Differentiation of alloreactive versus CD3CD28 stimulated T-lymphocytes using Raman spectroscopy

DifferentiationofAlloreactiveVersusCD3/CD28StimulatedT-LymphocytesUsingRaman

Spectroscopy:AGreaterSpecificityforNoninvasiveAcuteRenalAllograftRejectionDetection

KristianL.Brown,1,2*OlenaY.Palyvoda,2JagdishS.Thakur,3SandraL.Nehlsen-Cannarella,4OmarR.Fagoaga,4ScottA.Gruber,1,2GregoryW.Auner2󰀁Abstract

Acuterejection(AR)remainsproblematicinrenaltransplantation.Asamarker,serumcreatinineislimited,warrantingamoreeffectivescreeningtool.Ramanspec-troscopy(RS)candetectT-cellactivationwithhighsensitivity.Inthisstudyweexploreitsspecificity.Seventy-fiveinactivated,40alloantigen-activated,and75CD3/CD28-activatedTcellswereanalyzedusingRS.CD3/CD28-activatedpeakmagnitudes(PM)were4.3%to23.9%lowerthaninactivatedPMatpositions:903,1031,1069,1093,1155,1326,and1449cm21,withadifferenceinpeakratio(PR)observedatthe1182:1195cm21position(0.91Æ0.06vs.1.2Æ0.01,respectively:P50.006).Differ-encesinCD3/CD28-andalloantigen-activatedPMwereobservedat:903,1031,1093,1155,1326,and1449cm21,withnoPRdifferencesatthe1182:1195cm21position(0.9160.06vs.0.8660.09:P50.8).SpectralsignatureseparationofCD3/CD28—andalloantigen-activatedgroupswas100%specificandsensitive.WeconcludethatRScandifferentiateTcellsactivatedbydifferentstimuliwithhighsensitivityandspecificity.'2009InternationalSocietyforAdvancementofCytometry󰀁Keyterms

Ramanspectroscopy;renaltransplantation;acuteallograftrejection;humanT-cellacti-vationanddetection;cellsurfacereceptors;CD3/CD28stimulation

1

DepartmentofSurgery,SectionofTransplantSurgery,WayneStateUniversity,Detroit,Michigan

DepartmentofBiomedicalEngineering,SmartSensorandIntegrated

Microsystems,WayneStateUniversity,Detroit,Michigan

DepartmentofPhysicsandAstronomy,WayneStateUniversity,Detroit,Michigan

DepartmentofPathology,WayneStateUniversitySchoolofMedicine,WayneStateUniversity,Detroit,Michigan

2

3

4

Received22June2009;Revision

Received19August2009;Accepted20August2009

Grantsponsor:NationalInstitutesofHealth;Grantnumbers:5R01EB000741-05,2T32GM008420-14;Grantsponsor:DavidFrommResearchAward(WayneStateUniversityDepartmentofSurgery).*Correspondenceto:KristianL.Brown,DepartmentofSurgery,Suite6-C,WayneStateUniversitySchoolofMedicine,4201St.AntoineBLVD,Detroit,MI48201Email:kbrown6@dmc.org

Publishedonline14September2009inWileyInterScience(www.interscience.wiley.com)

DOI:10.1002/cyto.a.20797©2009InternationalSocietyforAdvancementofCytometry

RENALtransplantationhasbecomethepreferredtreatmentforthevastmajorityof

patientswithend-stagerenaldisease(1,2).However,itsapplicabilitytoallinneedhasbeenlimitedbythecontinuedshortageofavailableorgans(3).Giventhescar-cityofdonorkidneys,itisimperativethatthefunctionalityandsurvivalofeachofthesegraftsbemaximizedintherecipient.Acuterejection(AR),whichispredomi-nantlyT-cellmediated,hascontinuedtonegativelyimpacttheoutcomeofrenalal-lografts(4–6).DiagnosisofARfollowingtransplantationbasedonserumcreati-nineelevation(SCE)hasprovenproblematic.DespitethewidespreaduseofSCEasamarkerbymostcenters,itrepresentsalatefindingthatbecomesapparentonlyaf-tersignificanthistologicdamagetothetransplantedorganhasalreadyoccurred.Moreover,SCEhasalowspecificity,reflectingthefactthatotherconditionssuchasurinarytractinfection(UTI),dehydration,obstruction,andevenimmunosuppres-sivemedicationscancausefalsepositivesleadingtounwarrantedreferralsforcostlyandpotentiallydangeroustransplantbiopsies(7),whichmayalsobesubjecttosamplingerror.Inadditiontobeingalatenonspecificmarker,SCEhasalowsensi-tivity,nottakingintoaccountsubclinicalAR(SCAR)whichoccurswithnochangeinserumcreatinine(8).SCARhaspromptedagreaterimplementationofprotocolrenaltransplantbiopsiesinresponsetoconcernsovertheaccelerateddevelopment

