IMMUNOBIOLOGY
ReducedfrequenciesandsuppressivefunctionofCD4ϩCD25hiregulatoryTcellsinpatientswithchroniclymphocyticleukemiaaftertherapywithfludarabine
MarcBeyer,MatthiasKochanek,KamruzDarabi,AlexeyPopov,MarkusJensen,ElmarEndl,PercyA.Knolle,RomanK.Thomas,MichaelvonBergwelt-Baildon,SvenjaDebey,MichaelHallek,andJoachimL.Schultze
GloballysuppressedT-cellfunctionhasbeendescribedinmanypatientswithcancertobeamajorhurdleforthedevel-opmentofclinicallyefficientcancerimmu-notherapy.Inhibitionofantitumorim-muneresponseshasbeenmainlylinkedtoinhibitoryfactorspresentincancerpatients.Morerecently,increasedfre-quenciesofCD4؉CD25hiregulatoryTcells(Tregcells)havebeendescribedasanadditionalmechanismreducingimmu-nity.Weassessed73patientswithB-cellchroniclymphocyticleukemia(CLL)and42healthycontrolsanddemonstratedsignificantlyincreasedfrequenciesofcy-totoxicTlymphocyte-associatedprotein4(CTLA4؉)–,ForkheadboxP3(FOXP3؉)–,glucocorticoid-inducedtumornecrosisfactorreceptor-relatedprotein(GITR؉)–,CD62L؉–,transforminggrowthfactor1(TGF-1؉)–,interleukin10(IL-10؉)–TregcellsinpatientswithCLL,withhighestfrequenciesinuntreatedorprogressingpatientspresentingwithextendeddis-ease.Mostsurprisingly,inthemajorityofpatientswithCLLtreatedwithfludarabine-
containingtherapyregimenstheinhibi-toryfunctionofTregcellswasdecreasedorevenabrogated.Inaddition,frequen-ciesofTregcellsweresignificantlyde-creasedaftertherapywithfludarabine.Inlightofsimilarfindingsforcyclophospha-midethecombinationoffludarabineandcyclophosphamidemightbefurtherex-ploitedinstrategiesreducingimmunosup-pressionpriortocancerimmunotherapy.(Blood.2005;106:2018-2025)
©2005byTheAmericanSocietyofHematology
Introduction
HumanandmurineCD4ϩCD25ϩTcellscontaincellsthatsuppressantigen-specificT-cellimmuneresponses.1-5Thesenaturallyoccur-ringregulatoryCD4ϩCD25ϩTcellsoriginatefromthethymusandplayacentralroleinthemaintenanceofperipheraltolerancebysuppressionofautoreactiveT-cellpopulations.Inmurinemodels,regulatoryTcells(Tregcells)preventautoimmuneandinflamma-torydiseases1,6,7andinhibitantitumorimmuneresponses.8-12AlthoughatrulyuniquemarkerforTregcellsisstillnotavailable,severalmoleculeshavebeenassociatedwiththesecellsincludingcytotoxicTlymphocyte-associatedprotein4(CTLA4),13-16glu-cocorticoid-inducedtumornecrosisfactorreceptor-relatedprotein(GITR,TNFRSF18),17,18ForkheadboxP3(FOXP3),19-21L-selectin(CD62L,SELL),22,23andOX40antigen(CD134,TNFRSF4).23,24
Inhumans,TregcellsareenrichedwithintheCD4ϩCD25hipopulation,whereasCD4ϩCD25loTcellsrepresentmainlyprevi-ouslyactivatedThelpercells.25TheseCD4ϩCD25hiTregcellsinhibitproliferationandcytokinereleasebyconventionalCD4ϩCD25ϪTcells.26Decreaseofthesecellswasfoundinpatientswithautoimmunediseases,27-31whereasanincreaseofTregcellsinpatientsafterallogeneicbonemarrowtransplantationwasassociatedwithareducedgraft-versus-hostdisease.32-35Inpatientswithmalignantmelanoma,36Hodgkinlymphoma,37orovarian,38,39
FromtheMolecularTumorBiologyandTumorImmunology,UniversityofCologne,Cologne,Germany;ClinicIforInternalMedicine,UniversityofCologne,Cologne,Germany;andInstituteforMolecularMedicineandExperimentalImmunology,UniversityofBonn,Bonn,Germany.
SubmittedFebruary15,2005;acceptedMay15,2005.PrepublishedonlineasBloodFirstEditionPaper,May24,2005;DOI10.1182/blood-2005-02-0642.SupportedbytheSofjaKovalevskajaAwardoftheAlexandervonHumboldt-Foundation(J.L.S.)andtheWilhelm-SanderStiftung(J.L.S.andM.K.).M.B.B.issupportedbyaCarrerasFoundationFellowshipandaMaxEderAwardfromtheDeutscheKrebshilfe.P.A.K.andE.E.aresupportedinpartbygrantHBFG-109-517.
gastric,40,41lung,39,42breast,43,44andpancreaticcancer43inhibitoryCD4ϩCD25ϩTcellsarealsoincreased.Inanelegantstudy,Curieletal38demonstratedthatfunctionalTregcellswereenrichedinascitesfromwomenwithovariancancer,migratedtowardCCL22expressedbytumorcellsandtumor-associatedmacrophages,andspecificallyinhibitedantitumorimmunity.Moreover,withinthissetting,theincreaseofTregcellspredictedpoorsurvival.38Onlyrecently,studiesassessingapotentialinfluenceofchemotherapyonTregcellshavebeeninitiated.Inmice,low-dosecyclophosphamidedecreasedthenumberofTregcells.45
Basedontheseobservationswewereinterestedinunderstand-ingwhetherCD4ϩCD25hiTcellsarealsoincreasedandpossessinhibitorycapacitiesinB-cellchroniclymphocyticleukemia(CLL)and,ifso,toassessthefrequencyandfunctioninthecontextofstageofdiseaseandpriortherapy.CLL,themostcommontypeofleukemiaintheWesternhemisphere,46ischaracterizedbyclonalproliferationandaccumulationofneoplasticBlymphocytes.47-49CLLisaparticularlyinterestingmodelbecauseitisfrequentlyassociatedwithclinicallymanifestimmunedefects,suggestinganunderlyingimmunedysregulation.50-56Infact,decreasedT-cellresponsestomitogenicandT-cellreceptor-mediatedstimulationshavebeendescribedinpatientswithCLL57,58;however,theaccountingcellularandmolecularmechanismsarestillunclear.59
M.B.andM.K.contributedequallytothiswork.
