Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival

SHORT COMMUNICATIONOverexpression of Jagged-1combined with blockade of CD40pathway prolongs allograft survivalYingying Lin1,Weili Chen1,Jiali Li1,Guoliang Yan2,Chun Li1,Ning Jin1,Jie Chen2,Chang Gao1,Pengfei Ma1, Shuangyue Xu1and Zhongquan Qi1Dendritic cells(DCs)have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance.Here we investigated whether blockade of the CD40pathway could enhance the immune tolerance induced by DC2.4cells modified to express Jagged-1(JAG1-DC)in heart transplantation.Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody(mAb)administration significantly prolonged cardiac allograft survival in mice,with long-term survival (4110days)of50%of the allografts in the recipients.The therapy specifically inhibited the immune response,induced alloantigen-specific T-cell hyporesponsiveness,upregulated transforming growth factor-βsynthesis and increased the population of regulatory T cells(Tregs)driven by Jagged-1-Notch activation.These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1stimulation.Immunology and Cell Biology advance online publication,7October2014;doi:10.1038/icb.2014.84Dendritic cells(DCs)in the periphery are in an immature state and become mature on migration to the draining lymphoid tissues,where DCs present antigens to naive T cells and initiate specific immune responses.This process is also crucial for the induction of peripheral tolerance.1–3Various strategies have been used to induce specific T-cell hyporesponsiveness and prolong allograft survival by enhancing the tolerogenic potential,which include the application of specific DC subsets and genetically modified DCs that express immunosuppressive molecules,such as viral interleukin-10(IL-10)or cytotoxic T lymphocyte antigen4-immunoglobulin.4–7It is worth noting that the immature murine DC2.4cell line has the advantage over primary DCs of being suitable for long-term passage in cell culture and expressing high levels of major histocompatibility complex(MHC) class II molecules,CD80and CD86.8–10To date,four Notch receptors(Notch1–4)andfive Notch ligands (Delta-like l,3,4and Jagged1,2)have been identified,and these molecules have various functions in mammals.11,12Notch signalling has also been implicated as a key regulator of T-cell activation and T helper(Th)cell differentiation;in addition,Notch ligands are expressed on the surface of antigen-presenting cells.13,14Moreover, there are emerging data suggesting that the Jagged-1-Notch signalling pathway could induce specific regulatory T cells(Tregs)by over-expression of Jagged-1or interactions with transforming growth factor-β(TGF-β),and these Tregs could not only inhibit primary immune responses,but also transfer tolerance of antigenic specificity into the recipient.15–17It is well known that the CD40/CD40L pathway is one of the most important costimulatory pathways and controls the activation of antigen-specific T cells.18Blockade of this pathway has been reported to effectively disrupt T-cell activation and prolong survival of heart, kidney and islet allografts.19In this study,we are thefirst to demonstrate that combined administration of anti-CD40L mono-clonal antibody(mAb)with overexpression of Jagged-1(using the adenovirus,which is the most widely used vector in gene therapy applications)20,21blocks the activation of effector T cells,upregulates the levels of Tregs and leads to long-term allograft survival. RESULTSOverexpression of Jagged-1in the DC2.4cell lineThe pAd5/F35-GFP and pAd5/F35-GFP-Jagged-1viruses efficiently transduced DC2.4cells(abbreviated to GFP(greenfluorescent protein)-DC and JAG1-DC,respectively)at a multiplicity of infection of150plaque forming units per cell.More than90%of the DC2.4 exhibited strong greenfluorescence at48h(both GFP-DC and JAG1-DC,see Figures1a and c).Then,we observed that the Jagged-1protein(180kD)was only expressed in JAG1-DC (Figure1b).By contrast,no significant difference was found between JAG1-DC and GFP-DC in terms of the expression levels of CD11c, CD80,CD86and MHC-II(Figure1c).This result showed that neither transduction by control Ad vector nor overexpression of Jagged-1had any effect on DC expression of surface molecules.Jagged-1modulates the proliferation and activation of T cells by activating Notch-1To measure the effects of Jagged-1on immune responses,we isolated T cells from naive BALB/c mice and co-cultured them with GFP-DC1Organ Transplantation Institute,Xiamen University,Fujian Province,P.R.China and2Basic Medical Department of Medical College,Xiamen University,Fujian Province, P.R.ChinaCorrespondence:Professor Z Qi,Organ Transplantation Institute,Xiamen University,Fujian