Evaluation of vaccine efficacy in vitro

Evaluationofvaccineefficacyinvitro

usingBcells:Avalidapproach?

Name:MalteBorggreweSupervisors:AnkeHuckriede&GabrielaTapiaAbstract. Influenzavirus is the causeof annual “flu”epidemicsandoccasionalpandemics such as the H1N1 pandemic in 2009. Treatment of influenza isdifficult,which iswhy vaccination offers the best form of protection. Vaccinesconfer immunitymainlyby the inductionofahumoral response.ThishumoralresponseisfacilitatedbyBcells,whichproliferateanddifferentiatetomemoryBcells and antibody-secreting cells upon antigen exposure. Antigen-specificantibodies are able toprotect thehost froman infectionbybinding to surfaceproteins on the virus. Since influenza viruses alter their surface proteinsfrequently, there is a need for novel vaccines that protect from diverginginfluenzastrains.Vaccinecandidateandadjuvanttestingiscurrentlyperformedin animal models such as mice. However, animal experiments are laborious,expensive and translation to humans has always been a struggle. Here, thevalidityofanapproachoftestingvaccineefficacyinaninvitroBcellmodelwillbe discussed. To this end, the current status of research of in vitro B cellactivation and possible readouts will be reviewed and related to the B cellresponsefollowinginfectionandvaccinationinhumans.

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ListofabbreviationsAbbreviation MeaningAID Activation-inducedcytidinedeaminaseAPC Antigen-presentingcellASC Antibody-secretingcellBAFF BcellactivatingfactorBCR BcellreceptorCSR ClassswitchrecombinationDC DendriticcellELISA Enzyme-linkedimmunosorbentassayELISPOT Enzyme-linkedimmunospotassayHA HemagglutininHAI HemagglutinininhibitionIg ImmunoglobulinIIV InactivatedinfluenzavaccineIL InterleukinLAIV LiveattenuatedinfluenzavaccineNA NeuraminidasePB PlasmablastPC PlasmacellRT-qPCR Reverse-transcriptionreal-time

quantitativepolymerasechainreactionSHM SomatichypermutationTCR TcellreceptorTD TcelldependentTI TcellindependentTIV TrivalentinactivatedvaccineTLR Toll-likereceptorTNF Tumournecrosisfactor