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Figure1.MeanRamanspectraofalloreactive,inactivated,rest-ing,andCD3/CD28-activatedTlymphocytesatthe514.5nmwave-length.[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.interscience.wiley.com.]

ofchronicallograftnephropathyiftheconditionremainsundetectedanduntreated(9,10).

Arecentsurgeinproposedmethodologiesforthenonin-vasivediagnosisofARhasincludedRamanspectroscopy(RS).RSisalaser-basedtechnologythatutilizesamonochromaticincidentphotontoinducedetectablemolecularvibrationsinagivenmaterial.Thesevibrationsyieldaunique‘‘signature’’,whichweandothershaveshowninpriorstudiestobehighlyaccurateinthedifferentiationofactivatedfromnonactivatedTlymphocytesbaseduponmoleculardifferencesincellsur-facereceptors(11,12).DespitetheestablishmentofRSasamethodologytoidentifyT-cellactivationstates,thecapabilityofthesystemtodistinguishsignaturesofTcellsthatareacti-vatedbydifferentstimulistillrequiresexamination.Therefore,thepurposeofthisstudyistocomparespectralsignaturesofalloantigen-activatedandCD3/CD28activatedTcells.WehypothesizethattheRSsignaturesofTcellsactivatedviathesetwomethodologieswilldiffersignificantly.

MATERIALSANDMETHODS

Alloantigen-ActivatedT-CellPreparation

PriorapprovalforthestudywasobtainedfromtheWayneStateUniversityHumanInvestigationCommittee.Mononuclearcellswereobtainedusingsodium-heparinizedvenousbloodcollectedfromhealthyparticipantsandsepa-ratedviadensitygradientasdescribedbyBoyum(13).There-sultantcellswerewashedwithHank’sbalancedsaltsolution(HBSS,Invitrogen,Carlsbad,CA)andsuspendedincompletemedium.Cellularviabilitywas[98%ascheckedbyvitaldyeexclusion.

ThreedistinctT-cellsamplegroupswerecreatedforthealloantigen-activatedTcells:1.activated;2.inactivated;and3.restingTlymphocytes.TheactivatedTlymphocytesampleswerecreatedviaatwo-waymixedlymphocyteculture(MLC)asdescribedbyDupontetal.(14),whichbringsintoproxim-ityTlymphocytesfromnonrelatedindividuals,antigenpre-sentingcells(APC;monocytesandmacrophages),andthenecessarycomponentstomodelallograftrejection.T-cellacti-vationwasconfirmedbyCD69directedflowcytometryasdescribedelsewhere(15,16)Theinactivatedgroupwascreated

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byutilizingTlymphocyte/stimulatorsamplesfromtwononrelatedindividualswhichwerepretreatedfor20minwithMitomycinC(0.025mgMitomycinC[0.5mg/ml]addedtoeach1mlofcellsuspension)at378C.TheseinactivatedTlym-phocytesampleswerewashedtwicewithHBSSbeforethestartofMLCs.EfficacyofMitomycinCwasverifiedusingdoubleinactivationcontrolcultureswhichwereruninparallel.Finally,therestingTlymphocytegroupconsistedofTcellsthatwereneitherstimulatedviaMLCnorinhibitedbyMito-mycinCtreatment.ThesecellsweretheproductofCD69neg-ativesortingviaflowcytometry(15,16).FollowingMLCbutbeforeRSanalysis,BcellswereremovedbynegativeselectionusingFluoroBeads-Bimmunomagneticbeadsandmagneticsorter(OneLambda,CanogaPark,CA)whilemonocytes/macrophageswereremovedviasteelwoolcolumnstoensurethatthesesignatureswerenotobtainederroneously.Allcultureswereincubatedat378Cin5%CO2inparallelovera7-dayperiod,withRS,antibody,andviabilityanalysiscarriedoutdaily.

