Cellular Immunology 271 (2011) 73–77

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Cellular Immunology

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Dendritic cells transfected with PD-L1 recombinant adenovirus induces T cellsuppression and long-term acceptance of allograft transplantation

Wei Peng a,1, Boli Ran b,1, Yuanzheng Ma a, Xunwu Huang a, Qing Chang a, Xiangwei Wang c,⇑a Department of Orthopaedics, 309th Hospital of PLA, Beijing 100091, Chinab Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, Chinac Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China

a r t i c l e i n f o

Article history:Received 22 March 2011Accepted 8 June 2011Available online 29 June 2011

Keywords:Dendritic cellsPD-L1AdenovirusAllograft transplantation

0008-8749/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.cellimm.2011.06.007

⇑ Corresponding author. Fax: +86 23 68774419.E-mail address: winn0324@sohu.com (X. Wang).

1 These authors were regarded as co-first authors.

a b s t r a c t

The purpose of this study is to assess the potential of dendritic cells transfected with PD-L1 recombinantadenovirus induces CD8+ T cell suppression and kidney allograft tolerance. To prove it, DCs transfectedwith PD-L1 recombinant adenovirus (DC/Ad-PD-L1) were transferred into the MHC-mismatched rat kid-ney transplants. After kidney transplantation, the mixed lymphocyte reaction (MLR) assay and kidneyfunction were analyzed. The results demonstrated that after administration of DC/Ad-PD-L1, the prolif-eration, cytokines secretion and activation marker expression of CD8+ T cells were suppressed. In addi-tion, DC/Ad-PD-L1 could improve kidney function and survival of transplants. The findings suggested thatDC/Ad-PD-L1 could induce CD8+ T cell tolerance and lead to kidney allograft tolerance, which provided apromising finding for clinical application.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Costimulation is required for effector or regulatory T lympho-cytes to respond optimally following the engagement of theirT-cell receptor (TCR) [1–3]. Costimulation blockade has beenwidely investigated as an approach to control T-cell reactivity inautoimmunity and transplantation [4–6]. Programmed death-ligand 1 (PD-1) expressed on T cells is an important inhibitory mol-ecule, which binds to the APC ligands PD-L1. On T-cell activation,up-regulation of PD-1 expression also contributes to T-cell homeo-stasis [7–10]. Therefore, the negative co-stimulatory pathwayPD-1/PD-L1 suppresses T-cell-mediated alloimmunity and inducestransplantation tolerance [11–13].

Dendritic cells (DCs) are the most potent APCs responsible forpriming of naive T cells in initiation of the immune response. Recentstudies revealed that DCs were also involved in the maintenance ofimmunologic self-tolerance [14–16]. In vivo transfer of Ag-loadedDCs with a tolerogenic character is regarded as a promisingtherapeutic means to negatively manipulate immune response[17–20]. However, the function of PD-1/PD-L1 in kidney trans-plantation has not been fully described. In this study, DC/Ad-PD-L1was transferred into the MHC-mismatched rat kidney transplants,and its potential of inducing immune tolerance in kidney transplantswas assessed.

ll rights reserved.

2. Materials and methods

2.1. Construction of recombinant adenovirus encoding PD-L1

The recombinant adenovirus vector encoding PD-L1 was con-structed using the Adeno-XTM Expression System (Clontech, PaloAlto, CA, USA) according to the manufacturer’s instructions. Briefly,the PD-L1 cDNA was cloned into the shuttle vector pDC315 andsequenced. The desired replication-deficient adenovirus containingthe full-length cDNA of PD-L1 was generated by homologousrecombination through cotransfection of plasmids pDC315-PD-L1and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposomereagent (Roche, Germany).

After several rounds of plaque purification, the adenovirus con-taining the PD-L1 gene was amplified and purified from cell lysatesby banding twice in CsCl density gradients. Viral products were de-salted and stored at �80 �C in phosphate buffered saline (PBS) con-taining 10% glycerol (v/v). The infectious titer was determined by astandard plaque assay. A second recombinant El-, E3-deleted ade-novirus carrying the LacZ protein under the control of CMV pro-moter (rAd-LacZ) was used as a control vector for DC transduction.