Theonlineversionofthearticlecontainsadatasupplement.
Reprints:JoachimL.Schultze,MolecularTumorBiologyandTumorImmunologyClinicIforInternalMedicine,UniversityofCologne,Joseph-StelzmannStr9/Haus16,50931Cologne,Germany;e-mail:joachim.schultze@uk-koeln.de.Thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.Therefore,andsolelytoindicatethisfact,thisarticleisherebymarked‘‘advertisement’’inaccordancewith18U.S.C.section1734.©2005byTheAmericanSocietyofHematology
2018
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BLOOD,15SEPTEMBER2005⅐VOLUME106,NUMBER6
Moreover,chemotherapyappliedtopatientswithCLLincludesdrugssuchasfludarabine,cyclophosphamide,oralemtuzumab,whicharecytotoxicforTcellsandhavebeenshowntoalterratiosofCD4ϩtoCD8ϩTcellsinvitro.60
Overall,weassessedTregcellsin73patientswithCLLand42healthyindividuals.Inadditiontopresentingclearevidenceofastage-dependentincreaseofTregcellsinthisleukemia,weob-served,forthefirsttimeinhumans,asignificantimpactofchemotherapy,particularlyfludarabine-basedtherapyregimens,onthefrequencyandfunctionofCD4ϩCD25hiTcells.
Patients,materials,andmethods
Patientsandclinicalparameters
Peripheralbloodfrom42healthyindividualsand73patientswithCLLwasobtainedfollowingapprovalbyourinstitutionalreviewboard(UniversityEthicsCommittee,Cologne,Germany),including35patientsinwhomweassessedbloodsamplesatleastat2differenttimepoints.Informedconsentforblooddonationswasobtained,pertheDeclarationofHelsinki,fromallvolunteers.Peripheralbloodmononuclearcells(PBMCs)fromhealthydonors(controls)andpatientsmeetingdiagnosticcriteriaforCLLwereobtainedfromperipheralbloodusingFicoll/Hypaque(Amersham,Uppsala,Sweden)densitycentrifugationandstoredinliquidnitrogenuntilfurtheruse.Patientsincludedforphenotypicalorfunctionalanalysiswereeitheruntreatedorhadnotreceivedcytoreductivetreatmentforaperiodofatleast1monthbeforeinvestigation.StagingwasperformedaccordingtotheBinetclassificationforCLL.ThemeanageofthepatientswithCLLatfirstanalysiswas61.2Ϯ10.4yearsandthatforthecorrespondinghealthycontrols,43.0Ϯ14.0years.ClinicalcharacteristicsofthepatientsstudiedaresummarizedinTableS1,availableontheBloodwebsite(seetheSupplementalMaterialslinkatthetopoftheonlinearticle).VarianceanalysistoassessdependencyofageandfrequencyofCD4ϩCD25hiTcellsinhealthydonorsandpatientswithCLLdidnotrevealacorrelationbetweenthesevariables.
Antibodiesandfluorescence-activatedcellsortinganalysisCellphenotypeofTcellswithinPBMCswasdefinedbymulticolorflowcytometryusingthefollowingantibodies:fluoresceinisothiocyanate(FITC)–conjugatedCD4;phycoerythrin(PE)–conjugatedCTLA4(CD152);PE-cyanin5(Cy-5)–conjugatedCD25,–CD45RA,and–CTLA4;allophycocya-nin(APC)–conjugatedCD4;APC-Cy-7–conjugatedCD4(allfromBectonDickinson[BD]PharMingen,Heidelberg,Germany);PE-conjugatedCD25,CD62L;peridininchlorophyllprotein(PerCP)–conjugatedCD3;PE-Cy-7–conjugatedCD25(allfromBectonDickinsonBiosciences,Heidelberg,Germany);andthecorrespondingisotypecontrolantibodies(BDPharMin-gen).Cellswerestainedaccordingtothemanufacturer’srecommendations.ForintracellularstainingcellswerepermeabilizedusingCytofix/Cytopermsolution(BDPharMingen)aftersurfacestainingandincubatedwithinterleukin10(IL-10)–FITC,GITR-FITC(bothfromR&DSystems,Wiesbaden,Germany),transforminggrowthfactor1(TGF-1)–PE(IQ-Products,Groningen,TheNetherlands),andCTLA4-PE(BDPharMin-gen)orwiththeappropriateisotypecontrols(BDPharMingen).Sampleswerethenwashedandstoredat4°Cuntilacquisition.
SampleswereacquiredeitheronaFACSCaliburorFACSCanto(bothfromBDBiosciences)andanalyzedwithCELLQuestorFACSDivasoftware(bothfromBDBiosciences)orWinMDI2.8(http://facs.scripps.edu/software.html).CD25loandCD25hiTcellsweregatedasdemonstratedinFigure1Aforallsamplesanalyzedaccordingtopreviouslypublisheddata.25Theanalysiswasperformedindependentlyby2investigators(M.B.andJ.L.S.)withsimilarresults.FrequenciesofCD4ϩCD25hiTcellsinperipheralbloodareshownaspercentvaluesofCD4ϩTcells.TodeterminecellspositiveforadditionalTregcellmarkers,weusedstringentgatingcriteria,settinggatesatthe1%leveloftherespectiveisotypecontrol.