Province361005,P.R.China.E-mail:oti@Received14March2014;revised25August2014;accepted3September2014or JAG1-DC.Proliferation of the T cells alone was different from T cells+DC (P o 0.05,Figure 1d).JAG1-DC signi ficantly inhibited the proliferation of T cells compared with GFP-DC (P o 0.01,Figure 1d).However,there was no inhibition when JAG-DC were added to memory (CD44High CD62L Low )T cells (data not shown).In addition,the HES-1and Notch-1mRNA expression levels in T cells were also signi ficantly increased after co-culturing with JAG1-DC for 12h compared with GFP-DC (with a mean 4-fold upregulation of HES-1and 68-fold upregulation of Notch-1,Figure 1e).However,the expression of Deltex-1showed no signi ficant differences.Because HES-1or Deltex-1is the downstream transcrip-tional regulator induced by Notch-1receptor activation,our results con firm that the overexpression of Jagged-1protein was biologically active.22,23JAG1-DC signi ficantly enhances the immunosuppressive effect of CD40/CD40L costimulatory blockadeThe JAG1-DC group showed markedly prolonged graft survival compared with either the heart transplantation (HTx)control group or the GFP-DC group (median survival time =14vs 7.5or 8days,n =6per group,both P o 0.01,Figure 2a).But there was also no statistical difference between the control Ab group and the Ab+GFP-DC group (median survival time =44vs 50.5days,n =6per group,P 40.05).A signi ficant prolongation of graft survival was achieved in the Ab+JAG1-DC group,with long-term survival (4110days)of 50%of the recipients,compared with the Ab group or Ab+GFP-DC group (median survival time =103.5vs 44or 50.5days,n =6per group,both P o 0.05).These results suggest that the addition of CD40L blockade to the JAG1-DC treatment signi ficantly prolongs graft survival time.Combination treatment prevents in flammatory reactions in grafts and spleens of recipientsOn days 7and 100post transplantation,all grafts were collected and the expression of genes associated with graft rejection and tolerance were detected with quantitative real-time PCR (Figure 2b).Compared with the Ab+GFP-DC group,the expression of IFN-γwas signi ficantly reduced both on days 7and 100in the Ab+JAG1-DC group.However,the expression of IL-10and IL-4showed no signi ficant differences in the five groups (data not shown).Overall,the relative expressions of both TGF-βand Foxp3were signi ficantly higher in the Ab+JAG1-DC group on day 7(or on day 100)than that in the Ab+GFP-DC group.We observed that the JAG1-DC group had a reduced proliferative response on day 7(Figure 2d).Furthermore,the Ab+JAG1-DC group had a lower proliferative capacity than the JAG1-DC group.To determine whether this proliferation was donor speci fic,the spleno-cytes from C3H mice were used as third-party stimulators.The result revealed that the response of recipient spleens to third-party cells was still strong in the combined therapy group on day 100(Figure 2d).Combination treatment induces expressions of Foxp3and TGF-βTo determine whether the combination treatment resulted in the expansion of Tregs,the expression of CD4and Foxp3were examined (Figure 2c).The results revealed that CD4+Foxp3+T cells were signi ficantly expanded in the spleens and lymph nodes of the Ab +JAG1-DC group on day 7compared with the HTx control group (mean ±s.d.:2.14±0.07vs 1.51±0.11%in the spleens,5.21±0.24vs 3.86±0.27%in the lymph nodes,both P o 0.01).On day 100,the Ab +JAG1-DC group had a higher population of CD4+Foxp3+T cells in the spleens and lymph nodes compared with the Ab+JAG1-DC group on day 7(2.65±0.09vs 2.14±0.07%and 6.09±0.29vs 5.21±0.24%,P o 0.01and P o 0.05,respectively).But there was no difference among the HTx control group,GFP-DC group (spleens:1.59±0.08%,lymphFigure 1Overexpression of Jagged-1in the DC2.4cells.(a )At 48h after infection,the DC2.4cells were analysed for GFP expression and (b )Jagged-1protein (≈180kDa)was detected by western blot.(c )DC2.4cells transduced with Ad5/F35-GFP-Jagged-1(solid lines)showed the same expression of MHC class II and costimulatory ligands as control GFP-DC (dotted lines)by flow cytometry (the isotype controls were shown as filled histogram).(d )MLR cultures consisted of T-cell responders,and GFP-DC or JAG1-DC2.4stimulator cells were determined after 3days.