Download pdf - Loss of Memory B Cells During Chronic

Research article

TheJournalofClinicalInvestigation http://www.jci.org

Loss of memory B cells during chronic HIV infection is driven by Foxo3a- and

TRAIL-mediated apoptosisJulien van Grevenynghe,1,2,3,4 Rafael A. Cubas,4 Alessandra Noto,1,2,3,4

Sandrina DaFonseca,1,2,3,4 Zhong He,1,2,3,4 Yoav Peretz,1,2,3 Abdelali Filali-Mouhim,1,2,3,4 Franck P. Dupuy,1,2,3,4 Francesco A. Procopio,1,2,3,4 Nicolas Chomont,1,2,3,4 Robert S. Balderas,5

Elias A. Said,6 Mohamed-Rachid Boulassel,7 Cecile L. Tremblay,1,2 Jean-Pierre Routy,3,7 Rafick-Pierre Skaly,1,2,3,4,8 and Elias K. Haddad1,2,3,4,8

1Vaccine and Gene Therapy Institute Florida, Port St. Lucie, Florida, USA. 2Centre de recherche du Centre Hospitalier de lUniversit de Montral (CRCHUM), Hpital Saint-Luc, Qubec, Canada. 3Laboratoire dImmunologie, Dpartement de Microbiologie et dImmunologie, Universit de Montral,

Montral, Qubec, Canada. 4Institut National de la Sant et de la Recherche Mdicale U743, CRCHUM, Universit de Montral, Montral, Qubec, Canada. 5BD Biosciences, San Diego, California, USA. 6Department of Microbiology and Immunology, College of Medicine, Sultan Qaboos University,

Muscat, Oman. 7Immunodeficiency Service and Division of Haematology, Royal Victoria Hospital, McGill University Health Center, McGill University, Montral, Qubec, Canada. 8Department of Microbiology and Immunology, McGill University, Montral, Qubec, Canada.

LossofmemoryBcellsoccursfromtheonsetofHIV-1infectionandpersistsintothechronicstagesofinfec-tion.Lackofsurvivalofthesecells,eveninsubjectsbeingtreated,couldprimarilybetheconsequenceofanalteredlocalmicroenvironmentinducedbyHIVinfection.InthisstudyweshowedthatmemoryBcellsurvivalwassignificantlydecreasedinaviremicsuccessfullytreated(ST)subjectscomparedwithsubjectswhocontrolviralloadasaresultofnaturalimmunity(elitecontroller[EC])orwithuninfectedcontrol(HIV)subjects.ThelowersurvivallevelsobservedinmemoryBcellsfromSTsubjectsweretheresultofdisruptedIL-2sig-nalingthatledtoincreasedtranscriptionalactivityofFoxo3aandincreasedexpressionofitsproapoptotictargetTRAIL.Notably,memoryBcellsurvivalinSTsubjectswassignificantlyenhancedbytheadditionofexogenousIL-2inaFoxo3a-dependentmanner.WefurthershowedthatFoxo3asilencingbysiRNAresultedindecreasedexpressionofTRAILandapoptosislevelsinmemoryBcellsfromSTsubjects.OurresultsthusestablishadirectroleforFoxo3a/TRAILsignalinginthepersistenceofmemoryBcellsandprovideamecha-nismforthereducedsurvivalofmemoryBcellsduringHIVinfection.ThisknowledgecouldbeexploitedforthedevelopmentoftherapeuticandpreventativeHIVvaccines.

IntroductionInadditiontoprogressiveTcelldysfunctionandcelldeath,HIVinfectionitselfleadstoearlyandprofoundderegulationofBcellphysiology,homeostasis,andfunction.ThesearemanifestedbypolyclonalactivationofundifferentiatednaiveBcells(1),induc-tionofhypergammaglobulinemia(2),increasedexpressionofactivationmarkers(3),highfrequenciesofapoptoticcells(4),poorresponsiveness toantigenicandmitogenicstimulation(5,6),andaprogressivedepletionofperipheralCD27+memoryBcells(7).Ofnote,thislossofmemoryBcellsalreadyoccursfromtheonsetofacuteinfection(8,9).Interestingly,success-fullytreated(ST)subjects,withdrug-suppressedviremia,stillshowlowfrequenciesofCD27+memoryBcellsandlowproduc-tionofAbsthatarenotfullyrestoredbyhighlyactiveantiret-roviraltreatments(HAARTs)(1013).Ontheotherhand,elitecontroller(EC)subjects,whonaturallycontrolviralreplicationandmaintainCD4+Tcellcountscomparabletothoseofunin-fectedcontrol(HIV)subjectsintheabsenceofHAART,shownomemoryBcelllossanddisplaybroadandfunctionalTandBcellmemoryresponses(1316).STsubjectsthusprovideidealsubjectstoidentifydefectsinmemoryBcellsurvival,whereas

