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A novel plasmid DNA electroporation method allows transfection of murine DC

A novel plasmid DNA electroporation method allows transfection of murine DC
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  Research paper A novel plasmid DNA electroporation method allows transfectionof murine DC Matthias Bros ⁎ , Nadine Wiechmann, Verena Besche 1 , Timo Castor 1 , Stephan Sudowe,Stephan Grabbe, Angelika B. Reske-Kunz Clinical Research Unit Allergology, Department of Dermatology, Johannes Gutenberg-University, Obere Zahlbacher Str. 63, 55131 Mainz, Germany a r t i c l e i n f o a b s t r a c t  Article history: Received 19 June 2007Received in revised form 28 December 2008Accepted 6 January 2009Available online 30 January 2009 Understeadystateconditions dendritic cells(DC)exerttolerogenicfunction,but acquirepotentimmunogenic function due to strong upregulation of costimulatory molecules andproin fl ammatory cytokines. In numerous studies the potential of modi fi ed DC to inducetoleranceor immune reactionstowardsa distinct antigenhas been demonstrated.However,DCare refractory to transfection with plasmid DNA by non-viral methods. In this study we havetestedthesuitabilityofanewlydevelopedelectroporationdevicetotransfectimmaturemurinebone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules atconsiderable extent which renders this method suitable to perform all kinds of promoterstudies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants.However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4 + T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression constructencoding MOG ef  fi ciently stimulated MOG peptide-speci fi c Tcell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression andstrongly diminished allogeneic Tcell proliferation. Therefore, this method also allows to studyfunctional properties of genetically altered DC.© 2009 Elsevier B.V. All rights reserved. Keywords: ElectroporationDendritic cellsImmune responseTolerogenic phenotype 1. Introduction Dendritic cells (DC) reside as sentinels in almost everytissue and are specialized in the uptake of antigen (Lutz andSchuler, 2002). Under steadystateconditions, a small fractionof DC acquires a semi-mature state and migrates to thedraining secondary lymphoid organs. These semi-mature DCinduce apoptosis, anergy or even a regulatory state ininteracting autoreactive T cells as well as in T cells speci fi cfor harmless environmental antigens, thus exerting tolero-genic function (Akbari and Umetsu, 2004).Uponinfection,DCengulfandprocesspathogenicmaterialand are fully activated by pathogen-derived molecules likelipopolysaccharide (LPS), or by proin fl ammatory cytokinesproduced by cells in the micro-environment (Adams et al.,2005). Activated DC strongly upregulate expression of costimulatory molecules and production of proin fl ammatorycytokines and thus constitute the most potent antigenpresenting cells, capable of stimulating naive antigen-speci fi cT cells and thereby inducing a primary immune reaction.In numerous studies, aimed at either reconstitutingtoleranceorinducingpotentimmuneresponses,thepotentialof modi fi ed DC as vaccines for immunotherapy has beenevaluated (Figdor et al., 2004). More recently, in severalstudies genetic alteration of DC by transfection with messen-ger RNA in vitro and plasmid DNA in vivo was shown to allowfor processing and presentation of antigenic peptides derivedfrom a transgene (Van Meirvenne et al., 2002; Breckpot et al.,2004; Sudowe et al., 2003), as well as for production of immunomodulatory molecules with autocrine and/or para-crine effect on DC function and T cell polarization (Kikuchi,2006). However, DC were found to be largely refractory  Journal of Immunological Methods 343 (2009) 13 – 20 ⁎  Corresponding author. Tel.: +49 6131 3933473; fax: +49 6131 3933360. E-mail address: (M. Bros). 