TregCELLFREQUENCYANDFUNCTIONINCLL
2019
Figure1.FrequencyofCD4؉CD25hiTcells.(A)FlowcytometricanalysisofCD4andCD25onperipheralblood-derivedTcellsofahealthyindividualandapatientwithCLL.Cellswereeitherstainedwiththeappropriateisotypecontrols(leftpanel)orCD4andCD25mAbs(rightpanel).CD4ϩCD25ϩTcellsweredividedintoCD25loandCD25hicellsaccordingtopreviouslypublisheddata.25Numbersrepresentpercent-ageofeventswithintherespectiverectangle.Settingsshownherewereusedfortheanalysisofallsamplesunderstudy.(B)FrequencyofCD4ϩCD25hiTcellsin26controlandall73CLLsamples.Shownherearemedian,75percentile(box),SD(whiskers),andoutliers(dots)(*PϽ.001,Studentttest).(C)FrequencyofCD8ϩCD25hiTcells.
CD4؉T-cellisolationandculture
CD4ϩTcellswerepurifiedfromPBMCsusingCD4magnetic-activatedcellsorting(MACS)beads(MiltenyiBiotec,BergischGladbach,Germany)asdescribedpreviously.61ToassesspolyclonalCD4ϩT-cellactivation1ϫ105CD4ϩTcells/wellwereactivatedinAIM-V(GibcoInvitrogen,Karlsruhe,Germany)/EX-Cell610(JRHBiosciences,Lenexa,KS)withanti-CD3(0.2g/mL,OKT-3)andanti-CD28monoclonalantibody(mAb;0.2g/mL,kindgiftofDrL.M.Nadler,Dana-FaberCancerInstitute,Boston,MA)in96-wellplates.TheproliferationofTcellswasmonitoredbymeasuringincorporationof5-bromo-2Ј-deoxyuridine(BrdU;RocheDiagnostics,Mannheim,Germany)onday2to3ofculture.Cellswereharvested24hoursaftertheadditionofBrdU.BrdUincorporationwasassessedbyabsorbanceatawavelengthof450nmusingamultiwellenzyme-linkedimmunosorbentassay(ELISA)reader.IsolationofCD4؉CD25hiandCD4؉CD25؊Tcells
In18patientswithCLLwewereabletoobtainsufficientamountsofperipheralblood(Ͼ50mL)toisolateCD4ϩCD25ϪTcellsandCD4ϩCD25hiTcellsfromPBMCsforfunctionalanalysis.14Briefly,CD4MACSmultisortbeads(MiltenyiBiotec)wereusedforisolationofCD4ϩTcells.Afterdetaching,cellswerewashedandCD4ϩCD25hiTcellswerepositivelyselectedusingCD25microbeads(2Lbeads/107CD4ϩTcells).ThedescribedtechniqueisoptimizedfortheisolationofhumanCD4ϩCD25hiTcellswithhighpurity.62Useofhigherconcentrationsofmicrobeadsforisolationresultsinbetterrecoverybutdecreasesthepurity.ThenegativefractionofCD4ϩCD25ϪTcellswasusedaseffectors.Alternatively,CD4ϩTcellswereisolatedwithCD4MACSbeadsasdescribedearlierandstainedwithCD4-FITCandCD25-PE.CD4ϩCD25hiTcellswerepurifiedusingaFACSDiVacellsorter(BDBiosciences).Cellswerereanalyzedaftersortingandroutinelyshowedmorethan95%purity.Forsomeexperiments,CD4ϩCD25hiaswellasCD4ϩCD25ϪTcellswerepreacti-vatedwith0.5g/mLanti-CD3mAbat37°Cfor20hoursinthepresenceof10U/mLIL-2(Proleukin;Chiron,Munich,Germany)inX-VIVO15(BioWhittakker,Verviers,Belgium).
RNAextractionandreal-timereversetranscription–PCRforFOXP3
TotalRNAfrompurifiedCD4ϩCD25ϪandCD4ϩCD25hiTcellswasisolatedusingTRIzol(Invitrogen,Karlsruhe,Germany).First-strandcDNAwassynthesizedfrom100ngtotalRNAusingSuperScriptIIIkit(Invitrogen)accordingtothemanufacturer’srecommendation.ForFOXP3andglyceraldehydephosphatedehydrogenase(GAPDH)transcripts,real-timepolymerasechainreaction(PCR)wasperformedwithaLightCycler(RocheDiagnostics)basedonspecificprimersandgeneralfluorescencedetectionwithSYBRGreen.Thefollowingprimercombinationswere
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used:FOXP3forward,5Ј-CGGACACTCAATGAGATCTA-3Ј;FOXP3reverse,5Ј-ATCCTCCTTTCCTTGATCTT-3Ј;GAPDHforward,5Ј-TGATGACATCAAGAAGGTGGTGAA-3Ј;andGAPDHreverse,5Ј-TCCTTGGAGGCCATGTGGGCCAT-3Ј.AllPCRswereperformedusingLightCycler-FastStartDNAMasterSYBRGreenIkit(RocheDiagnostics).cDNAfromJurkatcellswasusedasastandardandnormalizationtoGAPDHwasperformedforeachsample.RelativefoldchangesofFOXP3expressioninCD4ϩCD25hiTcellswerenormalizedtoGAPDHasdescribed.38
Generationofdendriticcells