studyingmemoryBcellsinECsubjectscouldleadtotheidentifi-cationofuniquecharacteristicsofmemoryBcellsurvivalassoci-atedwithnaturalcontrolofHIVinfection.WepreviouslyidentifiedtheFoxo3apathwayasamajorregula-

torofcentralmemoryTcellsurvival(15,17).Foxo3aisatran-scriptionfactorthatisconstitutivelyexpressedinhematopoieticcellsandcaninducethetranscriptionofproapoptotictargetgenessuchasBim,FasL,andTRAIL(1820).PhosphorylationofFoxo3abyseveralexternalsignalsincluding-chainreceptorcytokinessuchasIL-2orTandBcellreceptors(17,21,22)leadstoitsdegra-dationinthecytoplasmandinhibitionofitstranscriptionalactiv-ity(18,23,24).EvidenceintheliteraturesuggeststhatFoxo3amightparticipateinnormalBcelldevelopmentandhomeostasis.Forexample,Foxo3ahasbeenshowntobeinvolvedinBcelldif-ferentiation,whereitcanlinkBcRsignalingwithrecombinationmachineryandaffectVDJrecombinationandaffinitymaturation(25,26).Inthatcontext,Foxo3a-deficientmiceshowreducednumbersofpreBcellsandrecirculatingBcellswhencomparedwithwild-typecounterparts(27).AlthoughtranscriptionfactorssuchasBcl-6andMcl-1havebeen

previouslyshowntoplaycriticalrolesinthegenerationofmemoryBcells(28,29),littleisknownabouttheimplicationofFoxo3ainthesurvivalorthedevelopmentofmemoryBcellsinthecontextofacuteandchronicviralorevenbacterialinfections.Inthisstudy,weinvestigatedthemolecularmechanismsresponsibleforthelack

Authorshipnote:Rafick-PierreSkalyandEliasK.Haddadarecoseniorauthors.

Conflictofinterest:Theauthorshavedeclaredthatnoconflictofinterestexists.

Citationforthisarticle:J Clin Investdoi:10.1172/JCI59211.

research article


ofmemoryBcellsurvivalinchronicallyHIV-infectedsubjects(whodisplayedadecreaseinthefrequencyofmemoryBcells)andinECandHIVsubjects(whomaintainedastatisticallysignificanthighernumbersofmemoryBcells).OurresultsidentifiedacriticalrolefortheFoxo3a/TRAILpathwayinmemoryBcellsurvival.

ResultsCD27+ memory B cells from ST subjects are short lived and apoptotic.Previousreportshave indicated thatHIV+patients, includingthoseundergoingHAART,showsignificantlylowerfrequenciesofmemoryBcellswhencomparedwithuninfecteddonors(1013).TobetterunderstandtheunderlyingmechanismsresponsibleforthedecreaseinthenumbersofmemoryBcells,wemeasuredthefrequenciesandabsolutenumbersofperipheralmemoryBcellsinchronicallyHIV-infectedsubjectsaswellasinuninfecteddonors(HIV).SupplementalTable1(supplementalmaterialavailableonlinewiththisarticle;doi:10.1172/JCI59211DS1)summarizestheclinicalandvirologicaldataofthe2groupsofaviremicchronicallyHIV-infected(ECandST)subjects.WefirstcomparedtheexvivofrequenciesandabsolutenumbersoftotalperipheralBcellsinST,EC,andHIVsubjects.TotalBcells(definedasCD3CD19+)weresimilarintheirfrequenciesandabsolutenumbersinallgroupstest-ed(Figure1AandSupplementalTable1).Incontrast,theabsolutenumbers(P=0.028;n=13)(Figure1B)andfrequencies(Figure1C)

ofCD27+memoryBcellsfromSTsubjects(16.2%9.8)weresignif-icantlylowerthanthosefromECsubjects(35.9%9.2%;P

research article


n=5)(Figure1E).Thiswasfurtherconfirmedbythehighlysta-tisticallyinversecorrelation(Spearmanr=0.8643;P

research article


theseresultsdemonstratedacentralroleforFoxo3aandmostlikelyimplicateditsproapoptotictargetsBimandTRAILinregu-latingmemoryBcellsurvivalduringHIVinfection.

Foxo3a-mediated memory B cell survival in ST subjects is TRAIL depen-dent.WethenmeasuredtheexpressionlevelsofFoxo3atargetmol-eculesendowedwithproapoptoticpropertiesinexvivopurifiedmemoryBcellsfromST,EC,andHIVsubjects(n=5).WefoundthatmemoryBcellsfromSTsubjectsshowedsignificantlyhigherexpres-sionlevelsofTRAIL(100%)whencomparedwithEC(21.7%21%;P

research article


paredtothoseobservedinECandHIVsubjects(SupplementalFigure5,AD).Ofnote,wedidnotobserveanydifferencesintheexpressionlevelsofTRAILreceptors(DR4andDR5)onexvivomemoryBcellsbetweenST,EC,andHIVsubjects(SupplementalFigure6,BandC).TofurtherconfirmtheinvolvementoftheTRAILpathwayin