NonspecificAntigenModelofActivation

TlymphocyteswereisolateddirectlyfromperipheralbloodandurineusingaDynalT-cellnegativeisolationkit(Invitrogen,CanogaPark,CA).Tomodelanonspecificpro-cessofactivation,aCD3/CD28-coatedDynalbeadsystem(Invitrogen,CanogaPark,CA)wasutilizedtocross-linkcellmembranemolecules.ThisconsistedofTlymphocytesincu-batedwithbeads(beadvolumetitratedtocorrespondto3beadsforeveryT-cellataconcentrationof13107cellspervolume)incompletemediacontainingrecombinantinterleu-kin2(50U/ml).TheCD3/CD28beadswereremovedfromTcellsviamagneticsorterbeforeRamananalysis.

IndependentVerificationofActivation/Inactivation

IndependentofRSanalysis,theactivationstatusofallsam-pleswasverifiedbyantibodystainingandflowcytometry.ForthealloreactiveandCD3/CD28stimulatedsamples,activationstatuswasassessedusingamonoclonalantibody,CD69(FastImmune,BectonDickinson,SanJose,CA),asdescribedelsewhere(17).

Figure2.SuperimposedmeanRamanspectrafrominactivated(red)andCD3/CD28-activatedTlymphocytesannotatedwith(*)fociofsignificantdifferencesand(–)markersofcellularviability(18).[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.interscience.wiley.com.]

RamanSpectroscopic-BasedT-CellAnalysis

ORIGINALARTICLE

-;eni1;;;;;;r0;;;1004;24r.....2.oe;)45287454.09.cg5E9...83241104n)768838029.63saF5(UL11241111snhO7v18ec(T.885.....1rMTVAsssss5sSv...vvv0.atPENACP;.pnRI39s8s9s25v909v7s8ec.DS38v8v0v67....94365011040n.....rV%..200502601.0508.208.06.0:.37v.o0.0ie5;.7;.;0;1;;9;8;i200.;dp6A0A9A2AA1A9A3At57;3Aet,1N8N18N55N81N01N11N91NaRp5A31N81NoDn%se;uedlausVtie;t3;;;;;;;;.nty3;;12880540c.ngo;202.9..3.ea)hE5560.2960...3..25198123)567951013.78mmpF07U(L10158119s51nkmO4(TAga.88.1..1v1.iesysplMSVLOEv..s.ssssvsv.62.ssPLRP;TA.SD2v949v8s80s2va,gV%56v9..908v03180v.0v1290........kM.n20210802.05.00430.:.0.03.09.0aPi0;.;.;0507.;5;40oe;ts6A8A4A9;0;1A4A2;iAt580;2;A3Apsle1N08N18N4A8A25N1N01N11N91NaRp61N81NdlerecidfiTncaee,pvesitv;it1tcc0;aa;;;;;;;;ea.sr;e)452810042;.29......eor5E9ll..874541.04o)70(U68833241.3pllF11248029s08l4Lma,aO(TVA.8851111v61sao,dMLONACPv.......61.sllss;lePLI22s5sv12ss9s87.s.01s8lAtA.DaS51v7v49v6v0v800v2v.......e;.csvV%202109802.05.71v...0490..0:o03...-lit0;.;.;0;50100.;4;i20Tlec6A8A4A9A0;A2;5A2At5800;A3;a1N0gc8N18N45N80A411N1N1N98A1NaRp61N1NnTnitisg,denritetdsanerv;ai;;;;,t,tc266;;84a3.;;57048;2.39es-ve;....i8)0290..51540..82tR20E)4Uc;;9.403242.DF6(L048001s0asCO4(.A11510.1149v3081ellsr/3MALOVALPvN8.80.s..013071.s..0ss...9.010.0s.oec;.lDPSNA;l.42v2s0v8s;v0sv0.;s0CSD2566.v;807v.%24v8v.;;%v0;ATV%:.2..,df32807%.08.8.3025%5.%o0.93..%..9..o605;607;00;7131i1detett660A312A9928A433a5022924tafavi118N7:5N31:9N9:1:Rp3511:1:hvitistccanananimI,t,a;;8cR3;;;22at3;12;5;4;50DnIn..C;a;20.;988003...6.3/).vSD5520.2529.1.2338e%E676.198.78D2lFV;109758513512eO)5)U5L.81051001514.CDr7s01099s110v.2s0fC.tfM7((v.80.s.0..0.01s.s.ss.9s0v0.o/oPATVA3n.aSSP4v2.0vv0v0.s.0;vv09.0v.030syNE29.67s.v;88;3;enDcrR..9a30807%0..08.%2%2;5;000:.;.uoCif..2.ls(im6;5;619;013.35%..73%o02.%48ainrnm6A0A173062i8374243ta5101251vaegi.1N8N32A97:5N31:9:9:1:Rp31:1:kpgssuasemittkeponoacanepvdhceiitecfg5agaicn9r;a1;;;;;;aeea1p0;88;84;69rh1..enpcom45;810042.2......4vist.oS;E2.37454103adneC.434.08FV)5U7L118058222615s61meod2tu8.O)5(.820..10021090v13100oant1u,in11M7TVAss(CPv.80.0.s.rflAsv.010..01.01s..s.s.s.fagoePA;v0v0lS41.;820s;dvSabNNADI27v9s8vv0;v0v096.0.6.v%.;2;33eeNmoita%...%vs;tT3080750..082;;00.%23%%5.%:.o01%ilka6;.;.;0.35329rln6A5629A920..231i362451eear1N0A31728N7:5N33847443ta501:9:9:1:Rp31:1:dceeopt1Tmn,seflogAdibNnrsoaes;Tbaepar5omkdsuer9fuatri)11anepno21tM:aogc,tC21935269DAaadPmta(88303695824nkD28000001134oaaPA67911111111pespCytometryPartA󰀁75A:917À923,2009