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Tableofcontents

1Introduction 4

2Bcellresponsetoinfectionsandvaccinationsinhumans 6

2.1Bcellresponseduringinfluenzainfection 6

2.2Vaccination-mediatedBcellresponse 8

3InvitroBcellactivation 9

3.1NaïveBcellactivation 9

3.2MemoryBcells,plasmacellsandplasmablasts 10

3.3Antibodyresponse 11

3.4Classswitchrecombinationandsomatichypermutation 12

4TowardsevaluatingvaccineefficacyusingBcellcultures 14

5Conclusion 17

6Acknowledgements 17

7References 18

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1IntroductionInfluenzaviruscausessevererespiratorydiseaseinhumansandisthecauseoftheannual “flu”epidemics.1Differentstrainsof the influenzavirusexist,whichare defined by differences in their surface proteins hemagglutinin (HA) andneuraminidase (NA) (Fig. 1).2 The virus possesses a negative-strand RNAgenome,whichisseparatedintoeightsegments(Fig.1).ReplicationoftheRNAgenome isprone toerrors,whichcauses rapidantigenicdrift leading tominoralterations in the surface proteins.2 Moreover, the genome segments of twodistinct influenza strains can recombine to formanovel strain.Thisprocess iscalledreassortmentandleadstoantigenicshift,whichisthecauseforoccasionalpandemics suchas theH1N1pandemic (Swine flu) in2009.3The treatmentofinfluenza infections is difficult, which is why vaccinations provide the mostreliableformofprotection.4Influenzavaccinesconsistofvirus-derivedantigensthatstimulate theadaptiveimmune system. Inactivated influenza vaccines (IIV) and live attenuatedinfluenza vaccines (LAIV) are most commonly used (Box 1).5 These vaccinesmainly target thehumoral immune response, causingproductionof antibodiesspecific for HA or NA. Antigen-specific antibodies are able to bind the viralsurface proteins, thereby inhibiting entry of the virus into host cells, orfacilitatingtheclearanceofvirus-infectedcells.6ThehumoralresponseismainlydrivenbyBcells,whichareabletodifferentiateintoantibody-secretingcells(ASCs)uponantigenexposure.7Afterdevelopmentinthebonemarrow,naïveBcellscanbefoundcirculatinginbloodorassociatedwith lymphoid tissues, such as lymph node or spleen. When naïve B cellsencounteranantigen,theywillgetactivatedinpresenceorabsenceofThelpercells.8 T-independent (TI) activation causes differentiation into plasma cells(PCs),whereasT-dependent(TD)activationleadstodifferentiationintomemoryBcellsandPCs.9Beforeactivation,Bcellsproducethenon-switchedantibodiesIgMandIgD.Duringdifferentiation,Bcellsundergoclassswitchrecombination(CSR), which leads to expression of the switched antibodies IgG, IgA or IgE.10These antibodies differ in function. IgG, for example, is the predominant Igisotype found in the body, whereas IgA is the main secretory antibody andthereforeimportantinmucosalimmunity.10,11IgEprotectsfromparasiticworminfections and is associated with allergic reactions.11 Furthermore, B cellsexperience somatic hypermutation (SHM) during differentiation in order todevelop high affinity antigen-specific antibodies.10 Antigen-specific memory BcellsaremaintainedinthebloodandthebonemarrowandcanrapidlyexpandanddifferentiateintoPCsaftersecondaryantigenstimulation.7,12Currently used influenza vaccines facilitate antibody-mediated protectionagainst the variable head domain of HA or NA.5 However, seasonal antigenicdriftsandoccasionalantigenicshiftscreateaneedforthedevelopmentofnovelvaccinesthatgeneratelonglastingandbroadimmunity.Todate,animalmodelssuch as mice are mainly used to evaluate the functionality of new vaccinecandidatesoradjuvants.However,animalexperimentsareexpensive,laboriousand translation frommice to humanshasbeenprovendifficult.13 Therefore, ahumanmodel that investigates theefficacyofnewvaccines ishighlydesirable.Naturally, the possibilities to test vaccine candidates in humans are restricted,due to ethical and safety reasons. A different approach could be the use ofprimary human cell cultures. Since B cells and the humoral response are the

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maintargetsofvaccinesandthemainreadoutsforvaccineefficacy,establishinganinvitromethodusingBcellscouldadvancevaccinedevelopment.Albeit, such a B cell culture method for testing vaccine efficacy has not beendescribed or established yet. Therefore, this review will discuss whether thisapproachcouldofferavalidformofvaccinetesting.Forthatpurpose,theroleofB cells during natural influenza infections and vaccinations in humanswill bereviewed. Moreover, the current status of research concerning in vitroB cellactivationandreadouts foractivationwillbeaddressed.Basedonthe findings,the capability of vaccine-mediated B cell activation in vitro will be discussed,regardingvaccinetestinginhumansandpossiblereadouts.

Lofanoetal.2015Figure1.StructuralcompositionoftheinfluenzaAvirus.(A)TheinfluenzaAviruspossessesanegative-senseRNAgenome,which isdivided intoeightsegments.Amongotherproteins,thesesegmentsencodeforviralsurfaceproteinshemagglutinin(HA)andneuraminidase (NA). (B) HA contains a variable head domain and a conserved stemdomain.