reducedmemoryBcellsurvivalofSTsubjects,wepretreatedpuri-fiedmemoryBcellsfromSTandHIVsubjectswithTRAILinhibi-tor(TRAILi)andthencoculturedthecellsfor7dayswithautolo-gousCD19PBMCs.ThelevelsofapoptosisinmemoryBcellsweredeterminedatday7usingannexinVstaining.TheconcentrationofTRAILiusedinourinvitroassaywasfirstdeterminedbyitsabil-itytoinhibitTRAIL-mediatedapoptosis(about95%inhibition)inBJABcells(SupplementalFigure6Aandref.33).OurresultsshowedthatpretreatmentofmemoryBcellswithTRAILiledtoasignifi-cantimprovementinthesurvivalofmemoryBcellsfromSTsub-jectssimilartothatobservedincocultureofHIVsubjects(P

research article


EC,andHIVsubjectsusingELISA(n11pergroupofsubjects).WefoundsignificantlyhigherlevelsofIL-2intheplasmaofEC(8.2%6.1%)whencomparedwithST(2.2%1.7%;P

research article


Exogenous IL-2 enhanced memory B cell survival from ST subjects by regulating Foxo3a and TRAIL pathways.WeinvestigatedwhethertheadditionofexogenousIL-2wouldenhancethesurvivalofmemoryBcellsfromSTsubjectsinthe7-daycocultureassayinaFoxo3a/TRAIL-dependentmanner.Wefirstdeterminedwhether IL-2 inducedphosphorylatedFoxo3a (pFoxo3a) inmemoryBcells.ThishasbeenshowntoblockFoxo3atranscrip-tionalactivitybyexcludingitfromthenucleus(refs.17,21,and22andFigure5A).TreatmentofmemoryBcellsfromSTsub-jectswiththe-chainreceptorcytokineIL-2for30minutesledtoasignificantincreaseinpFoxo3aatitsThr32residue(P

research article


untreatedmemoryBcellsinECandHIVsubjects(Figure5D).Moreimportantly,IL-2treatmentofmemoryBcellsfromSTsub-jectsledtoasignificantdecreaseinTRAILexpressionlevelsatday7ofcoculture(P

research article


WefurtherdeterminedwhethertheimprovementofactivatedmemoryBcellsurvivalwouldleadtoaconcomitantincreaseinthelevelsofAbproduction.TreatmentofmemoryBcellsfromSTsubjectswithZ-VAD(Pancaspaseinhibitor)orwithTRAILisignificantlyenhancedtheirsurvival(FI1.70.2[P

research article

0 TheJournalofClinicalInvestigation http://www.jci.org

viouslyshowntobeinvolvedintheactivationofmemoryBcells(53)andintheenhancedproductionofanti-HIVAbs(54,55).IL-2wasalsoshowntobeinvolvedinincreasedAbresponsestotetanusvaccinationinIL-2treatedSTsubjects(56),confirmingtheinvolvementofthiscytokineinthepersistenceanddevelop-mentofBcellresponses.However,despitethesignificantinvitroimprovementofmemoryBcellandASCsurvivalinSTsubjectsuponexogenousIL-2stimulation,therestorationwasnotcom-plete(around65%;Figure5D).TheseresultsstronglysuggestthatotherupstreamsignalsbesidesIL-2maycontributetothealtera-tionofFoxo3aactivityandtothealteredmaintenanceofmemoryBcellsinchronicHIV-infectedsubjects.Theextentof restorationof thehumoral immuneresponse

followingHAARThasbeenshowntobedependentontheearlyinitiationoftreatment.Recently,Moiretal.showedthatearlyini-tiationofantiretroviraltherapieswithinthefirstyearrestoredthefrequencyofmemoryBcellstolevelsobservedinhealthyindividu-als(57).Similarly,Chongetal.confirmedtheseresults,althoughthedataalsosuggestedthatwithtime,despiteearlytreatmentwithantiretroviraltherapy,therewasadeclineinthehumoralimmuneresponse(10).Ourownsubjects,whohadbeentreatedearlyandforatleast5years,showedregressionofmemoryBcellsthatwasnotfullyrestoredbyHAART.Altogether,theseresultsseemtosuggestthattheearlyinitiationofHAARTtemporallyrestoresmemoryBcellnumbersbutmaybeunabletohalttheslowprogressivedeclineovertime.Thisisinaccordancewiththefactthatprogressiveandpersistentstructuralandanatomicdamagesoccurinlymphnodeswithinthefirst2weeksofHIVinfection(siteofmemoryBcellgen-eration)andarenotfullyrestoredbyHAART(8,58).Inaddition,ourresultsalsohighlightthefactthatmemoryB