(e )To con firm the functionality of Jagged-1expressed on transduced DCs,T-cell populations were analysed for HES-1,Deltex-1and Notch-1mRNA expression after 12h in the MLR in vitro .Each reaction was carried out in triplicate.Data are representative of three independent experiments,which showed similar results (*P o 0.05,**P o 0.01,***P o 0.001).A full colour version of this figure is available at the Immunology and Cell Biology journal online.2Immunology and Cell Biologynodes: 4.04±0.28%)and JAG1-DC group (spleens: 1.73±0.11%,lymph nodes:4.52±0.15%)on day 7.There was also no difference among the Ab group (spleens: 2.0±0.06%,lymph nodes:4.57±0.23%),Ab+GFP-DC group (spleens: 2.02±0.11%,lymph nodes:5.02±0.12%)and Ab+JAG1-DC group either in the spleens or lymph nodes on day 7.The levels of the in flammatory or tolerogenic cytokines in animal sera were analysed (Figure 2e).Compared with the HTx control group,the JAG1-DC group exhibited signi ficantly upregulated expres-sion of TGF-βon day 7.In addition,the levels of TGF-βwere stably increased in the Ab+JAG1-DC group when compared with the Ab group or Ab+GFP-DC group on day 7.Although the level of TGF-βwas decreased at 100days,it remained at a high level compared with that of the HTx control group.However,there were no marked or persistent increases in the production of IL-4and IL-10in the Ab +JAG1-DC group (data not shown).DISCUSSIONNotch has an important role in regulating the differentiation of naive CD4T cells into distinct Th lineages while antigen-presenting cells express different Notch ligands.24When Notch is activated,the γ-secretase complex proteolytically releases the Notch intracellular domain,which subsequently traf fics to the nucleus and heterodi-merises with the DNA-binding transcription factor CSL,leading to Notch-dependent target gene transcription.25It has been reported that the NICD/CSL complex can bind to lineage-speci fic transcription factors such as T-bet,Gata3or the ROR γt promoter.These interac-tions have a role in directly modulating the expression of cytokines and the phosphorylation of signal transducer and activator of transcription family,26thereby regulating the differentiation of naive T cells into Th1,Th2or Th17cells.27–29It has also been reported that Jagged-1-Notch interaction can speci fically direct differentiation into Th2cells.Our results indicated that overexpression of Jagged-1in DC2.4cells inhibits T-cell proliferation in vitro ,as a result of Notch-1activation.However,there was no marked or persistent increase in the production of IL-4in vivo .Instead,JAG1-DC combined with anti-CD40L therapy markedly inhibited splenic T-cell proliferation and Th1immunity.In addition,the Jagged-1-Notch pathway regulates naive CD4T-cell differentiation into induced Tregs,which include Th3and Tr1cells.17,30We observed that the expression of TGF-β,but not IL-10,was increased in the grafts and sera in the Ab+JAG1-DC group.Meanwhile,this combination treatment induced a larger CD4+Foxp3+Treg population in the spleens and lymph nodes after 100days,as well as in the grafts.Th3cells have been shown to induce tolerance and downregulate both Th1and Th2responses.31,32Therefore,we presumed that the induced Tregs in the Ab+JAG1-DC group were mostly TGF-β-secreting Th3cells,causing suppression of Th1immunity and maintaining allograft acceptance,which is associated with the presence of the TGF-βsignalling pathway.16,33,34The tolerance induced by anti-CD40L therapy has been showed to depend mostly on the induction of hyporesponsiveness and Tregs rather than T-cell depletion,35,36matching the results we obtained for the combined therapy group in vivo .Another potential mechanism by which the Jagged-1-Notch signalling pathway functions in Th or induced Treg differentiation may be related to interaction withtheFigure 2The JAG1-DC combined with blockade of CD40pathway synergizes to prolong cardiac allograft survival.(a )Cardiac allograft survival was calculated by the Kaplan –Meier method and compared by a log-rank test (n =6mice per group).(b )The relative expression of the mRNA levels of IL-2,IFN-γ,TGF-βand Foxp3in grafts was detected by quantitative real-time PCR.(c )The proportion of CD4+Foxp3+Tregs in the recipients in indicated groups were analysed by flow cytometry.(d )MLR were used to test the proliferative responses of recipient splenic cells to donor cells of C57BL/6or C3H mice.