2.2. Animals

Naïve inbred male Lewis and Fisher 344 rats weighing200–250 g were purchased from the Experimental Animal Centerof Chinese Academy of Medical Science (Beijing, China). Animalswere housed under pathogen-free conditions. All experimental

74 W. Peng et al. / Cellular Immunology 271 (2011) 73–77

procedures were carried out following approval of the InstitutionalAnimal Care Committee.

2.3. DC generation from rat bone marrow

In brief, bone marrow was flushed from the tibias and femurs ofFisher 344 rats and depleted of erythrocytes with commercial lysisbuffer (Sigma, St.Louis, MO, USA). The cells were washed twice inserum-free RPMI-1640 medium and cultured in a six-well plateat 5 � 106 cells per well with RPMI-1640 medium supplementedwith 10% (v/v) FBS containing 10 ng/ml recombinant murine GM-CSF (R&D System, Inc., USA) and 10 ng/ml recombinant murineIL-4 (R&D System, Inc.). On days 3 and 5, half of the media were re-freshed without discarding any cells and fresh cytokine containing(mGM-CSF and mIL-4) media were added. On days 7 and 8 of cul-ture, mTNF-a (R&D System, Inc.) was added to the media. On day10, non-adherent cells obtained from these cultures were consid-ered mature bone marrow-derived DCs. DCs were isolated andused for transfection assays and T-cell tolerance experiments.

2.4. Adenovirus-mediated gene transduction of DCs

Transduction of DCs with Ad vector was conducted in 6-wellplates with 1 � 106 DCs/well in 3 ml of RPMI 1640 medium con-taining 10% FBS. Virus was added to the wells at a multiplicity ofinfection (MOI) of 200, and the DCs were harvested after 24 h ofincubation.

2.5. Western blot assay

For western blot assay, proteins of the cell extracts were sepa-rated by sodium dodecyl sulphate–polyacrylamide gel electropho-resis (SDS–PAGE) and then transferred onto a nitrocellulosemembrane. The membrane was incubated with 5% non-fat milkin PBS and then with anti-PD-L1 antibody (Santa Cruz Biotechnol-ogy, USA) for 2 h at room temperature. After washing, the mem-branes were incubated with an alkaline phosphatase conjugatedgoat anti-rat IgG antibody (Amersham Biosciences, UK) for 1 h atroom temperature. Immunoreactive bands were detected usingthe ECL western blotting analysis system (Amersham Biosciences).

2.6. Cell transfer assay

Transduced DCs were washed and injected to Lewis rats (with a2-day interval for three times) at a dose of 5 � 106 by i.v. in 1 ml ofPBS at 7 days before kidney surgery. As a control, Lewis rats wereinjected with the same procedure using PBS.

2.7. Kidney surgery

Operative procedures were performed under general anesthesiainduced by 10% chloral hydrate (0.03 ml/kg body weight) adminis-tered intraperitoneally. 7 days after the last cell transfer, the leftkidney of the donor rat (Fisher 344) was perfused through the aor-ta with 4 �C heparinized Ringer’s lactate solution and was isolated,removed, cooled and positioned orthotopically in the host rat (Le-wis), whose native kidneys had been removed and the left renalvessels isolated and clamped. The donor and recipient renal arteryand vein were then anastomosed end-to-end with 10–0 polypro-pylene (Prolene�) sutures. After releasing the vascular clamps,the donor and recipient ureters were anastomosed end-to-endwith 11–0 polypropylene (Prolene�) sutures. No ureteral stentwas used. Ischaemic time was about 30 min (range 20–45 min).Animals that died within 3 days of surgery were considered techni-cal complications and were excluded from the analysis.