Box1.InactivatedinfluenzavaccineandLiveattenuatedinfluenzavaccineInactivatedinfluenzavaccine(IIV)Inactivated vaccines are produced by growing viruses in embryonated chicken eggsand killing them using physical or chemical processes.14 This inactivation methodrenders thevirusnon-infectious.Usually, trivalentorquadrivalentvaccinesareused.Thismeans that the vaccine consists of two different influenzaA strains (H1N1 andH3N2) and one or twodifferent influenzaB strains.9 The use of different strains forvaccinesisnecessarytoprotectagainstthestrainthatcirculatesinaseason.14IIVsareinjectedintramuscularly.Liveattenuatedinfluenzavaccine(LAIV)As thename illustrates, liveattenuatedvaccinesconsistof livingviruses,whichhavebeenalteredinordertoreducetheirvirulence.14JustlikeIIVs,LAIVsusuallycomeastrivalent or quadrivalent vaccines. In contrast to IIVs, LAIVs are administeredintranasally,therebymimickingtherouteofanaturalinfluenzainfection.

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2Bcellresponsetoinfectionsandvaccinationsinhumans

2.1Bcellresponseduringinfluenzainfection

TheBcellresponseduringaninfluenzainfectioninhumanscanbedividedintothreemainreactions:aninnate-likeresponse,aTIresponseandaTDresponse.Theinnate-likeresponseismainlyfacilitatedbysecretionofnaturalantibodiesbyB1cells(Fig.2A).15B1cellsareinnateimmunecellsthatdonotdifferentiatetobecomeeffectorBcells.TheseinnatecellssecretepolyreactiveIgMantibodies,which are antigen-nonspecific, but are able to recognize a variety of differentmicrobialstructures includingviralsurfaceproteins.However, theclearanceofviruses by natural antibodies is not very efficient, which is why an adaptiveimmuneresponseisneededtodevelopvirus-specificantibodies.15The adaptive B cell response can be triggered in a TI and TD manner.9 TheprocessofTIactivationofnaïveBcellsusuallystartsbeforeTDactivationandyields the first antigen-specific antibodies. TI activation of B cells can befacilitatedthroughcross-linkingof theBCRe.g.byrepetitivepolysaccharides.16Such repetitive polysaccharides can be found in LPS and the influenza HAprotein.16,17 Moreover, B cells can get activated by toll-like receptor (TLR)stimulation (Fig. 2B), e.g. by single-stranded RNA or by unmethylated CpGDNA.16 TLRs are pattern recognition receptors that detect microbialsubstances.16 During TI activation, B cells can rapidly differentiate into PCs,which produce low affinity antibodies. TI activationhowever, does not lead todifferentiationintomemoryBcells(Fig.2B).9TDBcell activation leads to the inductionofhighlyantigen-specificantibodies(Fig.2C).9Thelpercellsgetactivated inthe lymphnodebydendriticcells thatcarryaviralantigen.ActivatedTcellsmigrateintotheBcellfollicle,wheretheyinteract with B cells that are specific for the same antigen. This interactionconsistsofbindingoftheTcellreceptor(TCR)totheMHCclassIImolecule,andCD40L to CD40 (Fig. 2C).9 After encountering T cell help, CSR takes place infollicularnaïveBcells.ThisallowsBcellstoaltertheirantibodyisotypefromIgDor IgM to IgG, IgE or IgA.10 Furthermore, T cell help induces SHM in variableregions of immunoglobulin DNA regions, leading to the development of highaffinityantibodies,specificfortheviralantigens.10The previously described processes that happen during an influenza infectionarewell established, butmainly based on animal experiments. In general, it isdifficult to study themechanismsof influenza infections inhumansbecauseofethicalandsafetyreasons.However, studieshavemadeaneffort toprofile thecytokineandantibody responsesof subjects suffering fromanatural influenzainfection,whichcangiveaninsightintothemechanismsofthehumanimmuneresponse.18 Itwasshownthatduring theacutephaseofan influenza infection,subjects developed fever and exhibited increased interferon (IFN) γ andinterleukin (IL) 6 responses.18 IFNγ primes the immune system against viralinfections and specifically induces B cells to undergo class switching to IgG-secreting cells.19 IL6 is a B cell stimulating cytokine that induces B cellproliferationanddifferentiation.19Furthermore, theamountof antigen-specificantibodies was measured.18 About 10 days after the onset of fever, antigen-specific IgM, IgAand IgGantibodies simultaneouslyand rapidly increased.Theantibodytitrespeaked2-3dayslater.18Withinthenext40days,antibodytitres