cellsfromECsubjectsweresignificantlydifferentthanthosefromSTsubjectsand,moreinterestingly,displayeduniquecharacteris-ticsthatwerenotalwaysfoundinHIVsubjects.WefoundthattheexvivofrequenciesofmemoryBcells(Figure1C),thelevelsofIL-2intheirplasma(Figure4A),andtheresponsivenesstoIL-2asmea-suredbypSTAT5levels(Figure4E)weresignificantlyhigherinECsubjectswhencomparednotonlywithSTsubjectsbutalsowithHIV,suggestinganincreasedsurvivalcapabilityofmemoryBcellswithinECsubjects.Ofnote,thehigherfrequenciesofmemoryBcellsobservedwithinECsubjectscouldnotbeexplainedbyprevi-ouslyreportedpolymorphismssuchasCCR5mutationsassoci-atedwithHIVprotection(SupplementalTable1andref.15).Overall,thesedataconfirmedthat,despiteHAART,thereisstill

arequirementtomaintaintheintegrityandsurvivalofperipheralCD27+memoryBcellstopreventHIVdiseaseprogressionandtherebyenhancethesuccessofpossibleantiviralvaccines.ThemanipulationorinductionofFoxo3aactivitybyanticancerdrugssuchasimatinibortheuseofrecombinantTRAILhasprovenefficientintreatingresistantcancersandautoimmunediseases(5961).Conversely,interferingwithFoxo3aandTRAILsignaling,aloneorincombinationwithHAART,couldrescuebothTandBcellfunctionsandcouldalsorestoreglobalAbresponsesincludingthoseelicitedagainstHIV,thusimprovingthesuccessoftherapeu-ticvaccinesagainstthisandotherinfections.

MethodsProducts.RPMI-1640media,FBS,and-mercaptoethanolwerepurchasedfromSigma-Aldrich.AllAbsusedforflowcytometrywerepurchasedfromBDBiosciences,exceptforanti-DR4andanti-DR5Abs,whichwereobtainedfromAbcam.AllprimaryAbsused forWesternblots (anti-phospho

formsofFoxo3a,anti-Bim,andanti-ERK)werepurchasedfromCellSig-naling.Anti-actinAbswerepurchasedfromSigma-Aldrich,anti-Foxo3afromAbcam,andanti-TRAILfromProSciInc.SecondaryHRP-conjugat-edgoatanti-mouseandanti-rabbitIgGAbswereobtainedfromBio-RadLaboratories.IL-2,IL-6,andIL-10cytokineswerepurchasedfromR&DSystems.Anti-CD40Ab(cloneG28.5;ATCChybridoma)wasprovidedbyWalidMourad(CentreHospitalierdelUniversitdeMontral).

Purification of CD27+ memory B cells.MemoryBcellswerepurifiedusingmagneticbeadseparation(MiltenyiBiotec).Weperformedaninitialnega-tiveselectionstepusingBCellIsolationKit2,whichallowedustoretaintotalBcells,followedbyapositiveselectionstepusingCD27microbeads(MiltenyiBiotec),whichallowedustopurifytheCD27+memoryBcellsubset.ToachievethehighestpurityofmemoryBcellsfromoursubjectcohorts,weperformed3roundsofelutionforboththenegativeandposi-tiveselectionsteps.Inaddition,purifiedmemoryBcellsfromallsubjectgroupswereanalyzedforpurity,andlevelsofapoptosisusingCD3CD19+andannexinVphenotype.ThepurityofmemoryBcellswasdeterminedataround93.6%usingflowcytometry.EqualnumbersofmemoryBcellsfromthe3subjectcohortswereusedinthecoculturesystem.Thiswasdonebycellcountingwithtrypanblueexclusion.

STAT5 phosphorylation assay.PBMCsfromST,EC,andHIVsubjectswerestimulatedwith10ng/mlofIL-2for15minutes.STAT5aphosphorylationwasmeasuredwithBDBiosciencesPhosflowusinganti-pSTAT5aspecificAbaspreviouslydescribed(17).

Western blots.HighlypurifiedsortedCD3CD19+CD27+memoryBcellsfromHIV+andHIVsubjectsweresubjectedtoSDS-PAGEandWesternblotanalysisaspreviouslydescribed(15,17).

Memory B cell coculture assay.PurifiedmemoryBcells(105)werecoculturedfor7dayswith9105autologousCD19-depletedPBMCs(purity>99.8%)inthepresenceof10MazidothymidinetoinhibittheproductionofHIVviralparticles.P24ELISAperformedonthesupernatantconfirmedthelackofviralreplication.AbsolutenumbersofviablememoryBcellsandthefre-quencyofapoptosis(annexinV+cells)weremeasuredatday7ofcoculture.