2.8. Coculture of DCs and CD8+ T cells by one-way MLRs assay

DCs were prepared using the method described above. Singlecell suspensions were treated with mitomycin C (50 lg/ml; Sig-ma–Aldrich) for 20 min at room temperature and were thenwashed twice with RPMI 1640. These cells were used as the stim-ulator cells in the assay. Spleen leukocytes of Lewis rats were asep-tically prepared by mashing the spleen and then lysing the RBC inNH4Cl buffer. Responder CD8+ T cells were harvested by negativeisolation using magnetic beads according to the manufacturer’sprotocol (Dynal T Cell Negative Isolation kit; Invitrogen Life Tech-nologies).The purity of the CD8+ T cells was determined by FACS(BD Biosciences); only CD8+ T cell populations with >95% puritywas used in this study. Responder (2 � 105, Lewis) and stimulator(4 � 105, Fisher 344) cells were added to round-bottom 96-wellplates to a final volume of 200 ll RPMI 1640 with 10% FCS. Eachexperiment was performed in triplicate.

2.9. Proliferation assays

Cells activated in an MLR were allowed to incubate for up to3 days before harvesting. [3H] Thymidine (0.5 lCi/well; MPBio-medicals) was added 24 h before harvesting (Skatron Instruments)using Type A filter mats (Perkin-Elmer Life and Analytical Sciences)and a beta plate scintillation mixture (Perkin-Elmer). Disintegra-tions per minute were determined using a liquid scintillation coun-ter (1205 Betaplate; Perkin-Elmer).

2.10. ELISA assay for quantitating cytokine production

ELISA was performed on supernatants obtained on day 3 of cul-ture. ELISAs for IL-2 and IFN-c (Ready, Set, Go! ELISA kit; eBio-science) were performed according to the manufacturer’sprotocol and analyzed on a Model 680 microplate reader (Bio-Rad).

2.11. Flow cytometry for detecting activation marker expression

CD8+ T cells were cocultured with DCs for 3 days. The cells werewashed with PBS and then stained with anti-CD25-FITC and anti-CD69-APC at 4 �C for 30 min. The cells were then washed againin PBS and analyzed on a FCM Calibur flow cytometer with Cell-Quest software for CD25 and CD69 activation marker expression.Isotype controls were also performed.

2.12. Assessment of proteinuria and survival

Every 2 days after transplantation, 24-h urine samples were col-lected using metabolite cages. Urine protein concentrations weremeasured using the Bradford method. In addition, the mean sur-vival was also monitored during this period of this time.

2.13. Statistical analysis

The statistical significance of differential findings betweenexperimental groups and controls were determined by t-test andwere considered significant for P < 0.05.

3. Results

3.1. Gene transfer and PD-L1 protein expression

Protein expression of PD-L1 was demonstrated by transienttransfection of adenovirus into DCs and was detected by Westernblot assay. In accordance with protocols mentioned above, DCswere transfected with Ad-PD-L1 or Ad-LacZ at MOI 100 for 24 h.

Fig. 1. Expression of PD-L1 protein in DCs by Western blot analysis. DCs weretransfected with Ad-PD-L1 or Ad-LacZ at an MOI of 200 for 24 h. The expression ofthe PD-L1 protein was detected after Ad-PD-L1 transfection. However, there was noexpression of PD-L1 protein after Ad-LacZ transfection or in non-treated DCs. Lane1: non-treated DCs; lane 2: DCs transfected with Ad-LacZ; lane 3: DCs transfectedwith Ad-PD-L1. Three repeated experiments showed consistent results.

Fig. 3. CD8+ T cell cytokine production. Transfected DCs were cocultured withallogeneic CD8+ T cells for 3 days. The supernatant was harvested and analyzed byELISA. Groups were defined as follows: untreated DC cells, DC/Ad-LacZ and DCs/Ad-

W. Peng et al. / Cellular Immunology 271 (2011) 73–77 75

As seen in Fig. 1, the expression of the PD-L1 protein was detectedafter Ad- PD-L1 transfection. However, there was no expression ofPD-L1 protein after Ad-LacZ transfection and non-treated DCs.

