, the concentra-tion of cytokines was determined for each sample.

Statistical analysis

All analyses were carried out using Statistical Package for theSocial Science (SPSS) Version 13.0. Continual variables wereexpressed as mean±standard deviation (SD). Student's t-test wasused for comparison between two groups. A statistically significantdifference was considered if Pb0.05.

Results

Construction of recombinant vector pcDNA3.1-HPV16mE7

The purpose of the site-directed mutagenesis of the HPV16 E7gene is to reduce its transforming activity and to increase the vaccinesecurity. HPV16 E7 was cloned into pcDNA3.1 and was confirmed byendonuclease cleavage. To make pcDNA3.1-HPV16E7 mutants, site-directed mutagenesis was performed using pcDNA3.1-HPV16E7 astemplate. HPV16E7 mutants were confirmed by sequencing. Theresults showed we obtained the two expected mutations, 58TGT→GGT, and 24 TGT→GGT.

Morphological of BMDC

Immature DCs were generated from BMDCs by culture in mediacontaining GM-CSF and IL-4 for 7 days as described in the Methodssection. These immature DCs were embedded within collagen gel andcultured in RPMI1640 for 48 h. Cells were observed by phase-contrastmicroscopy. The DCs extended characteristic processes from the cellmembrane within 24 h after initial culturing. As shown in Fig. 1, someDCs retained a round shape with many short processes, while some

DCs changed to a spherical shape and extended several long“dendritic” processes that were 20- to 50-Am long (Fig. 1).

Immunocytochemistry analysis of the expression of the E7 protein intransfected DCs

BMDCs were transfected with HPV16mE7-DNA-liposome com-plexes for 5 h in 3-D culture. Then cells were cultured in 3-D modelsfor additional 48 h. Immunocytochemistry staining showed that theHPV16mE7 protein was successfully expressed in DCs in both the 3-Dand 2-D models, and no differences were observed between cells inthe two models, suggesting that cells grown in the 3-D model cansubstitute for those grown in the 2-D model for testing HPV16 E7function in DCs (Fig. 2).

HPV16 E7 promotes maturation of BMDC

DCs transfected with the HPV16 E7 plasmid were cultured in the3-D model for 48 h and then were collected from collagen gels bydigestion with collagenase. The expression of antigen presentation-related molecules, such as MHCII, CD80, and CD40, was examined byFACS analysis. The data showed that expression of the antigenpresentation-related molecules CD80 and CD40 were significantlyincreased in the 3-D culture model (pb0.05) compared to those in the

Fig. 2. Immunocytochemistry stain for expression of the E7 protein in transfected DCs.(A) Expression of the E7 protein in transfected DCs in the 3-D model (10×40),(B) expression of the E7 protein in transfected DCs in the 2-D model (10×40).

Fig. 3. Expression of antigen presentation-related molecules on DCs in the 3-D modelcomparison with those in the 2-D model. Cells were collected and analyzed 48 h aftertransfection. DCs expression MHC-II, CD40 and CD80 were analyzed by FACS. Error barsindicate the mean±S.D; pb0.05, statistically significant.

Fig. 4. BMDCs secrete the cytokine IL-12p70 in the 3-D model and the 2-D model.BMDCs were transfected with HPV16mE7 for 48 h in both models. The supernatantswere collected, and IL-12p70 secretion was detected by ELISA analysis. Data indicatethat IL-12p70 secretion was enhanced by HPV16mE7-DCs in the 3-D model over thosein the 2-D model (pb0.05) or untransfected DCs in the 3-D model (pb0.05). Error barsindicate the mean±S.D; pb0.05, statistically significant.

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2-D culture model and untransfected DCs in 3-D culture. In addition,we also found no obvious difference in MHC II expression by DCsbetween the 3-D and 2-D models (pN0.05). However, increased MHCII expression was observed in DCs growing in the 3-D modelcompared to untransfected DCs in 3-D culture (pb0.05) (Fig. 3).These results showed that transfected DCs in the 3-D model couldpromote maturation of BMDCs and enhance DC antigen presentcharacteristics.

HPV16 E7 enhanced IL-12p70 expression in 3D culture

Using the 3-D collagen model, we investigated secretion of IL-12p70 by BMDCs. 48 h after DNA transfection, DCs were collectedfrom collagen gels by digestion with collagenase, and the concentra-tion of IL-12p70 was examined by ELISA analysis. We found thatHPV16mE7 could stimulate DCs to secrete IL-12p70, and the IL-12p70level in 3-D culture is higher than in 2-D culture (pb0.05) (Fig. 4).Secretion of IL-12p70 was enhanced in 3-D culture compared tountransfected DCs in 3-D culture (pb0.05). These results indicatedthat HPV E7 stimulates IL-12p70 secretion more efficiently in 3-D

culture than in 2-D culture, implying that it may also promote the Th1immune response.

