Loss of Memory B Cells During Chronic

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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.

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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

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

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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

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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

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

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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

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

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

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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

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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).

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Loss of Memory B Cells During Chronic