ForT-cellstimulationwegeneratedallogeneicdendriticcells(DCs)asdescribedpreviously.63Briefly,PBMCswereplatedinIscovemodifiedDulbeccomedium(IMDM)with5mMglutamineand25mMHEPES(N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonicacid;allfromPAALab-oratories,Coelbe,Germany)and1%autologousplasmafor2hoursat37°C.Subsequently,theadherentcellfractionwasculturedfor20hoursinRPMI1640(PAALaboratories)supplementedwith2mMglutamineand1%autologousplasma(DCmedium).Onday1,newDCmediumcontaining800U/mLgranulocyte-macrophagecolony-stimulatingfactor(GM-CSF;Leucomax;Novartis,Nuremberg,Germany)and1000U/mLIL-4(Immu-notools,Friesoythe,Germany)wasadded.Cytokineswereaddedonday3infreshDCmedium.Onday5,nonadherentcellswerereplatedinfreshDCmediumwithcytokines.Onday6,10ng/mLtumornecrosisfactor␣(TNF-␣),1g/mLprostaglandinE2(PGE2;bothfromSigmaAldrich,Taufkirchen,Germany),1000U/mLIL-6,and10ng/mLIL-1(bothfromR&DSystems)wereaddedandmatureDCswereharvestedonday7.AssessmentofinhibitoryfunctionofCD4؉CD25hiTcellsToassessthesuppressiveactivityofCD4ϩCD25hiTcellsonconventionalT-cellproliferation,amodifiedallogeneicMLRwasperformedasprevi-ouslydescribed.14,64,65Briefly,aftermagneticseparationbothCD4ϩCD25ϪandCD4ϩCD25hiTcellswereincubatedfor20hourswith10U/mLIL-2and0.5g/mLanti-CD3mAbinX-VIVO15.Subsequently,thesepreactivatedCD4ϩCD25ϪTcells(5ϫ104/well)werecoculturedwithirradiatedallogeneicPBMCs(2ϫ105/well)ormatureDCs(DCs/Tcells,1:20)inX-VIVO15supplementedwith10%fetalcalfserum(FCS),100U/mLpenicillin/streptomycin,and2mMglutamine(allfromGibcoInvitrogen).PurifiedallogeneicCD4ϩCD25hiTcellswereaddedatdifferentconcentrationsasindicated.TherewasnoinfluenceontheinhibitoryeffectofCD4ϩCD25hiTcellswhentheCD4ϩCD25ϪTcells(effectorTcells)werenotpreactivatedpriortotheinhibitionassay.Onday4thecellswerepulsedwithBrdUandBrdUincorporationwasanalyzed20hourslaterasdescribed(see“CD4ϩT-cellisolationandculture”).Assessmentofviabilityafterinvitroincubationwithfludarabine
Tomeasuretheeffectoffludarabine,PBMCsfromhealthycontrolswereculturedinRPMI1640supplementedwith10%FCS,100U/mLpenicillin/streptomycin,and2mMglutaminewithorwithout10Mfludarabine(Fludara;MedacScheringOnkologie,Munich,Germany)for48hours.Cellswerethenharvestedandstainedusingthefollowingantibodies:FITC-conjugatedCD3,APC-Cy-7–conjugatedCD4,PE-Cy-7–conjugatedCD25,7-amino-actinomycinD(7-AAD),andPE-conjugatedannexinV(allfromBDBiosciences)accordingtothemanufacturer’sinstructions.ViablecellsweredefinedasannexinVand7-AADdouble-negativecellsandlistedaspercentofparentalCD4ϩCD25ϪorCD4ϩCD25ϩTcells.Cytometricbeadarrayforchemokines
Theconcentrationofinterferon␥(IFN-␥)incellculturesupernatantswasmeasuredusingthehumanTh1/Th2cytokinekitII(BDPharMingen).Inbrief,capturebeadsweremixedwithculturesupernatantsandPEdetectionreagentandincubatedfor3hoursatroomtemperature.Thebeadswerethenwashedwithwashbufferandanalyzed.
BLOOD,15SEPTEMBER2005⅐VOLUME106,NUMBER6
Statisticalanalysis
ComparisonbetweenpairedorunpairedgroupswasperformedusingtheappropriateStudentttest.Pvaluesbelow.05weredefinedasstatisticallysignificant.Duetotheexplorativenatureofthisstudy,nomultiplicityadjustmentprocedureswereperformed.AllstatisticalanalyseswereperformedusingtheSPSSstatisticalsoftwarepackage(SPSS12.0forWindows,SPSS,Chicago,IL).Onlinesupplementalmaterial
FigureS1depictsproliferationofpurifiedCD4ϩTcellsfromhealthyindividualsandCLLpatientsactivatedwithanti-CD3andanti-CD28mAbs.TableS1containsinformationonthepatients.
Results
IncreasedfrequenciesofCD4؉CD25hiTcellsinpatientswithCLL
FrequenciesofCD4ϩCD25hiTregcellswereassessedusingmulti-colorflowcytometryandfollowingpreviouslypublisheddata.25AsdepictedinFigure1A,healthydonorsshowedasignificantnumberofCD4ϩCD25loTcellswithasmallerpercentageofCD4ϩCD25hiTcells,whereaswefoundanincreasedfrequencyofCD4ϩCD25hiTcellsinpatientswithCLL.Usingthesesettings,weanalyzedsamplesfrom73patientswithCLLand26healthyindividuals.ThefrequencyofTregcellsincontrols(4.5%Ϯ1.1%)wassimilartopreviouslypublishedresults(Figure1B).25Incontrast,patientswithCLLshowedsignificantlyincreasedfrequenciesofTregcells(10.4%Ϯ4.4%,PϽ.001).ControlexperimentsassessingcellsurfaceexpressionofCD25onCD8ϩTcellsdidnotrevealanoverallactivationofTcellsinpatientswithCLL,supportinganincreaseinCD4ϩCD25hiTregcells(Figure1C).