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ORIGINALARTICLE

Figure3.Ramanspectraofinactivated(red)andCD3/CD28-activatedTlymphocytesdemonstratingdifferencesinpeakmagnitudes(insetsA—E).Annotationswithininsets:InsetA-(*)903cm21;InsetB-(^)788cm21;InsetC-(*)1002cm21;InsetD-(*)1031cm21,(^)1069cm21,({)1093cm21,(l)1155cm21;InsetE-(*)1326cm21,(^)1449cm21.ThefocusedspectralregionsarefromTlymphocytesrepresentedinFigure1.[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.interscience.wiley.com.]

StainedcellswereviewedusingaNikonEclipseTE2000-Uinvertedmicroscope,withimagescapturedandprocessedwithMetavue6.2r5software(Downingtown,PA).

RamanSpectroscopy

BeforecollectionofRamanspectraldata,allsampleswerewashedwithPBS(Invitrogen,Carlsbad,CA).Tcellswereallowedtosettlewithinananalysisreservoirplacedonaleveledmicroscopestage.Onlynonapoptotic,rounded,non-adhered,isolatedandindividualizedTcellswereselectedforstudy.EachsamplewasanalyzedusingidenticalRamanspec-troscopicparameters.ToassurethatTcellswerealignedinthelasertargetareaandthatthelaserfocuspointwascenteredonthelymphocyte,anenhancedvideoimagingsystemwasemployed.Imageswerecapturedbeforeandaftereachmea-surementtoconfirmthatnocellshiftingordestructionoccurred.Toprovideforreference/controlspectralpeaks,bothpureCD3/CD28beadsandTcellsthatwerenotsubjectedtomagneticsorter(Tcellsstillboundwithbeads),were

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analyzed.Wire2software(Renishaw,OldTown,UnitedKing-dom)wasutilizedinconjunctionwiththeRSsystemforquantitativemeasurement.

Figure4.Ramanspectraofinactivated(red)andCD3/CD28-acti-vatedTlymphocytesdemonstratingdifferencesin(*)1182cm21and(^)1195cm21peaks.[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.interscience.wiley.com.]

RamanSpectroscopic-BasedT-CellAnalysis

Figure5.SuperimposedmeanRamanspectrafromCD3/CD28-activated(red)andalloreactive(blue)Tlymphocytesannotatedwith(*)fociofsignificantdifferencesand(^)markersofcellularviability(18).

