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Research article
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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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research article
TheJournalofClinicalInvestigation http://www.jci.org
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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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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