CD4؉CD25hiTcellsfrompatientswithCLLalsoexpressTregcell-associatedproteins
BeforeevaluatingTregcellfunction,weassessedexpressionofproteinsthathavebeenpreviouslyassociatedwithTregcells.TheseincludedCTLA4,GITR,CD62L,TGF-1,IL-10,andFOXP3.ThefrequencyofCD4ϩCD25hiCTLA4ϩTcellswassignificantlyincreasedinpatientswithCLLcomparedtohealthyindividualsbothforintracellularandextracellularstaining(Figure2A-B;PϽ.01andPϽ.05forpanelsAandB,respectively).Similarly,CD4ϩCD25hiGITRϩcellsfrompatientswithCLLweresignifi-cantlyaugmented(3.34%Ϯ1.67%inpatientsversus0.96%Ϯ0.29%incontrols,PϽ.001;Figure2C).GITRexpres-sioninconventionalCD4ϩCD25ϪTcellswassimilarlylowinbothpopulations(datanotshown).InpatientswithCLLtheCD4ϩCD25hiT-cellpopulationalsocontainedincreasedfrequenciesofCD62Lϩcells(PϽ.01;Figure2D).
IntracellularexpressionofTGF-1andIL-10wasassessedbecausethese2cytokineshavebeenassociatedwithTregcellfunction.66,67SimilarlytoCTLA4,GITR,andCD62L,TGF-1andIL-10weresignificantlyincreasedinCD4ϩCD25hiTcellsfrompatientswithCLL(Figure2E-F;PϽ.001andPϽ.05inpanelsEandF,respectively).WealsoobservedIL-10ϩaswellasTGF-1ϩcellswithinconventionalCD4ϩCD25ϪTcellsinthepatientsbutnotinhealthycontrols(datanotshown).BecauseFOXP3hasbeenshowntobeacrucialmoleculeformurineCD4ϩCD25ϩTregcells,
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TregCELLFREQUENCYANDFUNCTIONINCLL
2021
Figure2.ExpressionofproteinsassociatedwithTregcells.CTLA4,GITR,CD62L,TGF-1,andIL-10wereassessedonCD4ϩTcellscoexpressingCD25byeithercellsurfaceorintracellularmulticolorflowcytometry.Percentpositivecellsweredeterminedusingstringentgatingcriteriawithlessthan1%ofeventswithinthepositivegatewhenanalyzingrespectiveisotypecontrols.Atleast25,000eventsperanalysiswereacquired.Eachdotrepresentsasingleindividualassessedintherespectivegroup;meanexpression(line)ofallsamplesineachgroupisalsoshown.Significantdifferences(PϽ.05,Studentttest)betweencontrolsandCLLsamplesaremarkedbyanasterisk:(A)intracellularCTLA4(PϽ.01),(B)extracellularCTLA4(PϽ.05),(C)intracellularGITR(PϽ.001),(D)extracellularCD62L(PϽ.01),(E)intracellularTGF-1(PϽ.001),and(F)intracellularIL-10(PϽ.05).(G)ExpressionofFOXP3byhumanCD4ϩCD25hiTcells.CD4ϩCD25hiTcellsandCD4ϩCD25ϪTcellsweresortedbyMACSfromperipheralbloodofhealthycontrols(nϭ7)andpatientswithCLL(nϭ6).Real-timePCRforFOXP3wasperformedandrelativefoldchangesofCD4ϩCD25hiTcellstoCD4ϩCD25ϪTcellswerenormalizedtoGAPDHasdescribed.38
wealsoperformedreal-timePCRforFOXP3inasubsetofpatientswithCLLandhealthyindividuals.PCRsshowedstrongexpressionofFOXP3mRNAinCD3ϩCD4ϩCD25hiTcellsfromhealthyindividuals(nϭ7)andexpressionofFOXP3wasevenhigherintheCD3ϩCD4ϩCD25hiTcellsfrompatientswithCLL(nϭ6).Overall,CD4ϩTcellsexpressinghighlevelsofCD25,FOXP3,CTLA4,GITR,CD62L,TGF-1,andIL-10aresignificantlyincreasedinpatientswithCLL.
ReducedinhibitoryfunctionofCD4؉CD25hiTcellsfrompatientswithCLL
In18patientswithCLL,sufficientnumbersofhighlypurifiedCD4ϩCD25hiTcellswereisolatedtoanalyzetheirinhibitoryfunctionincomparisontoTregcellsfrom16healthycontrols.RegulatoryfunctionofCD4ϩCD25hiTcellswasassessedusinganallogeneicMLRofCD4ϩCD25ϪTcellsandallogeneicirradiatedPBMCs65orDCsasstimulators.14,64AutologousconventionalCD4ϩCD25ϪTcellsfrompatientswithCLLwerenotusedintheMLRbecausetheseTcellsmightthemselvesbeinhibitory.
Incontrols,significantinhibitionofallogeneicCD4ϩCD25ϪTcellsasexemplifiedinFigure3A(control,PϽ.01)wasdeter-minedat1:1ratiosofallogeneicconventionalTcellstoTregcells.Incontrast,patientswithCLLpretreatedwithfludarabine-containingchemotherapyshowedreducedorevenabrogatedTregcellfunction(Figure3A,flud).TregcellsfrompatientswithCLLnevertreatedwithfludarabine-containingtherapyregimensshowedinhibitoryfunctionsimilartohealthycontrols(Figure3A,w/oflud).AssessmentofIFN-␥productionbytheCD4ϩCD25ϪTcellsconfirmedtheproliferationresults.IFN-␥wasgreatlydiminishedinculturesinhibitedbyCD4ϩCD25hiTcellsfromhealthydonorsandpatientswithCLLnevertreatedwithfludarabine,butnotinculturesderivedfrompatientspreviouslytreatedwithfludarabine(Figure3B).ThesedatawerefurthercorroboratedatlowernumbersofTregcells(Figure3C).HealthycontrolsandpatientswithCLLnevertreatedwithfludarabineshowedinhibitionatlowerratiosofTregcellstoconventionalTcells,whereaspatientswithCLLpretreatedwithfludarabinedidnotshowanyinhibitionattheseconditions(Figure3C).InFigure3DinhibitionofallogeneicT-cellproliferationbyTregcellsisshownforallhealthycontrolsandpatientsata1:1ratioofTregtoconventionalTcells.Overall,therewasasignificantreductionofTregcell-inducedinhibitionwithinthefludarabinegroup(PϽ.001),whereaspatientswithCLLnevertreatedwithfludarabineshowedinhibitoryfunctionofTregcellsthatwasnotsignificantlydifferentfromhealthycontrols.
