Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1 BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMAIRES-m4-1BBL was 14.6% lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/ mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.


Introduction
Prostate-specific membrane antigen (PSMA) is an over-expressed membrane-bound cell surface protein on prostate cancer cells (1) and a well-defined tumor-associated antigen (TAA) (2).Because of these properties, PSMA has been proposed as an ideal target for a variety of therapeutic approaches in prostate cancer including the delivery of immunoconjugates, immunotherapy, and prodrugs (3)(4)(5).Dendritic cells (DCs) are highly efficient and specialized antigen-

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presenting cells (APC) that play a central role in immunity (6).DC-based vaccines have been shown to be well suited to induce significant immune responses against prostate cancer (7,8).Of course, DC signaling for a tumor-specific T-cell immune response involves a number of co-stimulatory molecules, which may amplify, sustain, and drive diversity in the ensuing immune response (9).4-1BBL is one of well-characterized co-stimulatory molecules expressed on APC (10).4-1BBL with its receptor 4-1BB expressed on T cells has profound effects on T cells, including activation of both CD4 + and CD8 + T cells, enhanced expansion (11,12), increased long-term survival (13,14), and anti-apoptosis of activationinduced CD8 + T cells (15).However, the effect of 4-1BBL on DCs is not clear.In the present study, we constructed a recombinant adenovirus (Ad) co-expressing a TAA and mouse 4 (m4)-1BBL genes and determined the effects of 4-1BBL on DCs during TAA processing.

Material
Female C57BL/6 (H-2 Kb) mice, 6-to 8-week-old, were obtained from Shanghai Slac Laboratory Animal Co., Ltd.(China).Animals were maintained at the Central Animal Facility of Wuhan University according to standard guidelines and experiments were conducted according to the guidelines of the China Council for Animal Care.HEK 293, a human embryonic kidney 293 cell line, was kindly provided by the Ministry of Education Key Laboratory of Virology (Wuhan, China).All cells were cultured in RPMI-1640 medium with 10% heat-inactivated FCS, 2 mM/L glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 .

Construction of recombinant co-expression adenovirus vector containing tPSMA and m4-1BBL genes
The Ad vectors used in the present study were derived from serotype 5.The AdMax™ Expression System (Microbix Biosystems, Inc., Canada) was used.The Ad-enhanced green fluorescent protein (Ad-eGFP) vector carries the cDNA of eGFP under the transcriptional control of the immediate early promoter of cytomegalovirus (CMV).
For Ad transduction, day 7 iDCs were washed and plated onto a 24-well plate containing 200 µL serum-free medium supplemented with 10 ng/mL GM-CSF and 10 ng/mL IL-4.Virus was added to the wells at 250 multiplicity of infection (MOI) and transduction was allowed to proceed for 2 h at 37°C in 5% CO 2 .Complete medium was then added and cells were cultured for an additional 48 h.The transduction efficiency of DCs was checked on the basis of eGFP expression by flow cytometry.

Western blot analysis
For the detection of tPSMA and m4-1BBL protein expression, 2 x 10 6 iDCs were infected with recombinant adeno-

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viruses (250 MOI).Two days later, cells were lysed and subjected to SDS-PAGE.Then, protein was transferred to a nitrocellulose membrane (Amersham, USA) and the transferred membrane was probed with polyclonal goat anti-PSMA antibody or goat polyclonal anti-4-1BBL antibody (USA), followed by a horseradish peroxidase-conjugated anti-goat IgG secondary antibody (USA).Antibodies on the membrane were visualized by chemiluminescence (Pierce, USA).Western blot for β-actin was used as an internal sample.

Surface marker analysis of DCs
Forty-eight hours after Ad transduction and another 24-h activation with LPS, DCs were stained for 30 min on ice with FITC-or PE-labeled monoclonal antibodies specific for CD11c, MHC II, CD80, CD86 (BD Pharmingen, Germany).After washing three times in PBS, the cells were analyzed by flow cytometry.Isotype-matched monoclonal antibodies were used as controls.

Determination of cytokine profile by ELISA
To determine the effect of Ad-tPSMA-IRES-m4-1BBL on the secretion of IL-6 and IL-12 from DCs, supernatants were harvested and the concentrations of IL-6 and IL-12 were measured by ELISA.A mouse IL-6 Quantikine ELISA Kit (R&D Systems) and a mouse IL-12 Quantikine ELISA Kit (R&D Systems) were used, respectively.All procedures and conditions were consistent with manufacturer instructions.

Apoptosis analysis by flow cytometry
For apoptosis analysis, 5 x 10 5 iDCs were cultured on a 12-well plate containing 500 μL complete medium supplemented with 10 ng/mL GM-CSF and 10 ng/mL IL-4.Forty-eight hours after Ad transduction, 1 µg/mL of the activating agent LPS and 0.1 µg/mL of the apoptosis inducer bortezomib (Velcade, UK) were added to each well.After an additional 16-h culture, DCs were collected and stained with FITC-conjugated annexin V and propidium iodide according to manufacturer instructions, for apoptosis analysis by flow cytometry (Apoptosis Kit, BD Pharmingen, Germany).