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graduallydecreased,butremainedelevatedcomparedtoinitialvalues.18Inmostsubjects,antigen-specificIgAtitreswerelowercomparedtoIgMandIgG.Summarized, the B cell response during influenza infections is a process thatinvolves several steps. Innate-like B cells can offer a first line of defence bysecretingnaturalantibodies,followedbyantigen-specificlowaffinityantibodiesduring T-independent activation. Germinal centre reactions finally result inhighlyspecificmemoryBcellsandASCs,whichsecretehighaffinityantibodiesresponsible for clearance of the infection. In infected humans, an increase ininflammatory cytokines and antigen-specific antibodies can be observed aftertheonsetoffever.

Figure 2. B cell response during influenza infection.A) B1 cells are innate-like Bcells, which secrete natural antibodies. These natural antibodies serve as a first linedefence against intruding viruses. B) The T-independent (TI) response is driven byspecific TI-antigens, such as highly repetitive hemagglutinin (HA) sequences. Thesesequences are able to crosslink the B cell receptor (BCR) to activate B cells. Other TIsignals include Toll-like receptor (TLR) agonists, which are able to activate B cells.ThesecellswillnotformmemoryBcells,butplasmacells(PCs)thatsecretelowaffinityantibodies.C)TheT-dependent(TD)responseis facilitatedbyproteinantigens,whichbindtheBCR.ThelpercellswillgiveanactivationsignaltoBcellsthroughMHC-Tcellreceptor (TCR) interaction and CD40-CD40L interaction. Activated B cells willdifferentiatetoformmemoryBcellsandPCsthatsecretehighaffinityantibodies.TLR=Toll-likereceptor,BCR=Bcellreceptor,TI=T-cellindependent,TD=T-celldependent,TCR=Tcellreceptor,MHC=Majorhistocompatibilitycomplex.

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2.2Vaccination-mediatedBcellresponse