SortingofCD4ϩCD25hiTcellsbyfluorescence-activatedcellsorting(FACS)insomeexperimentsdidnotrevealdifferentresults,furthersupportingthatthesecellshadlosttheirinhibitoryfunction(datanotshown).UseofallogeneicmatureDCsinsteadofPBMCsforT-cellstimulationledtosimilarresults,suggestingthattheobservationwasintrinsictoCD4ϩCD25hiTcellsderivedfrompatientswithCLL(datanotshown).Interestingly,whenassessingproliferationofCD4ϩTcellsuponstimulationwithanti-CD3andanti-CD28mAbs,weobservednormalT-cellproliferationin3of3patientspretreatedwithfludarabine,whereas7of11patientsuntreatedornottreatedwithfludarabineshowedsignificantlyreducedoverallCD4ϩT-cellproliferation(FigureS1).
CD4؉CD25hiTcellsareincreasedinpatientswithCLLwithextendeddisease
NextweassessedthefrequencyofTregcellsincontextofstageofdiseaseinpreviouslyuntreatedpatientswithCLL(Figure4).Ofall73patients,26werepreviouslyuntreated.ThisanalysisrevealedacorrelationbetweenTregcellfrequencyandstageofdiseasewithhighestnumbersofTregcellsinpatientswithextendeddisease(BinetC)followedbypatientswithBinetBandwithlowestbutstillincreasednumbersinBinetA.InpatientsinBinetstageBandespeciallyinBinetstageCwealsoobservedanincreaseofCD4ϩCD25hiTcellscoexpressingGITR,CD62L,andintracellularCTLA4comparedtohealthycontrols(datanotshown).
CD4؉CD25hiTcellsarereducedparticularlyaftertherapywithfludarabine
Next,weinvestigatedtheassociationofTregcellfrequenciesandtherapy(Figure5).Forthisanalysis,weonlyincludedsamplesfrompatientsinBinetstagesBandCbecausemostpatientswithBinetstageAhadnotreceivedtherapy.Again,untreatedpatients(BinetB/C)showedanincreasedfrequencyofCD4ϩCD25hiTcells(Figure5A,notx)coexpressingGITR,CD62L,andintracellularCTLA4(datanotshown).Patientstreatedwithchemotherapyexcludingfludarabine(w/oflud)showedasimilarlyincreasedfrequencyofTregcellsandtherewasnocorrelationoffrequencyofTregcellsandtimefromtherapytophenotypicTreg-cellassessment(datanotshown).Incontrast,assessmentofTregcellcountsinpatientsreceivingfludarabinetreatmentshowedsignificantlyre-ducedTregcellfrequenciesonlyinpatientswhohadreceivedlastfludarabinetreatmentlessthan18monthspriortoTregcellassessment(PϽ.01).ThenumberofcellscoexpressingGITR,
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Figure3.FunctionalanalysisofCD4؉CD25hiTcells.HighlypurifiedCD4ϩCD25ϪTcellswerestimulatedbyallogeneicirradiatedPBMCsorDCseitherinthepresenceorabsenceofhighlypurifiedCD4ϩCD25hiTcellsderivedfrompatientswithCLLorhealthydonors(bothallogeneic).Tregcellsfrom16controls,10patientspretreatedwithfludarabine,and8patientsnevertreatedwithfludarabinewereassessedintheseMLRs.AsafunctionofT-cellinhibition,proliferation(A)andIFN-␥production(B)weremeasured.PanelAshowsrepresentativeexperimentsfromacontrol,aCLLpatientpretreatedwithfludarabine(flud),andapatientnevertreatedwithfludarabine(w/oflud).Openbars(PB)indicatebackgroundproliferationofirradiatedallogeneicPBMCs;lightgraybars(PBϩTconv),alloantigeninducedproliferationofCD4ϩCD25ϪconventionalTcells;darkgraybars(PBϩTreg),backgroundproliferationofCD4ϩCD25hiTregcells;andblackbars(PBϩTconvϩTreg),proliferationofCD4ϩCD25ϪconventionalTcellsinthepresenceofCD4ϩCD25hiTregcellsata1:1ratio(errorbarsrepresentSD;*PϽ.01Studentttest).(B)MeasurementofIFN-␥bycytokinebeadarrayinthesupernatantsfromculturesdescribedinpanelA.(C)InhibitionofproliferationofCD4ϩCD25ϪconventionalTcellsbyCD4ϩCD25hiTregcellsatdifferentratios(responderstosuppressors)fromahealthydonor(E),afludarabine-treatedCLLpatient(F),andaCLLpatientnevertreatedwithfludarabine(f).Representativeexperimentsareshownhere,errorbarsrepresentSD.(D)PercentagesofinhibitionofproliferationofCD4ϩCD25ϪconventionalTcellsbyCD4ϩCD25hiTregcellsata1:1ratiofromallhealthydonors(nϭ16),fludarabine-treatedpatients(nϭ10),orpatientswithCLLnevertreatedwithfludarabine(nϭ8)(*PϽ.001Studentttest).