Transfected DCs in 3D culture does not increase IL-10 secretion

We investigated secretion of IL-10 by BMDCs using the 3-Dcollagen gel model. 2 days after culture initiation, DCs were collectedfrom collagen gels with collagenase, and the concentration of IL-10was examined with ELISA analysis. The results showed that the levelof IL-10 decreased in transfected DCs in 3-D culture compared withthose in 2-D culture (pN0.05). (Fig. 5). However, our results indicatedthat expressing IL-10 may inhibit T-cell proliferation and cytotoxicactivity.

Stimulation of T lymphocytes by HPV16mE7-DCs in 3-D culture

It was found that anti-tumor T cells were generated by a singlestimulation with BMDCs transfected with HPV16mE7-DNA. TheHPV16mE7-DCs in the 3-D model were more potent stimulators ofT lymphocytes than those in the 2-D model (pb0.05). The effect wasenhanced with a higher ratio of HPV16mE7-DCs to T cells. In addition,HPV16mE7-DCs in 3-D culture could enhance the stimulation index(SI) more than untransfected DCs (pb0.05) (Fig. 6).The MTT assayshowed that transfected DCs in 3-D culture could enhance T cellproliferation and the T cell immune response.

Fig. 5. Cytokine IL-10 secretion by BMDCs in the 3-D model compared with those in the2-D model. BMDCs were pulsed with HPV16mE7 for 48 h in both models, thesupernatants were collected, and IL-10 secretion was detected by ELISA analysis. Dataindicate that secretion of IL-10 was decreased in transfected DCs in 3-D culturecompared to those in 2-D culture (pN0.05). Error bars indicate the mean±S.D; pb0.05,statistically significant.

Fig. 7. Cytokine IFN-γ secretion was notably enhanced by HPV16mE7-DCs in the 3-Dmodel over those in the 2-D model (pb0.05) or untransfected DCs (pb0.05). BMDCstreated with HPV16mE7 for 48 h were co-cultured with T cells for 96 h, and IFN-γsecretion in the supernatant was analyzed by ELISA. Error bars indicate the mean±S.D.

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Cytokine IFN-γ secretion by BMDCs in the 3-D model compared with the2-D model

To investigate cytokine IFN-γ secretion by BMDCs in the 3-Dmodel, BMDCs pulsedwith HPV16mE7 for 48 hwere collected and co-cultured with T cells for 96 h. The supernatants were collected, andIFN-γ secretion was detected by ELISA. The results showed thatcytokine IFN-γ secretion was enhanced by HPV16mE7-DCs in the 3-Dmodel over those in the 2-D model (pb0.05). In addition, theconcentration of IFN-γ in HPV16mE7-DCs in the 3-D model washigher than that in untransfected DCs in the 3-D model (pb0.05).(Fig. 7). These results suggest that HPV16mE7-DCs in the 3-D modelcould induce a strong CTL antitumor immune reaction.

Discussion

A few studies have showed that DCs treated with HPV DNA caninduce specific CTL immune reactions (Yin et al., 2009; Bellone et al.,2009). However, compared with the VLP vaccine, the immunomod-ulatory mechanism of the HPV DNA vaccine is poorly understood. Ithas been reported that immunization using HPV-L1 VLPs induces arobust and effective immune response. VLP interaction with DCsresults in the up-regulation of co-stimulatory molecules and theproduction of the cytokines IL-6, IL-8, IL-10 and IL-12p40 (de Witte et

Fig. 6. Lymphocyte proliferation assays. BMDCs transfected with HPV16mE7 for 48 hwere collected and co-cultured with T cells for 96 h. Specific CTLs were detected byMTTassay. Data indicate that HPV16mE7-DCs in the 3-D model were more potentstimulators of lymphocytes than those in the 2-D model (pb0.05) or untransfectedDCs (pb0.05). Error bars indicate the mean±S.D; pb0.05, statistically significant.

al., 2007). DCs treated with HPV16 VLPs could induce Th1-dominatedprimary T cell responses in vitro (Lenz et al., 2001, 2005). DCs, asprofessional APCs, are specialized for the initiation and regulation of Tcell immunity, especially the potent antitumor immunity. However, itis still not known whether the HPV DNA vaccine can induce keycytokine IL-12p70 secretion or what mechanism initiates the cellularimmune reaction.

To address these issues, we used a 3-D culture model to study theeffect of the E7 DNA vaccine on DC activation. DC monolayer cellculture models neglect the interactions between DCs and the impactof the stromamicroenvironment, however DCs growing in 3-D culturesystems can mimic in vivo environments (Tasaki et al., 2004; Gunzeret al., 2000). In addition to exploring the immune effect of the E7 DNAvaccine, we also tested whether DCs growing in a 3-D model are aneffective antigen presenting system for improving the immunepotency of the DNA vaccine.