Transfection of siRNA in the 7-day coculture assay.PurifiedmemoryBcells(6106)wereelectroporatedusingNucleofectorIItechnologyaccordingtotheAmaxaBiosystemsmanufacturersprotocol.SpecificFoxo3asiRNAandsiRNAnegativecontrolwereobtainedfromInvitrogen(Foxo3aVali-datedStealthDuoPak).siRNA(3g)wastransfectedforeachcondition.Followingtransfection,cellswereculturedinRPMImediawithoutantibi-oticsfor6hours.Cellswerethenwashedat224gfor10minutesatroomtemperature(RT)toremovedeadnecroticcells.Transfectedcellswerethenwashed,counted,andcoculturedwiththeirautologousCD19PBMCsfor7days.Twodaysontothecocultureassay,cellswerepurifiedbynegativeselectionusingaBcellisolationkitandassayedforFoxo3a,BimEL,TRAIL,totalERK,and-actinexpressionthroughWesternblotanalysis.

Polyclonal activation.CFSE-labeledpurifiedmemoryBcellswerefirstpolyclonallyactivatedfor2hourswith5g/mlanti-CD40,0.1g/mlanti-IgGplusanti-IgM(JacksonImmunoResearch;catalogno.109-005-044),10ng/mlcytokines(IL-6plusIL-10)inthepresenceorabsenceofIL-2(10ng/ml),TRAILis(20ng/ml),orZ-VAD(25M).ActivatedmemoryBcellswerewashedandcoculturedfor3dayswithautologousCD19PBMCstogenerateASCsinthepresenceofAZT.P24ELISAperformedonthesupernatantconfirmedthelackofviralreplication.Apoptosiswasperformedatday3ongatedactivatedmemoryBcellsusingannexinV.DMSO(0.01%)wasincludedintheseexperimentsasnegativecontrolandgavesimilarresultstountreatedconditions.Insomeexperiments(Figure7,AD),cellsweretransfectedasdescribed

aboveandthencoculturedwithautologousCD19depletedPBMCsfor2days.ThisensuredefficientFoxo3asilencingwithinmemoryBcellspriortotheiractivation.MemoryBcellswerethenpurifiedatday2usingaB

research article


cellisolationkitandassessedforFoxo3a,BimEL,TRAIL,totalERK,and-actinexpressionbyWesternblotanalysis.TransfectedpurifiedmemoryBcellswerealsopolyclonallyactivated(asdescribedabove)foranextra3daysinELISPOTplatestomeasureASCgeneration.

ELISPOT assay.ThenumbersofASCsweremeasuredonday3activatedmemoryBcellsbyELISPOTassayaccordingtothemanufacturersproto-col(ELISPOTplusforhumanIgMandIgG,3840-2AW-Plusand3820-2AW-Plus,respectively;MABTECH).SFCsgeneratedfromactivatedandtreatedmemoryBcellsfromST,EC,andHIVsubjectswereenumeratedwithImmunoSpotS5UVAnalyzer(CellularTechnologyLtd.)andnormalizedtotheirnegativecontrol(restingmemoryBcellsfromthesamepatient).

HIV-specific ELISA.PBMCsfromST,EC,HIV(n=5),andprimaryinfected(PI;n=2)subjectswerepolyclonallyactivatedwithorwithout1g/mlPWMfor3days.Supernatants(200l)werecollectedtomeasureHIV-1specificIgGAbtitersusingELISA.Tothataim,1gofHIV-1Gag(HIV-121)and1gofENV(HIV-101)(ProspectProteinsInc.)dilutedincoatingBuffer(eBioscience)wereincubatedovernightat4Con96-wellplates.Thenextday,plateswerewashed10timesinPBS/0.05%Tween-20.Thiswasfollowedby30minutesofsaturationinPBS/10%FBS.Plateswerethenincubatedwithculturesupernatantsfor2hoursatRT,washed10times,andincubatedfor2hoursatRTwith100l1g/mlanti-IgGbiotin (ELISPOT kit for human IgG, 3850-2HW-Plus;Mabtech).Plateswerewashed10timesandincubated1houratRTwith100ldilutedstreptavidin-HRP(1:1,000inPBS)(3850-2HW-Plus;Mabtech).FollowingafinalwashstepinPBS/0.05%Tween-20,100l1TMBsubstrate solution (eBioscience)wereaddedperwelluntilappearanceofcolor.Enzymaticreactionswerestoppedbyadding50l1MH3PO4.Finally,ODat405nMwasmeasuredwithSpectraMaxplus384-platereader(MolecularDevices).

Statistics.Allstatisticalanalyseswereperformedusingthenonparamet-ricMann-WhitneyUtest,assumingindependentsamples.Thistest,whichusestherankofthedataratherthantheirrawvaluestocalculatethesta-tisticalsignificance,isanalternativetothettestwhentheassumptionof

normalityisnotsatisfiedorcouldnotbetestedinthecaseofsmallsamplesize.Inthisstudy,Pvaluesoflessthan0.05wereconsideredsignificant.

Study approval.AllHIV-infectedsubjectsprovidedwritteninformedcon-sent.ThisresearchwasapprovedbytheOfficeofResearchEthics,RoyalHospital,McGillUniversityHealthCenter,andbytheComitdEthiquedelaRechercheetleCentreHospitalierdelUniversitdeMontral.Virologi-calandimmunologicalprofilesforallHIV+subjectsincludedinthisstudyaresummarizedinSupplementalTable1.