1 In partial ful fi llment of the requirements of their doctoral thesis.0022-1759/$  –  see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2009.01.006 Contents lists available at ScienceDirect  Journal of Immunological Methods  journal homepage:  towards non-viral transfection with plasmid DNA in vitro(Breckpot et al., 2004).In this study we have evaluated the suitability of a newlydeveloped commercially available electroporation device totransfect murine bone marrow-derived DC (BM-DC) whichare as potent in stimulating T cells as primary DC isolatedfrom spleen (Garrigan et al., 1996), and therefore arefrequently used to study DC function. Despite a moderatetransfection rate, BM-DC showed well detectable expressionof reporter molecules. Electroporated BM-DC displayed alower increase in stimulation-dependent allostimulatorypotency, but were not impaired in their peptide-speci fi c Tcellstimulatorycapacity.Moreover,BM-DCtransfectedwithaMOG expression construct elicited strong proliferation of MOG speci fi c T cells. BM-DC engineered to overexpress IL-10largelypreventedallogenicTcellproliferationprobablyduetoef  fi cient autocrine and paracrine activity of this potenttolerance-inducing cytokine. 2. Materials and methods  2.1. Mice FemaleC57BL/6,BALB/c,and2D2Tcellreceptortransgenicmicespeci fi cally recognizing MOG 35 – 55 peptide (Bettelli et al.,2003) were bred and maintained in the Central AnimalFacilities of the University of Mainz under speci fi c pathogen-free conditions on a standard diet. The  “ Principles of Laboratory Animal Care ”  (NIH publication no. 85-23, revised1985) were followed.  2.2. Generation of BM-DC  BM-DC were generated from bone marrow progenitors of C57BL/6 mice as described (Scheicher et al., 1992) with minormodi fi cations (Lutz et al., 1999) using BM-DC medium (IMDMwith 10% FCS [both PAA, Cölbe, Germany], 2 mM  L  -glutamine[CarlRothGmbH&Co,Karlsruhe,Germany],100U/mlpenicillinand 100 µg/ml streptomycin [both Gibco, Paisly, UK]) supple-mentedwith5%ofmGM-CSFcontainingcellculturesupernatantderivedfromX63.Ag8-653myelomacellsstablytransfectedwithmurineGM-CSFexpressionconstruct(Zaletal.,1994;akindgiftby Dr. B. Stockinger, National Institute for Medical Research,London). Cultures harvested on days 7 to 9 of culture contained N 85% CD11c + BM-DC as assessed by FACS analysis.  2.3. Viability assay Viability of BM-DC was determined by staining of non-viable cells with trypan blue. At least 200 cells were counted,and the proportion of viable cells was determined.  2.4. Plasmids Toquantifytransgeneexpression,BM-DCweretransfectedwith a reporter construct encoding  fi re fl y luciferase undercontrol of the human EF1a promoter (pGL3-EF1a) describedpreviously (Bros et al., 2003). The EGFP encoding reporterconstruct pEGFP-N1 (Clontech, Mountain View, CA) was usedto assess transfection ef  fi ciency. To obtain an expressionconstruct for murine interleukin 10 (IL-10), its open readingframe (ORF) was ampli fi ed from cDNA derived from LPS-stimulated BM-DC (see below) by PCR using primers mIL10-s(5 ′ -CTTGCAGAAAAGAGAGCTCCA-3 ′ ) and mIL10-as (5 ′ -TGGAGTCCAGCAGACTCAAT-3 ′ ). The ampli fi ed IL-10 ORF wascloned into Ecl136-digested pZero 2 (Invitrogen, Groningen,Netherlands). A positive clone was double-digested withAcc65I and NotI, and the excised IL-10 minigene was clonedinto likewise double-digested pCI (Promega, Heidelberg,Germany), thereby generating pCI-IL10. In order to generatean expression construct encoding murine myelin oligoden-drocyte glycoprotein (MOG), the MOG ORF was ampli fi ed byPCR using primers mMOG-s (5 ′ -ACTCGAGATGGCCTGTTTGTGGAGC-3 ′ ) and mMOG-as (5 ′ -AGTCGACACAACCATCACTCAAAAG-3 ′ ) and murine brain cDNA as template. Ampli fi edPCR product was double-digested with XhoI and SalI(recognitionsitesareunderlinedinmMOGprimersequences)and was cloned undirected into SalI-digested vector pCDH1-MCS1 (SBI, Mountain View, CA), thereby obtaining pCDH1-MOG. Plasmid DNA was prepared by alkaline lysis, followedby incubation of cleared lysate with an LPS removing buffer(Cotten et al.,1994), and subsequent puri fi cation using anion-exchange columns.  