RSmeasurementswereconductedusingaRenishawInVia2000microscope-spectrometerwithaLeica63Xwaterimmersionobjective(NA51.20).T1lymphocyteswereana-lyzedusingthe514.5nm(green)Arexcitationwavelength.Laserpowerwassetat50%(8–12mW)fora2–4lmlaserspotsize.Spectrawerecollectedinthebackscatteringgeome-trywitha10secondintegrationtimeoverarangeof500to1500cm21withspectralresolutionof4cm21.

StatisticalAnalysis

Beforestatisticalanalysis,alldatawerecheckedforvari-oustypesofnoise.SpectraldatademonstrateduniformlyincreasingfluorescenceovertheentirerangeofRamanshift.Thiswassubtractedoutusingamodifiedcubic-splinealgo-rithmrequiringnoaprioriknowledgeofthespectra.Inaddi-tion,amedianfilterwasappliedtotherawdatawhichelimi-natedcosmicrayandspikes.Principalcomponentanalysiswasusedtoreducedimensionalityinthedata.ChisquareorStudent’sttestwasutilizedasappropriatetofurtherquantifydifferencesinpeakratiosandpeakmagnitudeswithadditionalquantitativedifferencesdeterminedbydiscriminantfunctionanalysis(DFA).GraphicalandquantitativeanalysisofdatawereaccomplishedusingSPSSversion15software(StatisticalSoftware,Chicago,IL).

RESULTS

Figure1summarizesRamanspectraforinactivated,rest-ing,alloreactive,andCD3/CD28-activatedT-cellgroups.CD3/CD28-ActivatedVersusInactivatedTLymphocytes

Atotalof75CD3/CD28-activatedand75inactivatedTcellswereanalyzedusingthe514.5nmexcitationwavelength.Systematicreviewofsamplesrevealednocellulardisruptionfollowinglaserexposure.Qualitativecomparisonsofactivatedandinactivatedspectrademonstrated2differences1at903,1031,1069,1093,1155,1326,and1449cmRamanshifts(Fig.2).Quantitativeanalysisoftheseshiftsdemonstrateddifferencesinpeakmagnitudesatallpositionsexceptfor628,788,and

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ORIGINALARTICLE

1002cm21(Table1,Fig.3A–E),whichhavebeenshowninpreviousstudiestocorrelatewithcellularviability(18).More-over,theCD3/CD28-activatedandinactivatedgroupsshowedsignificantdifferencesinpeakratiosatthe1182:1195cm21position(Table1,Fig.4).

CD3/CD28-ActivatedVersusAlloantigen-ActivatedTLymphocytes

Atotalof46CD3/CD28-activatedand40alloantigen-activatedTcellswereanalyzedwithsummarycomparisonspectrashowninFigure5.Whencomparingthesegroups,theydifferedsignificantlyatthe903cm21,1031cm21,1093cm21,1155cm21,1326cm21,and1449cm21positions.However,therewasnodifferencewhenanalyzingthepeakra-tioatthe1182:1195cm21position(Table1).DiscriminantfunctionanalysisoftheRamanspectraldataforCD3/CD28-activatedandalloantigen-activatedT-cellgroupsissummar-izedinFigure6.Therewas100%accuracywhenusingtheactivationsignaturetoseparatethesegroups.

DISCUSSION

Theimmunesystemreflectsadualityofrequirementsdictatedbytheneedtoremainrelativelyconservedinmanyofitsactivationpathways,whilemaintainingahighlevelofspec-ificityforaparticularantigen.Thisnotionhasbeenthor-oughlydemonstratedthroughstudiesinvolvingthemajorhis-tocompatibilitycomplex,theT-cellreceptor,andothercellsurfacereceptorsinvolvedintheactivationprocess(19).OurdatasuggestthatwhenexaminingthearrayofreceptorsexpressedduringactivationbytwodifferentmethodologiesusingtheRSsystem,thisdualityofconservationandspecific-ityisarticulatedviauniquepeakdifferences.Whenanalyzingtheratiosofthe1182:1195peaks,wefoundsimilarpatternsin

Figure6.DiscriminatefunctionanalysisofalloreactiveversusCD3/28-activatedTlymphocytes.[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.intersciece.wiley.com.]