Influenzavaccinationsattempttomimicanatural infection inhumans, therebyinducing an adaptive immune response that protects the host.2 This vaccine-mediated immune response is mainly driven by antigen-specific antibodies,whichoriginatefromBcells.Severalstudiesinvestigatedthehumoralresponsefollowingvaccination.ThesestudiesmainlyaimedtoprofiletheexpansionofBcells and theproductionof antigen-specific antibodies.Here, the findingsofexvivostudiesarepresentedandassociatedwiththemechanismofvaccinationandpossiblereadoutstodeterminevaccineefficacy.Exvivostudieshaveshownthatpriortovaccination,adultsandchildrenalreadyexhibit lowpercentages(>1%)of influenza-specificIgGmemoryBcells.20Aftervaccinationwith LAIV or TIV, all subjects showed a significant increase in IgGASCsandsomedevelopedanIgAASCresponse.IgAisofparticularinterest,sinceit is themajormucosal antibodyand therefore the first lineofdefenceagainstintruding influenza viruses.10 Furthermore, vaccination increased titres ofneutralizing antibodies significantly. Neutralising antibodies are able to bindviruses directly to facilitate their clearance or inhibit entry into host cells.10These findings indicate thata lownumberof influenza-specificmemoryBcellsexists prior to vaccination, which can expand rapidly to form ASCs.10,20Furthermore, antigen-specific and neutralising antibody titres appear to be anindicatortoassessvaccineefficacy.A different study investigated the clonal expansion of B cells followingvaccination.21 Here, ELISPOT assays and flow cytometry showed that antigen-specificIgGASCshavetheirorigininafewantigen-specificBcellclonesthatarepresent prior to vaccination.Although antigen-specificASCs arise rapidly aftervaccination, their presence is only transient. Specific memory B cells peakedapproximatelyoneweekafterASCsariseandweremaintainedforseveralweeksafter vaccination, shownby antigen-specific IgGELISA.22MeasuringmemoryBcells over a time period of 17 years revealed that some memory B cells aremaintainedseveralyearsfollowinganinfection.22BesidesantibodytitresandBcelldifferentiation,geneexpressionprofileswerefound recently to be a validmeasure and predictor of vaccine efficacy. It wasshownthatmRNAandproteinexpressionprofilesofsingleBcellschangeuponvaccination.23,24 The authors usedmicroarray datasets of PBMCs derived fromvaccinated subjects. For analysis, the fold change in expressionwas calculatedusingtheratiobetweenexpressionatday0(pre-vaccination)andday7(post-vaccination). Generally, immunoglobulin and proliferation gene sets wereenriched among PBMCs after vaccination, shown by single sample gene setenrichment analysis (ssGSEA).Moreover, up-regulationof these gene sets inBcellsthatwereisolatedaftervaccinationsuccessfullypredictedvaccineefficacybasedonhemagglutinininhibition(HAI)titresinthesubjects.24In summary, there are various factors that contribute to efficient vaccine-mediated protection from infection. Vaccines induce a rapid and transientproliferationofantigen-specificIgGASCs,andalaterandmorestableincreaseinspecific memory B cells. Furthermore, antigen-specific antibody titres can beusedtoassessvaccineefficacy.Interestingly,theabilityofavaccinetoinduceahumoral response can be predicted using the expression profiles of B cells.Therefore, this approach could be used as a predictive method to evaluatevaccineefficacy.

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3InvitroBcellactivation

3.1NaïveBcellactivation

To evaluate whether an in vitro B cell model can be used as method to testvaccine efficacy, the current status of research on B cell activation wasinvestigated.AlthoughtheroleofBcellsininfectionandvaccinationinhumansis largely understood, only a small number of studies has examined theactivationofBcellsinvitro.Lanzavecchia and colleagues published several studies that shed light on themechanismsofnaïveBcellactivationinculture.25-27Inthelate20thcentury,theauthorsestablishedafirstmodelofinvitronaïveBcellactivation.Accordingtothis study, a CD40L protein that mimics the T cell signal for B cell activationinduced moderate naïve B cell proliferation.26 This proliferation was largelyincreasedbyBCRengagementusinganti-Igantibodies.Basedontheseresults,atwo-signalmodelofnaïveBcellactivationwasputforward,consistingofCD40LandBCRstimulation.Severalyearslater,theauthorsfoundthatuponBCRactivationnaïveBcellsup-regulated the expression of TLR9 and TLR10, whichwasmeasured using RT-qPCR.25 Based on their findings, Lanzavecchia and colleagues investigated theroleofTLRsignallinginnaïveBcellactivation.27Proliferationassaysusingflowcytometry suggested that BCR stimulation and co-stimulation by either T cellsdirectlyorbyusingCD40Lmimicsgivesasignalforinitialproliferation,butnotforsurvivalanddifferentiation.27AfterBCRstimulation,TLRagonistsandTcellcognatehelpareneededinordertodriveextensivesurvivalandproliferationofnaïve B cells. Thus, BCR stimulation seems to be a requirement for TLRexpressionandstimulationinnaïveBcells.NaïveBcellsrespondtostimulationofTLRs1,2,6,7and9,whichisinlinewiththeTLRexpressionbyhumanBcells.TLR3/4 agonists however, fail to induce B cell proliferation.27 Nevertheless, BcellscanalsobestimulatedindirectlybyusingsupernatantsofDCcultures.Here,supernatants of TLR3/4 stimulated DCs (poly:IC and LPS) induced B cellproliferation.27 The addition of neutralizing antibodies against IL6 and IL12abrogatedthestimulatoryeffectofDCsupernatants.Furthermore, theadditionofIL6andIL12increasedBcellproliferation,indicatingthatcytokinessecretede.g.byDCscansimulateBcells.27Incontrasttothepreviousfindings,otherstudiessuggestthatnaïveBcellscanbeactivated invitro in theabsenceofBCRstimulation.28,29Theresultsof thesestudies indicate that naïve B cells exhibit a constitutive TLR9 expression,independentofBCRstimulation.28Hence,theauthorswereabletoactivatenaïveB cells in the presence of CD40L and the TLR9 agonist CpG alone.28,29 ELISAmeasurements showed that IL6, IL10 and tumour necrosis factor (TNF) αsecretion was enhanced in naïve B cells stimulated with CD40L and CpG.Additionally,stimulationofnaïveBcellswiththeTLR7agonistresiquimodledtoweakproliferationandstronginductionofIgMandIgGsecretion,measuredbyELISA.30 This suggests that TLR7 is constitutively expressed as well and thatTLR7stimulationisalsosufficientforBcellactivation.TheopposingfindingscanbeexplainedbyadifferentdiscriminationofnaïveBcell populations and by the use of different readouts for B cell activation.Lanzavecchia et al. isolated CD19+ B cells and used CD27, IgG and IgA for adoubleFACSsorting,whichyieldedanextremelyhighpurityofnaïveBcellsof