2.5. Transfection BM-DC were suspended in Resuspension buffer (Peqlab,Erlangen, Germany) at a densityof 5×10 6 cells/mland plasmidDNAwas added(100 µg/ml). Aliquots of cell suspension (10 µl)were electroporated employing the MicroPorator MP-100(Peqlab) under varying conditions as indicated. BM-DC(5×10 4 ) transfected with pGL3-EF1a were transferred towells of 48-well tissue culture plates (Greiner, Frickenhausen,Germany) containing 250 µl of BM-DC medium. In case of transfection with pEGFP-N1, a total of 5×10 5 transfected BM-DC(pooled fromtentransfections) wereseededperwellof24-well tissue culture plates (Greiner) containing 1 ml culturemedium. For subsequent real time PCR analysis and T cellproliferation assays, a total of 10 6 cells each were transfectedand seeded on 24-well tissue culture plates in 2 ml culturemedium. Eighteen hours later, cells were split by half and onefractionofBM-DCwasstimulatedover-nightbyadditionofLPS(1 µg/ml). For subsequent coculture with 2D2 T cells,stimulated untransfected or with pCDH1 empty vectortransfected BM-DC were pulsed with 10 µM MOG 35 – 55  peptide(Neosystem, Strasbourg, France) for 4 h.  2.6. Luciferase assay Luciferase expression was assayed 24 h post transfection.BM-DC were harvested and lysed in 50 µl of cell culture lysisreagent (Luciferase Assay System, Promega). Ten µl of celllysate was used for luciferase detection in a Turner DesignsTD-20/20luminometer(Promega;detectionrange10 − 1 to10 4 relative light units, RLU).  2.7. Flow cytometry OnedayaftertransfectionwithpEGFP-N1,cellswerewashedinstainingbuffer(PBS/2%FCS),and fl owcytometricanalysiswasperformed using a FACScan  fl ow cytometer equipped withCellQuest Software (BD Biosciences, San Jose, CA). 14  M. Bros et al. / Journal of Immunological Methods 343 (2009) 13 –  20  Fig.1. Optimizationof BM-DCelectroporationconditions. ImmatureBM-DC(5×10 4 )were transfected with pGL3-EF1a (1 µg)(A) at varying currentandatconstantpulse time (30 ms) with one pulse, (B) at constant current (1450 V) with one pulse and varying pulse time, (C) at constant current (1450 V) and pulse time (30 ms)and varying number of pulses, and (D) with varying amounts of plasmid DNA. Luciferase activities are given as percentage of the value obtained for the referencesetting (dashed line). BM-DC mixed with plasmid DNA but not electroporated served as negative control ( – ). Data represent mean±SD of three (A, B, D) or two(C)experimentsperformedintriplicate.(E)CategorizedluciferaseactivityofBM-DCtransfectedatreferencesettingcompiledof32experiments.(F)Datarepresentthe mean±SD of 32 assays each. (G) Viability of untransfected (white bar) and electroporated (black bar) BM-DC was monitored by trypan blue exclusion assay(mean±SD of   fi ve experiments). Luciferase activities (A – F) and (G) viability of BM-DC were assessed 24 h after transfection. Statistically signi fi cant differences in(A – D, F) luciferase activities obtained for the various conditions versus the reference setting and (F) viability between untransfected and transfected BM-DC areindicated ( ⁎  p b 0.05;  ⁎⁎  p b 0.01;  ⁎⁎⁎  p b 0.001).15 M. Bros et al. / Journal of Immunological Methods 343 (2009) 13 –  20   2.8. Real time PCR Total RNA was isolated using the RNeasy MiniPrep kit andperforming on-column DNase treatment (both from Qiagen,Hilden, Germany) and was reverse-transcribed applying a 1:1mix of Oligo-dT and random hexamer primers. Reactionmixtures for real-time PCR included 200 ng of cDNA andreactions were performed using SYBRGreen mastermix(ABgene, Hamburg, Germany) under conditions recom-mended by the manufacturer. Primer sequences have beendescribed (Bros et al., 2007). The house-keeping geneubiquitin C served as internal control. Speci fi city of productampli fi cation was con fi rmed by melting curve analysis andrelative quanti fi cation of gene expression was performedaccording to the comparative threshold cycle method.  2.9. Cytokine assay Cytokines were quanti fi ed using a sandwich-ELISA aspreviously described (Sudowe et al., 2003). ELISA captureantibody (JES052A5) and biotinylated detection antibody formurine IL-10 (BAF417) (both R&D Systems, Wiesbaden,Germany) were used as recommended by the manufacturer.Recombinant murine IL-10 (R&D Systems) was used asstandard.  