PD-L1. Histogram numbers represent the mean ± S.E.M. for each experiment.Experiments were repeated three times with consistent results. Compared withcontrols, ⁄P < 0.05.

3.2. DC/Ad-PD-L1 attenuates CD8+ T cell proliferation

To test whether DC/Ad-PD-L1 could attenuate CD8+ T cell pro-liferation, DC/Ad-PD-L1 were cocultured with CD8+ T cells for3 days using the methods described above. [3H] Thymidine(0.5 lCi/well; MP Biomedicals) was added to detect and quantifyproliferation. As seen in Fig. 2, CD8+ T cells cocultured with DC/Ad-PD-L1 exhibited decreased proliferation compared to DC/Ad-LacZ and untreated DC controls (P < 0.05). These results indicatethat DC/Ad-PD-L1 could attenuate CD8+ T cell proliferation.

3.3. DC/Ad-PD-L1 inhibits cytokine release of CD8+ T cells

To test whether DC/Ad-PD-L1 could attenuate CD8+ T cell cyto-kine production, after cocultured with CD8+ T cells for 3 daysas described above, the supernatant was harvested and analyzedby ELISA according to standard methods. As seen in Fig. 3, DC/Ad-PD-L1 could attenuate IL-2 and IFN-c production of CD8+ Tcells (73.1 ± 9.1 and 48.5 ± 5.9 pg/ml, respectively) compared toIL-2 and IFN-c released from the DC/Ad-LacZ control cells(115.7 ± 12.1 and 94.6 ± 7.8 pg/ml, respectively) (P < 0.05). Theseresults demonstrate that DC/Ad-PD-L1 can inhibit cytokine releasefrom CD8+ T cells.

Fig. 2. CD8+ T cell proliferation assay. Transfected DCs were treated withmitomycin C (50 lg/ml) for 20 min at room temperature to prepare them asstimulator cells. Allogeneic responder CD8+ T cells were harvested by CD8 negativeisolation with magnetic beads. Responder (2 � 105, Lewis) and stimulator (4 � 105,Fisher 344) cells were cocultured for 3 days. [3H] Thymidine (0.5 lCi/well) wasadded 24 h before harvesting using Type A filtermats and a beta plate scintillationmixture. Disintegrations per minute were determined using a liquid scintillationcounter (1205 Betaplate; Perkin-Elmer). The groups include the following:untreated DC cells, DC/Ad-LacZ and DCs/Ad-PD-L1. Histogram numbers representthe mean ± S.E.M. for each experiment. Three repeated experiments showedconsistent results. Compared with controls, ⁄P < 0.05.

3.4. DC/Ad-PD-L1 inhibits expression of CD8+ T cells activationmarkers

To test whether DC/Ad-PD-L1 could inhibit CD8+ T cells activa-tion marker expression, after cocultured with CD8+ T cells for3 days using the methods described above, the cells were washedwith PBS and stained with anti-CD25-FITC and anti-CD69-APC at4 �C for 30 min. The cells were then washed with PBS and analyzedon a FCM Calibur flow cytometer with CellQuest software. As seenin Fig. 4, DC/Ad-PD-L1 could decrease expression of the CD8+ T cellactivation markers CD25 and CD69 (8.3 ± 1.2% and 11.8 ± 2.5%,respectively) compared to the percentage of Ad-LacZ-transfectedcontrol cells (14.5 ± 2.7% and 19.4 ± 3.7%, respectively) (P < 0.05).These results demonstrate that DC/Ad-PD-L1 could inhibit CD8+T cell activation marker expression.