CD62L,andintracellularCTLA4wasalsolowerthaninpatientsnevertreatedwithfludarabine(datanotshown).Thosepatientsreceivingtheirlastfludarabinetreatmentmorethan18monthspriortoTregcellmeasurementshowedTregcellfrequenciescomparabletopatientstreatedwithoutfludarabine-containingregimensorevenuntreatedpatients.Itneedstobementionedthatthepatientgroupstreatedwithfludarabinewereenrichedforpatientswithlargertumorburdenandprogressivedisease,whereasmanyoftheuntreatedpatientsshowedaslowerdiseaseprogression.In21patientsweobtainedbloodsamplesatleastat2differenttimepointsmorethan6monthsapart(range,7-128months).Atthesecondtimepointweobservedin5of6untreatedCLLpatientsslightlyincreasedorsimilarfrequenciesofTregcells(Figure5B,no
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Figure4.FrequencyofCD4؉CD25hiTcellsincontextofstageofdiseaseinpreviouslyuntreatedCLLpatients.PatientswereclassifiedaccordingtotheBinetclassification:BinetA(nϭ9),BinetB(nϭ13),andBinetC(nϭ4).Shownherearemedian,75percentile(box),SD(whiskers),andoutliers(dots)ofdataobtainedbymulticolorflowcytometry.
tx).Inpatientstreatedwithnon–fludarabine-basedchemotherapy(w/oflud),therewasadiverseresponsewith2patientsshowingincreasedand4patientslowerfrequencies.Incontrast,for8of8patientstreatedwithfludarabine,frequencieswerelowerafterfludarabinetherapy(Figure5B,flud).Overall,frequencyofTregcellsinpatientswithCLLwasassociatedwithstageofdiseaseandwithsignificantlyreducedfrequenciesafterfludarabine-basedchemotherapy.
ToassesswhetherfludarabinemightpreferentiallyinducecelldeathinCD4ϩCD25ϩTcellsweincubatedPBMCswith10M
Figure5.CorrelationoftherapyandfrequencyofCD4؉CD25hiTcellsinpatientswithCLL.(A)Patientstreatedwithfludarabinelessthan18monthspriortoT-cellanalysis(fludϽ1.5a)werecomparedwithhealthycontrols(control),untreated(notx),otherwisetreatedpatientswithCLL(w/oflud)orpatientswithCLLtreatedwithfludarabinemorethan18monthpriortoanalysis(fludϾ1.5a)forthefrequencyofCD4ϩCD25hiTcellsasassessedbymulticolorflowcytometry.Shownherearemedian,75percentile(box),SD(whiskers),andoutliers(dots);*PϽ.01forfludϽ1.5aversusfludϾ1.5abyStudentttest.(B)SerialanalysisofCD4ϩCD25hiTcellsforthe3CLLtreatmentsubgroupsat2differenttimepointsseparatedbyatleast6months.
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Table1.Fludarabine-inducedapoptosisinCD4؉CD25؉andCD4؉CD25؊TcellsExperiment1Experiment2MediumplusMediumplusMediumfludarabineMediumfludarabineCD4ϩCD25Ϫ,%91669052CD4ϩCD25ϩ,%69237930CD4ϩCD25ϩTcellsaremoresusceptibletofludarabine-inducedapoptosisthanCD4ϩCD25ϪTcells.PBMCsfrom2differentdonorswereincubatedinvitrowithmediumaloneorwithmediumcontaining10Mfludarabine.After48hourscellswerestainedwith7-AADandannexinVandviablecellsweredefinedas7-AADandannexin-Vdouble-negativecells.Viablecellsarelistedaspercentageoftherespectivecelltype.
fludarabineandmeasuredapoptosisandcelldeathbyflowcytometryusingannexinVand7-AAD.Whereasmorethan50%ofCD4ϩCD25ϪTcellswereviableafterexposureto10Mfludarabinefor48hours,morethan70%ofCD4ϩCD25ϩTcellsunderwentapoptosis(Table1).Incontrast,morethantwothirdsofCD4ϩCD25ϩTcellsculturedintheabsenceoffludarabinewerestillaliveatthistimepoint.
Discussion
MostrecentlyalossoffunctionofCD4ϩCD25hiTcellshasbeenassociatedwithautoimmunediseases,28whereasarelativeincreaseofCD4ϩCD25ϩTcellswasreportedinpatientswithsolidcancers.36,39-44Ananalysisof73patientswithCLLand42healthycontrolsrevealedasignificantlyincreasedfrequencyofCD4ϩCD25hiFOXP3ϩCTLA4ϩGITRϩCD62LϩTGF1ϩIL-10ϩTregcellsinthepatientswithCLL.FrequencyofTregcellswasparticularlyincreasedinuntreatedpatientswithintermediatestage(BinetB)orextendeddisease(BinetC).However,whenpatientsweretreatedwithfludarabine-containingchemotherapy,thefre-quencyofTregcellswassignificantlyreduced,whichwasalsoshowninindividualpatientsassessedbeforeandaftertreatmentwithfludarabine.AlthoughthereducedfrequencyofTregcellsaftertherapyespeciallywithfludarabinewassurprising,thereducedinhibitoryfunctionofhighlypurifiedCD4ϩCD25hiTcellsfromthesepatientswasanunexpectedfinding.First,invitroexperi-mentsdemonstratedapreferentialinductionofapoptosisinCD4ϩCD25ϩTcellsafterincubationwithfludarabine.
Inovariancancer,acorrelationbetweentumorstageandTregcellswithinascitesbutnotwithfrequencyinperipheralbloodwasrecentlyestablished.38Incontrasttoovariancancer,TregcellsinpatientswithCLLwereevenincreasedinperipheralbloodmostlikelyreflectingthedisseminatedcharacterofthismalignancy.AclearcorrelationbetweenTregcellfrequencyandtumorstagewasonlyobservedinuntreatedpatients(Figure4),whereaspatientstreatedwithfludarabine-containingregimenspresentedwithsignifi-cantlyreducedTregcellsindependentofstageofdisease.
WithinourdatasetitisnotyetpossibletodeterminewhetherTregcellfrequencyisapredictivemarkerforsurvivalbecauseobservationtimesforpatientswithCLLarestilltooshortandmostpatientswithinthisstudyarestillalive.Nevertheless,ourdatastronglysuggestthatTregcellnumberscannotserveasapredictivemarkerincancerpatientstreatedwithchemotherapy,particularlywithdrugsthatinfluencefunctionandfrequencyofthesecells.WhetherthenumberofTregcellsinuntreatedpatientswithCLLisanindependentpredictorforsurvivalneedsfurtherexploration.