Allogeneic mixed lymphocyte reactions
Mixed leukocyte reaction (MLR) was performed using three types of LPS-matured DCs (Ad-tPSMA-IRES-m4-1BBL-transduced DCs, Ad-eGFP-transduced DCs and normal control DCs) as stimulator cells and T lymphocytes as responder cells.Stimulator cells were incubated with 50 ng/mL mitomycin C at 37°C for 30 min and then washed twice with PBS.Nylon wool-purified naive T cells derived from the spleen of allogeneic BALB/c mice were plated onto a 96well round-bottomed culture plate (Costar, USA) at 4 x 10 5 cells per well.Stimulators were then added and co-cultured with responders at ratios of 1:10, 1:10 2 , 1:10 3 , and 1:10 4 in complete RPMI 1640 medium.DCs and T cells incubated in medium alone served as stimulator control and responder control, respectively.After incubation for 4 days, 10 µL of the Cell Counting Kit-8 (Dojindo, Japan) solution was added to each well containing 100 µL medium for 4 h.Absorbance was measured at 450 nm on an automatic ELISA reader (TRITURUS, Spain).All determinations were carried out in triplicate and repeated three times.

Statistical analysis
Data are reported as mean ± SD and were analyzed by ANOVA or the Student t-test, with the level of significance set at P < 0.05.The SPSS13.0 software was used for statistical analysis.

Construction of recombinant adenoviruses and transduction of DCs with adenovirus
A replication-deficient adenovirus vector carrying the human tPSMA gene and m4-1BBL gene (Ad-tPSMA-IRES-m4-1BBL) or Ad-eGFP was constructed.The tPSMA and eGFP genes were driven by the CMV promoter, and the translation of the m4-1BBL gene was initiated from an IRES (Figure 1A).DCs were transduced with the adenovirus at 250 MOI to analyze the transduction efficiency.About 65% of DCs were positive for eGFP expression by flow cytometry (Figure 1B).250 MOI was considered optimal for gene transduction because cell viability was 93% (Figure 1B).

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Analysis of DC phenotype iDCs were infected with Ad-tPSMA-IRES-m4-1BBL, Ad-eGFP for 48 h, or not infected, and activated with LPS for another 24 h.Then, the expression of CD11c, MHC II, CD80, and CD86 was analyzed by flow cytometry.Ad-tPSMA-IRES-m4-1BBL-infected DCs enhanced the rate of expression of MHC II, CD80, and CD86 (Table 1).

Cytokine analysis
The culture supernatants of DCs transfected with Ad-tPSMA-IRES-m4-1BBL, Ad-eGFP, or uninfected DCs were collected for the analysis of IL-6 and IL-12 production by ELISA.The concentration of IL-6 and IL-12 in Ad-tPSMA-IRES-m4-1BBL-transfected DC culture supernatants was higher than in DCs transfected with Ad-eGFP and untreated DCs (Figure 3).

Analysis of DC apoptosis
iDCs transfected with Ad-tPSMA-IRES-m4-1BBL, Ad-eGFP, or uninfected DCs were treated with the activating agent LPS and the apoptosis inducer bortezomib.After 16 h of culture, DCs were collected and stained with FITC-conjugated annexin V and propidium iodide for detecting apoptosis.The rate of apoptosis of Ad-tPSMA-IRES-m4-1BBL-transduced DCs was lower than that of Ad-transduced and uninfected DCs (Figure 4).

Proliferation
DCs are potent stimulators of primary MLRs and are able to induce the proliferation of allogeneic T lymphocytes in vitro.We compared the abilities of our DC populations to stimulate primary MLRs among allogeneic T lymphocytes.The data demonstrated that DCs transduced with Ad-tPSMA-IRES-m4-1BBL induced stronger allogeneic T-cell proliferative responses in vitro than untreated DCs and DCs transduced with Ad-eGFP (Figure 5).