AcknowledgmentsWearegratefultothepatientsparticipatingintheCanadiancohortofHIV-infectedsubjects,theirphysicians,andattendingstaffmem-bers.WearethankfultoM.LegaultfromtheRseauSidaMI/FRSQforadministrativeandclinicalcoordinatingsupport.WethankV.Lafontaine,N.Sawyer,L.Lejeune,andY.Chouikhfortheirtechnicalexpertise.J.vanGrevenyngheisafellowoftheCanadianInstitutesofHealthResearch(CIHR).ThisstudywassupportedbyresearchfundsfromtheNIH,theCIHR,GenomeQuebec,GenomeCanada,FondsdeRechercheenSantduQuebec(FRSQ),andtheCana-dianNetworkforVaccinesandImmunotherapeutics.R.-P.SkalyisaholderoftheCanadaResearchChairinHumanImmunology.J.-P.RoutyandC.L.Tremblayareclinician-scientistssupportedbyFRSQ.Moreover,wewouldliketothankP.Ancuta,Y.Peretz,andJ.Schatzleforcriticallyreviewingthemanuscript.

ReceivedforpublicationMay27,2011,andacceptedinrevisedformAugust3,2011.

Addresscorrespondenceto:EliasK.HaddadorRafick-PierreSkaly,11350SWVillageParkway,3rdFloor,PortSt.Lucie,Florida34987,USA.Phone:772.971.5099;Fax:772.345.3675;E-mail:[email protected](E.K.Haddad).Phone:772.345.4785;Fax:772.345.3675;E-mail:[email protected](R.-P.Skaly).

1.MoirS,FauciAS.BcellsinHIVinfectionanddisease.Nat Rev Immunol.2009;9(4):235245.

2.DeMilito A, et al.Mechanisms of hypergam-maglobulinemiaand impairedantigen-specifichumoralimmunityinHIV-1infection.Blood.2004;103(6):21802186.

3.AmadoriA,etal.BcellactivationduringHIV-1infection.II.Cell-to-cellinteractionsandcytokinerequirement.J Immunol.1991;146(1):5762.

4.SamuelssonA,BrostromC,vanDijkN,Sonner-borgA,ChiodiF.ApoptosisofCD4+andCD19+cellsduringhumanimmunodeficiencyvirustype1infection--correlationwithclinicalprogression,viralload,andlossofhumoralimmunity.Virology.1997;238(2):180188.

5.JiangW,etal.ImpairednaiveandmemoryB-cellresponsiveness toTLR9 stimulation inhumanimmunodeficiencyvirusinfection.J Virol.2008;82(16):78377845.

6.MoirS,FauciAS.PathogenicmechanismsofBlymphocytedysfunctioninHIVdisease.J Allergy Clin Immunol.2008;122(1):1219.

7.DeMilitoA,MorchC,SonnerborgA,ChiodiF.Lossofmemory(CD27)BlymphocytesinHIV-1infection.Aids.2001;15(8):957964.

8.LevesqueMC,etal.PolyclonalBcelldifferentiationandlossofgastrointestinaltractgerminalcentersintheearlieststagesofHIV-1infection.PLoS Med.2009;6(7):e1000107.

9.TitanjiK,etal.PrimaryHIV-1infectionsetsthestageforimportantBlymphocytedysfunctions.AIDS.2005;19(17):19471955.

10.ChongY,etal.SelectiveCD27+(memory)Bcellreduction and characteristic B cell alteration

indrug-naive andHAART-treatedHIV type 1-infectedpatients.AIDS Res Hum Retroviruses.2004;20(2):219226.

11.HartM,etal.LossofdiscretememoryBcellsubsetsisassociatedwithimpairedimmunizationrespons-esinHIV-1infectionandmaybeariskfactorforinvasivepneumococcaldisease.J Immunol.2007;178(12):82128220.

12.LongweH, Gordon S,Malamba R, FrenchN.CharacterisingBcellnumbersandmemoryBcellsinHIVinfectedanduninfectedMalawianadults.BMC Infect Dis.2010;10:280.

13.TitanjiK,etal.LossofmemoryBcells impairsmaintenanceoflong-termserologicmemorydur-ingHIV-1infection.Blood.2006;108(5):15801587.

14.CaoY,QinL,ZhangL,SafritJ,HoDD.Virologicandimmunologiccharacterizationoflong-termsurvivorsofhumanimmunodeficiencyvirustype1infection.N Engl J Med.1995;332(4):201208.

15.vanGrevenynghe J, et al. Transcription factorFOXO3a controls the persistence of memoryCD4(+)TcellsduringHIVinfection.Nat Med.2008;14(3):266274.

16.WangQ,etal.Highlevelserumneutralizinganti-bodyagainstHIV-1inChineselong-termnon-pro-gressors.Microbiol Immunol.2008;52(4):209215.