2.10. T cell proliferation assay BALB/c or 2D2 splenocytes, depleted of erythrocytes byincubation in lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 ,100 µM EDTA-sodium, pH 7.4) for 1 min, were passed overnylon wool columns to enrich T cells. T cells (3×10 5 ) werecocultured for four days on 96-well tissue culture plates(Corning, Cambridge, MA) with graded numbers of irradiated(30 Gy) BM-DC in 200 µl of BM-DC medium. Cell proliferationwas assessed by the uptake of [ 3 H] thymidine (0. 5 µCi/well)for the last 16 h of culture. Cells were harvested onto glass fi ber  fi lters and retained radioactivity was measured in aliquid scintillation counter (1205 Betaplate, LKB Wallac,Turcu, Finnland).  2.11. Statistical analysis Data were analyzed for statistically signi fi cant differencesby applying Student's  t   test. 3. Results  3.1. Optimization of BM-DC electroporation conditions Given the lack of ef  fi cient non-viral transfection methodsfor murine DC, we evaluated the suitability of a newlydeveloped electroporation device totransfect murine BM-DC.In contrast to conventional electroporation cuvettes, cells areelectroporated in a micropipette tip supposed to allowformation of a more homogenous electric  fi eld resulting inreduced heating and oxide formation. In general, electro-poration conditions are de fi ned by strength of current as themost critical parameter, by pulse time and by number of pulses applied.In a  fi rst set of experiments, the current was varied,whereas the other parameters were kept at values previouslyshown to yield optimal results in various cell types (Manual,Peqlab). We transfected immature BM-DC with a constitu-tively active  fi re fl y luciferase encoding expression vector andmonitored luciferase activity 24 h after transfection. Reporteractivities were highest (  p b 0.01) at a current of 1450 V (Fig. 1A). At this current, the initially chosen pulse time of 30 ms proved toresult in signi fi cantly higherreporteractivitythanshorterorlongerpulsetimes(Fig.1B).Moreover,asinglepulse mediated optimal transfection (Fig. 1C). Luciferaseactivity correlated with the amount of plasmid DNA used fortransfection (Fig.1D). However, the ratio of luciferase activitydivided by used amount of plasmid DNAwas highest for 1 µg.In order to minimize the risk of unwanted side effectsmediated by recognition of DNA via TLR-9 dependent and -independent pathways (Ishii and Akira, 2006), we used aplasmid DNA concentration of 1 µg per 5×10 4 cells in furtherexperiments.At reference settings, luciferase activities resided in thecategoryof10 – 50RLUorhigherinmostcases(Fig.1E)andthe Fig. 2.  Transfection rate of electroporated BM-DC. Immature BM-DC (5×10 5 )were transfected with pEGFP-N1 (10 µg) at reference setting. EGFPexpression was monitored 24 h post transfection. (A) Morphology of BM-DC electroporated with pEGFP-N1. Images were taken at room temperatureat 20× magni fi cation using an Olympus IX70 inverted microscope (OlympusOptical,Hamburg,Germany)equippedwithaDXC-950P digitalcamera(SonyElectronics,NJ,USA).BM-DCmixedwithplasmidDNAbutnotelectroporatedshowed no EGFP expression (data not shown). (B) The transfection rate of electroporatedBM-DCwasassayedbyFACSanalysis.ThepercentageofEGFP-positive BM-DC only mixed with plasmid DNA (left panel) or electroporated(right panel) is indicated. (A, B) The data shown are representative of fourexperiments each.16  M. Bros et al. / Journal of Immunological Methods 343 (2009) 13 –  20  mean luciferase activity was equal to 33.9±36.5 RLU (mean±SD;  n =32) (Fig. 1F) and was always within the lineardetection range, demonstrating reliability of this transfectionmethod. In contrast, BM-DC mixed with plasmid DNA but notsubjected to electroporation displayed only backgroundactivity. However, as shown in Fig. 1G, electroporationsigni fi cantly impaired viability of BM-DC (44.5±9.6%,  n =5)as compared with the control group (82.8±3.4%,  n =5).In order to quantify transfection ef  fi ciency on single celllevel, we employed an EGFP encoding expression construct.By  fl uorescence microscopy we noted strong expression of EGFP in transfected BM-DC (Fig. 2A). FACS analysis revealedthat while mere incubation of BM-DC with plasmid DNAwas insuf  fi cient to yield EGFP positive cells (Fig. 2B, leftpanel), electroporation resulted in successful transfection(Fig. 2B, right panel), which amounted to a mean of 3.8±0.3% ( n =4).  