The immunomodulatory functions of DCs are highly associatedwith the mature state of the cell. Immature DCs have high phagocyticcapacity and down-express surface adhesion molecules and co-stimulation molecules. In contrast, mature DCs express high levels ofadhesion molecules and co-stimulation molecules. Co-stimulatorymolecules on the surface ofmature DCs are one of the prerequisites foractivation of T-cells. The co-stimulatory molecules identified on DCs,with their respective T-cell receptors, are CD54, CD80, CD83, CD86,CD40, and CD40 ligands (Rakesh and Austyn, 1998).We found that thelevel of co-stimulatorymolecules such as CD80 and CD40 on DCswereincreased significantly when cells treated with HPV16mE7-DNA weregrown in 3-D culture comparedwith those grown in the 2-Dmodel. Inaddition, cells in 3-D culture exhibited a maturing phenotype,suggesting that treatment with HPV16mE7 DNA in the 3-D modelhad a notable effect on DC maturation and the enhancement of DCantigen-presenting capacity.

IL-12p70 plays a key role during induction of Th1 immune responses.DCs are one of themain producers of the cytokine IL-12p70, which playsa direct role in thedevelopment IFN-γ secreting Th1 cells, co-stimulationof CTL differentiation and NK-cell activation (Bekeredjian-Ding et al.,2006). Previously studies have demonstrated that full-length E7-treatedDC can induce both E7-specific CD4(+) T-cell proliferative responsesand strong CD8(+)CTL responses capable of lysing autologous naturallyHPV-infected cancer cells in patients with cervical cancer, andimmunization with soluble E6 and E7 fusion Igs in mice resulted inantigen-specific activation of T helper 1 (Th1) cells (Santin et al., 1999;Kim et al., 2011). We found that compared to 2-D culture, DCs treatedwith HPV16mE7 in the 3-D model significantly increased the secretionof IL-12p70. This implies that DCs in the 3-D model could effectivelyup-regulate the level of IL-12p70. As we expected, high expressionof IL-12p70 induced strong IFN-γ secretion and CTL responses.When DCs were treated with E7 and co-cultured with T cells for96 h, IFN-γ secretion was notably enhanced by HPV16mE7-DCs in

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the 3-D model over those in 2-D model (pb0.05). It is known thatactivated lymphocytes secrete the cytokine IFN-γ, which not only has adirect antiviral effect but also has a strong immune regulatory andmodifying effect (Delneste et al., 2003). Our research confirmed that ahigh level of IL-12p70 not only increased T cell proliferation, but alsoled to high IFN-γ secretion, as well as a strong CTL immune response.Moreover, DCs in the 3-D model induced stronger T-cell proliferativeresponses and CTL immune reactions than those in the 2-D model.These results suggest that DCs treated with HPV DNA in the 3-D modelmay provide an appropriate and effective way to study the DC-basedHPV DNA vaccine.

Interestingly, our data showed that IL-10 secretion was down-regulated byE7-pulsedDCs in the3-Dmodelmore than those in the 2-Dmodel. Some studies have found that DCs that stably express IL-10 canmarkedly inhibit T-cell proliferation and cytotoxic activity and induce T-cell apoptosis. Moreover, IL-10 also balances Th1/Th2 in vivo byrestraining IL-12 and the transformation from T cells to Th1 cells. Ourresults support the evidence that IL-10 can inhibit T-cell proliferationand cytotoxic activity. In addition, clinical studies have also shown that,in women infected with HPV, there is a positive correlation betweenlesion grade and a high level of IL-10. IL-10 as an immunosuppressivecytokine might play an important role in creating a microenvironmentthat favors progressive cervical disease and immuneevasionbyHR-HPV(Syrjänen et al., 2009; Bhairavabhotla et al., 2007, and Bermudez-Morales et al., 2008; Bolpetti et al., 2010). However, our data provideevidence that IL-10 has a negative connection to IL-12 expression andT-cell proliferation as well as CTL activity. Moreover, DCs in the 3-Dmodel may inhibit IL-10 secretion, enhancing Th1 immunity andantiviral CTL activity. This could be beneficial for improving the DNAvaccine's immune effect.

In summary, our studyshowed thatDCs treatedwithE7 ina3-Dmodelenhanced the representative phenotypes of mature DCs, promoted IL-12p70 and IFN-γ secretion, and induced a strong CTL immune reaction.The results demonstrated that DCs in a 3-D model could enhance HPV16DNA vaccine antitumor immune characteristics, suggesting a DCs-basedHPV16E7 vaccine in a 3-D model is a new and promising approach.

Conflict of interest statement

Authors declare that there are no conflicts of interest.

Acknowledgment

This research was supported by the Natural Science Foundation ofShaanxi Province, China. (2007C252).

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