Discussion
DCs are among the most potent APCs for induction of antitumor immune responses currently known and have been recognized as potentially important tools for cancer vaccine strategies (17).Bone marrow-derived DCs have been successfully employed in vitro both in animal models and in clinical trials (18,19).Moreover, genetic material can be introduced into DCs with varying levels of efficacy, using techniques such as electroporation, lipid-mediated transfection, calcium phosphate precipitation, and virally mediated gene transfer (20)(21)(22).Recombinant adenovirus is the most efficient vector for gene transfer to DCs due to its two unique features.First, adenovirus performs a transgene delivery to DCs of higher magnitude than other available systems (23).In addition, adenovirus infection alone, without the addition of a therapeutic transgene, causes a high degree of DC maturation in terms of phenotype and function (24).Our results showed that the expression of eGFP in transfected DCs reached a high level of 65% (Figure 1B), which indicated efficient gene transduction.
PSMA is a well-defined prostate-restricted TAA whose expression is significantly elevated in carcinoma of the prostate, especially in advanced stages.It has been shown that a PSMA-encoding adenovirus-transfected DC vaccine induced a specific and strong immune response against prostate cancer cells (25).4-1BBL, with its receptor 4-1BB, forms a pair of co-stimulatory molecules with profound effects on T cells, including enhancement of T cell expansion, and increased T cell effector function (16,26,27).4-1BBL gene-modified DCs could enhance effector and memory cytotoxic T-lymphocyte responses (28,29).Recently, immature DCs have been reported to constitutively express 4-1BB (30), raising the possibility of DC-DC reciprocal and/or autocrine stimulation via the 4-1BB/4-1BBL pathway.Thus, 4-1BBL may be involved in DC activation in a T cell-independent, as well as a T cell-dependent, fashion (30).
In the present study, we tried to analyze the immunogenicity of DCs transduced with recombinant adenovirus carring a TAA gene and m4-1BBL gene.Our results demonstrated the higher expression of surface markers (MHC II, CD80, CD86) on DCs transduced with Ad-tPSMA-IRES-m4-1BBL than on DCs transfected with Ad-eGFP and non-transfected DCs (Table 1).This higher expression of surface molecules may enhance antigen presentation to T cells by DCs.Ad-tPSMA-IRES-m4-1BBL-transduced DCs significantly increased cytokine (IL-6, IL-12) production (Figure 3) and T-cell proliferation.We also used the apoptosis inducer bortezomib (0.1 µg/mL) to treat Ad-transfected DCs and the results indicated that Ad-tPSMA-IRES-m4-1BBL-transduced DCs showed higher anti-apoptosis ability (Figure 4).The present results demonstrate that recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and m4-1BBL gene-modified DCs enhanced immunogenicity, which may induce a specific and strong immune response against prostate cancer cells.Further studies are needed to address this possibility.For other abbreviations, see legend to Table 1.  1.For other abbreviations, see legend to Table 1.

Figure 1 .
Figure 1.Construction of recombinant (Ad) and transduction of dendritic cells (DCs) with adenovirus.A, Schematic overview of Ad5, Ad-eGFP recombinant adenovirus, and Ad-tPS-MA-IRES-m4-1BBL recombinant adenovirus.B, Transduction efficiency in DCs.eGFP expression was evaluated by flow cytometry 48 h after gene transduction (65.6% of eGFP-positive cells).The percentage of viable cells was evaluated by Trypan blue staining.CMV = cytomegalovirus.For other abbreviations, see legend toTable1.

Figure 2 .
Figure 2. Detection of tPSMA protein and m4-1BBLprotein by Western blot.Total cell lysates were prepared and the presence of tPSMA protein and m4-1BBL protein was detected using the anti-PSMA polyclonal antibody and anti-4-1BBL polyclonal antibody, respectivly.A tPSMA-specific band and a 4-1BBL-specific band were detected in dendritic cells (DCs) transfected with Ad-tPSMA-IRES-m4-BBL but not in DCs transfected with Ad-eGFP or untreated DCs.For other abbreviations, see legend to Table1.

Figure 3 .Figure 4 .
Figure 3. Cytokine (IL-6/IL-12) production by dendritic cells (DCs).The culture supernatants of DCs transfected with Ad-tPSMA-IRES-m4-1BBL, Ad-eGFP, or not transfected were collected for the analysis of IL-6 and IL-12 production by ELISA.Data are reported as means ± SD.Similar results were obtained from three independent experiments.*P < 0.05 compared to DCs transfected with Ad-eGFP and non-transfected DC (t-test).For other abbreviations, see legend to Table1.

Figure 5 .
Figure 5. Mixed lymphocyte reaction.T lymphocytes were stimulated with Ad-tPSMA-IRES-m4-1BBL-infected dendritic cells (DCs), Ad-eGFP-transfected DCs, and non-transfected DCs.DCs transfected with Ad-tPSMA-IRES-m4-1BBL for 48 h and matured with lipopolysaccharide for another 24 h were collected and co-cultured with T cells for 96 h.Specific cytotoxic T lymphocytes were detected by the cholecystokin-8 assay.Data are reported as means ± SD of three replicates.The data indicate that Ad-tPSMA-IRES-m4-1BBL-infected DCs were potent lymphocyte stimulators compared to Ad-eGFP-transfected DCs and non-transfected DCs (*P < 0.05 compared to Ad-eGFP-transfected DCs and non-transfected DCs; t-test).For other abbreviations, see legend to Table1.