(e )The concentrations of TGF-βin the sera were detected.Each reaction was carried out in triplicate.All data are representative of three independent experiments,which showed similar results (n =3mice per group,*P o 0.05,**P o 0.01,***P o 0.001).A full colour version of this figure is available at the Immunology and Cell Biology journal online.3Immunology and Cell BiologyNF-κB signalling pathway,which has crucial roles in inflammation, immune responses,cell survival and cytokine production.37–39 In conclusion,we showed that the combination therapy had critical effects on the regulation of immune response(it markedly inhibited Th1immunity and induced induced Tregs,but did not shift the balance towards Th2immunity)and promoted long-term allograft survival.This is thefirst report of the effect of JAG1-DC administration in combination with CD40pathway blockade in vivo.This combination strategy,which promotes the induction of Tregs through Jagged-1 stimulation,is highly attractive for gene therapy and has potential as gene modification for treating immunological diseases or as an adjunct to clinical transplantation.METHODSAnimalsFemale C57BL/6,BALB/c and C3H mice,6–8weeks of age,were purchased from Slac Laboratory Animal Co.Ltd(Shanghai,China)and maintained in a specific pathogen-free environment in accordance with the guidelines provided in the‘Guide for the Care and Use of Laboratory Animals’(US Department of Health and Human Services).Cell linesThe DC2.4cell line(H-2K b,ShangHai HuiYing Biotechnology Co.Ltd, ShangHai,China)was cultured in RPMI1640medium supplemented with 10%fetal calf serum(Gibco,Grand Island,NY,USA),50μmβ-mercaptoetha-nol(Sigma-Aldrich,St Louis,MO,USA),2m M L-glutamine and10m M HEPES (Life Technologies,Grand Island,NY,USA)and penicillin(100U ml−1)–streptomycin(100μg ml−1)at37°C in a humid environment with5%CO2. Adenoviral vectorCytomegalovirus promoter DNA,the complementary DNA corresponding to full-length Jagged-1(GeneBank:BC058675.1)and GFP was cloned into the chimaeric adenoviral vector pAd5/F35.A human embryonic kidney293cell line was transduced with these Ad5/F35vectors using Lipofectamine2000(Life Technologies)and expanded the viruses.The viruses were extracted using three consecutive freeze/thaw cycles and then were concentrated and purified by double caesium chloride gradient ultracentrifugation and desalting with a dialysis cassette(Pierce,Rockford,IL,USA).DC2.4cells were transduced with either pAd5/F35-GFP-Jagged-1or Ad5/F35-GFP adenovirus at37°C in serum-free RPMI1640medium.After2h,these cells were washed twice and resuspended in fresh complete medium.Western blotCell extracts were prepared by lysing them with lysis buffer(Beyotime Institute of Biotechnology,Jiangsu,China)supplemented with a protease inhibitor ‘cocktail’(Roche Diagnostics,Basel,Switzerland).Protein concentrations were determined by bicinchoninic acid assay(Pierce,Rockford,IL,USA).Immuno-blot analysis was performed as previously described.40Adoptive transfer and heart transplantationThe JAG1-DC or GFP-DC cells were transferred to BALB/c mice via the tail vein(5×106cells per recipient).On the day following the adoptive transfer, hearts from C57BL/6mice were transplanted into the neck vessels of the BALB/c recipients as described previously.41Graft survival was monitored by palpation daily,and rejection was defined as the loss of palpable cardiac contractions.Mixed lymphocyte reactionT lymphocytes were isolated from the spleens of BALB/c mice using nylon wool columns(Wako,Osaka,Japan)and used as responder cells.The purity of T cells was485%(as determined byflow cytometry)and their viability was 490%(as assessed by trypan blue exclusion).JAG1-DC or GFP-DC cells pre-treated with mitomycin C(50μg ml−1,Amresco,Solon,OH,USA)were used as stimulator cells in the mixed lymphocyte reaction(MLR)in vitro(DC: T cells=2×104cells:1×106cells=1:50).After HTx,the lymphocytes were isolated from the spleens of recipient mice and used as responder cells(purified by nylon wool columns)on day7or100. Splenic cells obtained from C57BL/6or C3H mice(also pre-treated with 40μg ml−1mitomycin C)were used as stimulator cells in the MLR ex vivo (stimulator cells:responder cells=1×105cells:1×106cells=1:10).All MLRs were cultured for3days at37°C in a humid environment with5%CO2,and the cell proliferation was measured using a BrdU kit(Roche,Mannheim, Germany).Monoclonal antibodiesThe anti-CD40L mAb(MR-1)and isotype control antibodies were produced from Bio X Cell(West Lebanon,NH,USA)and were administered(0.25mg per dose)by intraperitoneal injection on days0,2,4and6after HTx. Flow cytometryCells(1×106cells per100μl)were labelled using antibodies against murine CD11c,Jagged-1,MHC class II,CD80,CD86and isotype control antibodies (eBioscience Inc.,San Diego,CA,USA).CD4+Foxp3+Tregs were labelled using the Mouse Regulatory T cell staining kit(eBioscience Inc.).Flow cytometry was carried out as previously described.42Quantitative real-time PCRThe mRNA from cells or grafts was extracted using TRIzol(Invitrogen, Carlsbad,CA,USA).Reverse transcription and quantitative real-time PCR were performed using a StepOne Real-Time PCR System(ABI,Foster,UK). Measurement of cytokine productionThe concentrations of IL-4,IL-10and TGF-βwere detected using commercially available kits(NeoBioscience Technology Co.Ltd,Shenzhen,China). 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