3.2. Electroporation does not affect functional activity of immature BM-DC, but impairs allostimulatory potency of stimulated BM-DC  Next we tested for electroporation-associated functionalalterations of BM-DC. Non-transfected immature BM-DCexerted moderate allogeneic T cell stimulatory capacitywhich was signi fi cantly increased (  p b 0.01) upon maturationinduced by treatment with LPS (Fig. 3A). BM-DC transfectedwith an EGFP reporter construct and left unstimulatedinduced allogeneic T cell proliferation at comparable extentas non-transfected BM-DC, demonstrating that electropora-tionwithplasmidDNApersedidnotalterthefunctionalstateof immature BM-DC. In addition, electroporated BM-DCresponded to LPS-mediated stimulation as re fl ected bysigni fi cantly (  p b 0.05) enhanced T cell stimulatory capacity,althoughsigni fi cantly(  p b 0.01)lessthannon-transfectedBM-DC. Due to the lower stimulation-dependent increase inallostimulatorypotencyoftransfectedBM-DCversusuntrans-fectedstimulatedBM-DC,wemonitoredtranscriptexpressionof costimulatory molecules and cytokines involved in DC/Tcell interaction in either BM-DC population in response tostimulation by RT-PCR, for which only low cell numbers areneeded. As shown in Fig. 3B, some mRNA species wereupregulated to the same extent in both untransfected andelectroporatedBM-DC,whileupregulationof other moleculesappearedattenuatedinthelattergroup,althoughthiswasnotstatistically signi fi cant except for CD40. Thus the electropora-tion procedure appears to impair LPS-induced maturation of BM-DC.  3.3. Electroporated BM-DC display unaltered potency ininducing syngeneic peptide-speci  fi c immune responses Given the reduced capability of stimulated transfectedBM-DC to induce an allogeneic T cell response, we assessedtheir potential to induce MOG 35 – 55  peptide-speci fi c T cellresponses. As depicted in Fig. 4A, both transfected anduntransfected BM-DC stimulated with LPS and pulsed withMOG 35 – 55  peptide were equally potent in inducing prolifera-tion of MOG-reactive T cell receptor transgenic 2D2 T cells,thereby excluding strong impairment of DC function uponelectroporation. In addition, we assessed the potency of BM-DC electroporated with an expression construct encodingMOG protein to stimulate MOG-speci fi c T cells. Despite themoderate transfection rate of the method applied, BM-DC Fig.3. EffectsofelectroporationonprimaryallogenicTcellstimulatorycapacityandgeneexpressionofBM-DC.ImmatureBM-DCwereleftuntransfected(Ctrl)or were transfected with pEGFP-N1 and aliquots were stimulated with LPS.(A) The allogeneic T cell stimulatory capacity of differentially treated BM-DCwas assessed by mixed leukocyte reaction. Titrated numbers of irradiated BM-DCwere coculturedwith 3×10 5 nylonwool-enrichedTcells ofBALB/c mice for4daysintriplicateculturesandTcellproliferationwasassessed.Datarepresentmean±SD of triplicate cultures and are representative of two independentexperiments.Statisticallysigni fi cantdifferencesinTcellstimulatorycapacityof untransfected or pEGFP-N1 transfected BM-DC at mature versus immaturestate ( ⁎ ), and between untransfected and pEGFP-N1 transfected BM-DC atstimulated state (+) are indicated ( ⁎  p b 0.05;  ⁎⁎ , ++  p b 0.01;  ⁎⁎⁎ , +++  p b 0.001).(B) Stimulation-dependent changes in mRNA expression of transfected versusuntransfected BM-DC. Relative mRNA expression levels of the indicated mRNAspecies encoding costimulatory receptors and cytokines involved in DC/T cellinteraction were assessed. Aliquots of cells were stimulated with LPS directlyafter transfection, and mRNA expression was monitored 24 h later. Relativechanges in mRNA expression are given as fold of expression at unstimulatedstate. Statistically signi fi cant differences in mRNA expression between bothgroups are indicated ( ⁎⁎  p b 0.01).17 M. Bros et al. / Journal of Immunological Methods 343 (2009) 13 –  20
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