17.RiouC,etal.ConvergenceofTCRandcytokinesignalingleadstoFOXO3aphosphorylationanddrivesthesurvivalofCD4+centralmemoryTcells.J Exp Med.2007;204(1):7991.

18.CalnanDR,BrunetA.TheFoxOcode.Oncogene.2008;27(16):22762288.

19.DabrowskaA,KimN,AldoviniA.Tat-inducedFOXO3aisakeymediatorofapoptosisinHIV-1-

infectedhumanCD4+Tlymphocytes.J Immunol.2008;181(12):84608477.

20.SakoeY,SakoeK,KiritoK,OzawaK,KomatsuN.FOXO3Aasakeymoleculeforall-transreti-noicacid-inducedgranulocyticdifferentiationandapoptosisinacutepromyelocyticleukemia.Blood.2010;115(18):37873795.

21.BilancioA,etal.Keyroleofthep110deltaisoformofPI3KinB-cellantigenandIL-4receptorsignal-ing:comparativeanalysisofgeneticandpharma-cologicinterferencewithp110deltafunctioninBcells.Blood.2006;107(2):642650.

22.vanGrevenyngheJ,etal.LymphnodearchitecturecollapseandconsequentmodulationofFOXO3apathwayonmemoryT-andB-cellsduringHIVinfection.Semin Immunol.2008;20(3):196203.

23.BrunetA,etal.Aktpromotescellsurvivalbyphos-phorylatingandinhibitingaForkheadtranscrip-tionfactor.Cell.1999;96(6):857868.

24.HuMC,etal.IkappaBkinasepromotestumorigen-esisthroughinhibitionofforkheadFOXO3a.Cell.2004;117(2):225237.

25.HerzogS,etal.SLP-65regulatesimmunoglobu-linlightchaingenerecombinationthroughthePI(3)K-PKB-Foxopathway.Nat Immunol. 2008;9(6):623631.

26.HerzogS,RethM,JumaaH.RegulationofB-cellproliferationanddifferentiationbypre-B-cellrecep-torsignalling.Nat Rev Immunol.2009;9(3):195205.

27.HinmanRM,NicholsWA,DiazTM,GallardoTD,CastrillonDH,SatterthwaiteAB.Foxo3/micedemonstratereducednumbersofpre-Bandrecircu-latingBcellsbutnormalsplenicBcellsub-popula-tiondistribution.Int Immunol.2009;21(7):831842.

research article


28.ScheerenFA,etal.STAT5regulatestheself-renewalcapacityanddifferentiationofhumanmemoryBcellsandcontrolsBcl-6expression.Nat Immunol.2005;6(3):303313.

29.VikstromI,etal.Mcl-1isessentialforgerminalcenterformationandBcellmemory.Science.2010;330(6007):10951099.

30.SanzI,WeiC,LeeFE,AnolikJ.Phenotypicandfunctional heterogeneity of humanmemoryBcells.Semin Immunol.2008;20(1):6782.

31.KleinU,KuppersR,RajewskyK.EvidenceforalargecompartmentofIgM-expressingmemoryBcellsinhumans.Blood.1997;89(4):12881298.

32.WellerS, etal.HumanbloodIgMmemoryBcellsarecirculatingsplenicmarginalzoneBcellsharboringaprediversifiedimmunoglobulinreper-toire.Blood.2004;104(12):36473654.

33.SprickMR,etal.FADD/MORT1andcaspase-8arerecruitedtoTRAILreceptors1and2andareessen-tialforapoptosismediatedbyTRAILreceptor2.Immunity.2000;12(6):599609.

34.AveryDT,etal.BcellintrinsicsignalingthroughIL21receptorandSTAT3isrequiredforestablish-inglonglivedantibodyresponsesinhumans.J Exp Med.2010;207(1):155171.

35.BiancottoA,etal.Abnormalactivationandcytokinespectrainlymphnodesofpeoplechronicallyinfect-edwithHIV-1.Blood.2007;109(10):42724279.

36.IannelloA,etal.DynamicsandconsequencesofIL-21productioninHIV-infectedindividuals:alon-gitudinalandcross-sectionalstudy.J Immunol.2010;184(1):114126.

37.LitjensNH,HuismanM,HijdraD,LambrechtBM,StittelaarKJ,BetjesMG.IL-2producingmemoryCD4+TlymphocytesarecloselyassociatedwiththegenerationofIgG-secretingplasmacells.J Immunol.2008;181(5):36653673.

38.HarariA, Petitpierre S, VallelianF, PantaleoG.Skewedrepresentationoffunctionallydistinctpopu-lationsofvirus-specificCD4TcellsinHIV-1-infectedsubjectswithprogressivedisease:changesafteranti-retroviraltherapy.Blood.2004;103(3):966972.

39.YounesSA,etal.HIV-1viremiapreventstheestab-lishmentofinterleukin2-producingHIV-specific

memoryCD4+Tcellsendowedwithproliferativecapacity.J Exp Med.2003;198(12):19091922.