3.5. DC/Ad-PD-L1 suppresses proteinuria and improves survival

To assess the effect of DC/Ad-PD-L1could suppress proteinuriaand improves survival, after kidney transplantation, proteinuriaand survival were monitored. As seen in Fig. 5A, proteinuria in-creased slowly in the DC/Ad-PD-L1 group after kidney transplanta-tion. However, proteinuria increased quickly in the control group(P < 0.05). In addition, as seen in Fig. 5B, the percent survival ofcontrol rats began to decrease after kidney transplantation, andafter 12 days, nearly all of the rats died. On the contrary, in theDC/Ad-PD-L1 group, the survival rate remained 60% after 20 days(P < 0.05). Therefore, the results revealed that DC/Ad-PD-L1 coulddelay the development of severe proteinuria and prolong the meansurvival of rats.

4. Discussion

Organ transplantation is currently the only therapeutic choicefor the treatment of end-stage organ failure, but it requires thecontinuous administration of immunosuppressive drugs to abro-gate the host immune response against the graft [21–23]. Further-more, despite the efficacy of immunosuppression in preventingand reverting acute episodes of rejection, chronic rejection stilloccurs and more research must be carried out to fully understandthe mechanistic basis of this pathological process. Numerous evi-dences demonstrated that CD8+ cells played a crucial role in thepathogenesis of immune disorder diseases such as autoimmunedisease or organ transplantation, thus inducing tolerance of CD8+T cells is regarded as a promising therapeutic means to negatively

Fig. 4. CD8+ T cells activation marker expression. Transfected DCs were coculturedwith allogeneic CD8+ T cells for 3 days. The cells were stained with anti-CD25-FITCand anti-CD69-APC and analyzed on a FCM Calibur flow cytometer using CellQuestsoftware. The groups include the following: untreated DC cells, DC/Ad-LacZ and DC/Ad-PD-L1. (A) Activation marker expression was determined by counting thepercentage of CD25 or CD69 positive cell numbers. (B)The representative FACSgraphs of CD8+ T cells activation marker expression. Histogram numbers representthe mean ± S.E.M. for each experiment. Experiments were repeated three timeswith consistent results. Compared with controls, ⁄P < 0.05.

Fig. 5. Assessment of proteinuria and survival. (A) Every 2 days after transplanta-tion, 24-h urine samples were collected using metabolite cages. Urine proteinconcentrations were measured using the Bradford method. (B) Every 2 days aftertransplantation, the mean survival of rats was also monitored.

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manipulate immune response [24–27]. Activation of T cellsrequires specific signaling by the interaction between the T-cellreceptor and Ag presented by antigen-presenting cell (APCs), andis also regulated by positive and negative co-stimulatorysignals [28,29]. As a consequence therapeutic modulation ofco-stimulatory molecules for instance with CTLA4Ig can lead toCD8+ T tolerance [30–32]. In addition, much evidence suggests thatDCs are the most potent APC responsible for priming of naive CD8+T cells in initiation of the immune response and are also involvedin the maintenance of immunologic self-tolerance, promotingCD8+ T cells with regulatory functions or inducing anergy ofCD8+ T cells [33–35].

Therefore, to explore the potential of PD-1/PD-L1 pathway inthe kidney allograft tolerance, the dendritic cells transfected withPD-L1 recombinant adenovirus was prepared, and the effect wasassessed. Firstly, the Ad/PD-L1 was prepared and transduced intoDCs. The Western blot assay demonstrated that Ad/PD-L1 couldtransduce into DCs and mediate PD-L1 expression successfully.Secondly, DC/Ad-PD-L1 was transferred into the MHC-mismatchedrat kidney transplants. After kidney transplantation, the MLR assaywas performed. The results demonstrated that DC/Ad-PD-L1 couldsuppress the proliferation, cytokines secretion and activation mar-ker expression of CD8+ T cells. Lastly, the results also demonstratedthat DC/Ad-PD-L1 could improve kidney function and survival oftransplants.

Taken together, our findings suggested that DCs transfectedwith Ad-PD-L1 could induce T-cells tolerance, which provided a

promising therapeutic means to negatively manipulate immuneresponse in allograft transplantation.

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