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boththealloreactiveandtheCD3/CD28-activatedsamplesthatwerenotpresentintheinactivatedorrestingT-cellsam-ples.ThisRamanshiftlikelyrepresentsaconservedchangeincellsurfacemoleculessharedbyactivatedTcellsregardlessofmethodologyofactivation.Whenapplyingestablishedpeakassignmentdata(20–23),thesepeaksarerepresentativeofty-rosine/phenylalanineandadenine/thyminechanges.Theseconservedpeakchangescanbecontrastedwiththoseshiftsoccurringat903,1031,1093,1155,1326,and1449cm21whichwereobservedintheCD3/CD28-activatedbutnotinthealloantigen-activatedT-cellsamples.ThesefociofshiftslikelyrepresentaspecificchangeincellsurfacemoleculesreflectingaresponsetotheparticularCD3/CD28stimulus.Whencrossreferencingthesefociwithestablishedpeakassignmentdata(20–23),theyrepresentchangesinnucleicacids(903,1093,and1449cm21),aminoacids(1031and1155cm21),orboth(1326cm21)-changeswhichareconsist-entwithmolecularprocessesresponsibleforthetranscription,translation,andexpressionofcellsurfacereceptors.Whenfur-theranalyzingthesepeakdifferencesthatwereuniquetotheCD3/CD28-activatedTcells,therewasareductioninpeakmagnitudesobservedinallsixoftheaforementionedpeakpositions.Althoughtheprimaryetiologyofthispatternremainscryptic,itmostlikelyrepresentsareshufflingratherthanadown-regulationofcellsurfacebio-molecularmaterial.

Basedupontheaforementionedobservations,itispossi-bletodetectT-cellactivationandcharacterizetheparticularmodeofthisactivationusingRS.Thisrepresentsafounda-tionalsteptowardthedevelopmentofaRS-basedsystemforthenoninvasivedetectionofAR.Otherproposednoninvasivedetectiontechniqueswhichhaveshownefficacy,suchasmetabolomics(24),proteomics(25,26),andDNAmicroarrayprofiling(27,28),arecostly,timeintensive,computationallyexpensive,andrequireextensiveinfrastructuretocarryoutanalysis.ARS-basedsystemoffersadecidedadvantagebyfocusingondeterminedfociofspectraldifferencesthuspro-vidingalow-cost,rapidanalysiswhichcanbeoutfittedforportableuseintheclinicalsetting.TheRSsystemcandetectT-cellactivationwithinbothbloodandurinebiologicmatri-ces.ThisflexibilityisaffordedbythecomponentsubtractionofRamanspectralcontributionsmadebyothercells,fluids,andfreefloatingmacromolecules.Thesystemcanuse‘‘learned’’T-cellactivationsignaturestoselectTcellsthatmatchthestoredcellularprofilesevenfromwithinamulti-cellularfluid.Moreover,theabilityofRStodifferentiatebetweenthesignaturesofTcellsactivatedbydifferentmeansaffordsapotentialforhighspecificity.Thevalueofthishighspecificityisthreefold.First,itprovidesanavenuetomoveawayfromtheparadigmofneedinganinvasivebiopsytoconfirmthediagnosisofARandalsoprovidesanalternativetoprotocolbiopsiestodetectSCAR.Second,RSdetectionofARoffersamodalitythatcouldsignificantlyreducethedelayinARdiagnosis.Underthecurrentalgorithm,delaysintreat-mentarecreatedduetotheneedtoeliminateotherpotentialcausesofSCE,thuspotentiallyallowingfurtherhistologicdamagetothenephronsoftransplantedgraft.Third,acom-922

prehensivepanelofclinicallyrelevantsignaturesbaseduponviral(CMV),fungal(Candida),andbacterial(E.coli)stimulicouldbeinvestigatedwhichwillfurtherimprovetherapiddiagnosisandmanagementofinfectionsinthepost-trans-plantperiod.

CONCLUSION

BaseduponRSanalysisofthecellsurfacesofalloreactiveandCD3/CD28-activatedTlymphocytes,weconcludethatthereceptorexpressionandresultingspectralsignaturesofthesetwoactivatedT-cellpopulationsdiffersignificantly.TheseuniqueRSsignatureswillallowforamorerefinedapproachtowardthedevelopmentofanoninvasiveARdetec-tionsystemthathasahighspecificityandsensitivity.

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