40.FerreAL,etal.HIVcontrollerswithHLA-DRB1*13andHLA-DQB1*06alleleshavestrong,polyfunc-tionalmucosal CD4+ T-cell responses. J Virol.2010;84(21):1102011029.

41.MalaspinaA,etal.CpGoligonucleotidesenhanceproliferative and effector responses of B cellsin HIV-infected individuals. J Immunol. 2008;181(2):11991206.

42.MehtaN,etal.ImpairedgenerationofhepatitisBvirus-specificmemoryBcells inHIVinfectedindividualsfollowingvaccination.Vaccine.2010;28(21):36723678.

43.Herbeuval JP, et al. Differential expression ofIFN-alpha and TRAIL/DR5 in lymphoid tis-sueofprogressor versusnonprogressorHIV-1-infectedpatients.Proc Natl Acad Sci U S A.2006;103(18):70007005.

44.HerbeuvalJP,ShearerGM.HIV-1immunopathogen-esis:howgoodinterferonturnsbad.Clin Immunol.2007;123(2):121128.

45.StahlM,etal.Theforkheadtranscriptionfac-tor FoxO regulates transcription of p27Kip1andBim inresponse to IL-2. J Immunol.2002;168(10):50245031.

46.BreenEC.Pro-andanti-inflammatorycytokinesinhumanimmunodeficiencyvirusinfectionandacquiredimmunodeficiencysyndrome.Pharmacol Ther.2002;95(3):295304.

47.DecrionAZ,DichampI,VarinA,HerbeinG.HIVandinflammation.Curr HIV Res.2005;3(3):243259.

48.VicenziE,BiswasP,MengozziM,PoliG.Roleofpro-inflammatory cytokines and beta-chemo-kinesincontrollingHIVreplication.J Leukoc Biol.1997;62(1):3440.

49.EstesJD,etal.Simianimmunodeficiencyvirus-inducedlymphatictissuefibrosisismediatedbytransforminggrowthfactorbeta1-positiveregula-toryTcellsandbeginsinearlyinfection.J Infect Dis.2007;195(4):551561.

50.BrenchleyJM,etal.MicrobialtranslocationisacauseofsystemicimmuneactivationinchronicHIVinfection.Nat Med.2006;12(12):13651371.

51.ParryRV,etal.CTLA-4andPD-1receptorsinhibitT-cellactivationbydistinctmechanisms.Mol Cell Biol.2005;25(21):95439553.

52.TitanjiK,etal.Acutedepletionofactivatedmem-oryBcellsinvolvesthePD-1pathwayinrapidlyprogressingSIV-infectedmacaques.J Clin Invest.2010;120(11):38783890.

53.RoyMP,KimCH,ButcherEC.CytokinecontrolofmemoryBcellhomingmachinery.J Immunol.2002;169(4):16761682.

54.BeniguelL,etal.SpecificantibodyproductionbybloodBcellsisretainedinlatestagedrug-naiveHIV-infectedAfricans.Clin Dev Immunol. 2004;11(2):121127.

55.PinnaD,CortiD,JarrossayD,SallustoF,Lanzavec-chiaA.ClonaldissectionofthehumanmemoryB-cellrepertoirefollowinginfectionandvaccination.Eur J Immunol.2009;39(5):12601270.

56.LevyY,etal.Effectsofinterleukin-2therapycom-binedwithhighlyactiveantiretroviraltherapyonimmunerestorationinHIV-1infection:arandom-izedcontrolledtrial.Aids.2003;17(3):343351.

57.MoirS,etal.BcellsinearlyandchronicHIVinfec-tion:evidenceforpreservationofimmunefunctionassociatedwithearlyinitiationofantiretroviraltherapy.Blood.2010;116(25):55715579.

58.SchackerTW,etal.Lymphatictissuefibrosisisasso-ciatedwithreducednumbersofnaiveCD4+Tcellsinhumanimmunodeficiencyvirustype1infection.Clin Vaccine Immunol.2006;13(5):556560.

59.CretneyE,ShankerA,YagitaH,SmythMJ,Say-ersTJ.TNF-relatedapoptosis-inducingligandasatherapeuticagentinautoimmunityandcancer.Immunol Cell Biol.2006;84(1):8798.

60.EssafiM,BaudotAD,MouskaX,CassutoJP,Tic-chioniM,DeckertM.Cell-penetratingTAT-FOXO3fusionproteinsinduceapoptoticcelldeathinleu-kemiccells.Mol Cancer Ther.2011;10(1):3746.

61.KikuchiS,NagaiT,KunitamaM,KiritoK,OzawaK,KomatsuN.ActiveFKHRL1overcomes ima-tinib resistance in chronicmyelogenous leuke-mia-derivedcelllinesviatheproductionoftumornecrosisfactor-relatedapoptosis-inducingligand.Cancer Sci.2007;98(12):19491958.