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ThereductionofCD4ϩT-cellcountshasbeendescribedasasideeffectoffludarabine.68Wehaveextendedthesefindings,bydemonstratingapreferentialinductionofapoptosisofCD4ϩCD25ϩTcells.Itwillbeinterestingtodeterminewhethertheremainingcellsarestillinhibitory.Experimentsaddressingthisimportantaspectarecurrentlyongoing.Inlightofthesefindings,thesuccessfuluseofadoptivelytransferredautologoustumor-specificT-cellclonesinpatientswithmalignantmelanomaafterinductiontherapywithcyclophosphamideandfludarabineisintriguing.69AlthoughrecentdatahaveestablishedaroleforcyclophosphamideinreducingTregcells,11theuseoffludarabineismainlyassociatedwiththereductionofcellularitypriortoT-celltherapy.ThereducedTregcellfrequenciesandlossofinhibitoryfunctioninpatientswithCLL,however,wouldsuggestamorespecificeffectoffludarabinebyreducingparticularlyinhibitorycircuitswithintheimmunesystem.Sofar,ourdatasetisstillnotlargeenoughtodeterminewithsufficientstatisticalpowerapotentialcorrelationbetweenthenumberofTregcellsandtheclinicalobservationofautoimmunephenomenainCLL.IncorporatingassessmentofTregcellswithinlargerclinicaltrialswillbenecessarytoanswerthisimportantquestion.
GITRisespeciallyexpressedathighlevelsonrestingCD4ϩCD25hiTregcells.18Sofar,increasedexpressionofGITRonTregcellshasnotbeenreportedforcancerpatients.70ByflowcytometryweidentifiedanincreasedexpressionofGITRonTregcellsinpatientswithCLL.InmicetheligandforGITR(GITR-L)hasbeenmainlyidentifiedonimmatureDCs.71Inhumans,GITR-Lisexpressedinavarietyoftissues72includingPBMCs.StimulationofGITRonmurineCD4ϩCD25ϩTcellsabrogatedTregcellsuppression,therebybreakingimmunetolerance.18,73AnincreasedexpressionofGITRonTregcellsincancerpatientsmightbeaninterestingtargetfortherapeuticexploitation.WhetherGITRexpressioniselevatedinothermalignanciesshouldthereforebestudiedinmoredetail.
Inwomenwithovariancancer,TregcellsuniformlyexpressedhighlevelsofCCR4andmigratedtoCCL22expressedbytumorcellsandmacrophageswithinascites.38BecauseCLLcellsalsoexpressCCL22andattractCD4ϩCD40LϩTcells,74weinitiatedapreliminarystudyontheexpressionofCCR4onTregcellsinpatientswithCLL.In17patients,mostofwhomwerepretreatedwithfludarabine,CCR4expressionvariedinintensityandrangedfromonly30%to96%(mean,67.7%)oftheCD4ϩCD25hiTregcells(M.B.andJ.L.S.,unpublishedresults,January2005),whichisslightlylowerthanpreviouslypublisheddata.75Sofar,thereisnosignificantdifferenceofCCR4expressiononTregcellsbetweenuntreatedandchemotherapy-treatedpatientssothatthereducedexpressioninpatientswithCLLisunlikelyduetotherapy.
ThesignificantlyincreasedexpressionofTGF-1andIL-10inTregcellsfrompatientswithCLLwasalsoaccompaniedbyasignificantincreaseofconventionalCD4ϩCD25ϪTcellsexpress-ingthesecytokines(datanotshown).BothcytokinesplayanimportantrolefortheinhibitoryfunctionofTregcells.66IncontrasttoCLL,IL-10wasreportednottobeproducedbyTregcellsfrompatientswithinvasivebreast,lung,orpancreaticcancer39,42albeitotherstudiessuggestthatTregcellsmightexpressIL-10insomepatientswithsolidtumors.43CD4ϩCD25ϩTregcellsinpatientswithlung,colorectal,andovariancancerproducelargeramountsofTGF-1thanconventionalCD4ϩCD25ϪTcells.39,76InlightofTGF-1asanemergingtargetforantitumortherapy,77itneedstobefurtherevaluatedifTGF-1productionbyTregcellsisalso
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increasedinpatientswithsolidtumorsincomparisontohealthycontrolsaswehaveshownhereforpatientswithCLL.
Takentogether,wehaveelucidatedanimportanteffectoffludarabine-basedtreatmentregimensonCD4ϩCD25hiTregcells.ItwillbeinterestingtoseeiffludarabineincombinationwithcyclophosphamideisalsoreducingTregcellsafteradoptiveT-celltherapy69andallogeneicnonmyeloablativestemcelltransplanta-tion.78MonitoringfrequencyandfunctionofTregcellswithinsuchsettingswillhelptodefinethesignificanceofthesecellsforclinicalefficacyofcancerimmunotherapy.
Acknowledgment
Weareindebtedtoourpatientsfortheircommitmenttothisstudy.WethankA.Draube,E.Ja¨ger,C.Schweighofer,andC.Pallaschforreferralofpatients;B.Gathofforprovidinguswithbloodsamplesfromhealthyindividuals;andH.AbkenandJ.Chemnitzforcriticallyreadingthemanuscript.WethankI.Bu¨chmann,J.Claasen,andC.Hoyerforexcellenttechnicalassistance.
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From www.bloodjournal.org by guest on December 29, 2015. For personal use only.
2005 106: 2018-2025
doi:10.1182/blood-2005-02-0642 originally published onlineMay 24, 2005
Reduced frequencies and suppressive function of CD4+CD25hi
regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine
Marc Beyer, Matthias Kochanek, Kamruz Darabi, Alexey Popov, Markus Jensen, Elmar Endl, PercyA. Knolle, Roman K. Thomas, Michael von Bergwelt-Baildon, Svenja Debey, Michael Hallek and Joachim L. Schultze
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