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>99.99%.27 In contrast, other studies used a negative selection procedureemployingmagneticbeadsandthusworkedwithanaïveBcellpurityof>98%.ThismeansthatthenaïveBcellpopulationcouldhavebeencontaminatedwithothercellssuchasmemoryBcells.AcontaminationwithmemoryBcellswouldexplainthefindingsofconstitutiveTLRexpressioninnaïveBcells.Furthermore,Lanzavecchia and collegues employed proliferation assays rather thanmeasuringcytokineorantibodysecretiontodeterminenaïveBcellactivation.AlthoughnaïveBcellactivationinvitrohasnotbeenstudiedextensively,severalpublications indicate that TLR signalling plays a crucial role in proliferation,differentiationandsurvivalofnaïveBcells. Insummary,uponBCRstimulationnaïveBcellsup-regulateTLRs.Antigen-specificBcellsthenneedtogetfurthersignals from T cells (CD40L) and TLR agonists, which leads to survival,proliferationanddifferentiation(Box2)(Fig.3).InabsenceofBCRstimulation,TLR9agonistsandCD40Ltreatmentresultsinproductionofcytokines,duetoaconstitutive TLR9 expression in naïve B cells (Fig. 3). Moreover, TLR7stimulationincreasesantibodysecretionbynaïveBcells(Fig.3).

Figure3.NaïveBcellactivation invitro. InpresenceofBCRsimulation,TLRagonistandCD40Ltreatmentleadstoproliferation,differentiationandsurvivalofnaïveBcells.InabsenceofBCRstimulation,TLR7agonistorTLR9agonistandCD40Laloneareabletoinduceanantibodyorcytokineresponse,respectively.TLR=Toll-likereceptor,Ag=Antigen,BCR=Bcellreceptor.

3.2MemoryBcells,plasmacellsandplasmablasts

In contrast to in vitro naïve B cell activation, memory B cells have differentactivation requirements in culture.MemoryB cells are antigen-specificB cells,which can expand rapidly in the presence of an antigen. It is also worthmentioning thatmost studies focused on using the TLR9 agonist CpG DNA tostudyinvitroBcellactivation.Therefore,mainlyCpG-mediatedBcellactivationwillbediscussedinthefollowingchapters.JustlikenaïveBcells,memoryBcellsrespondtoCpGinitiallybysecretingIL6.28In contrast to naïve B cells however, RT-qPCR revealed that memory B cells

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express TLRs constitutively, which is therefore independent of BCRstimulation.25,28 Thus, memory B cells started proliferating in the presence ofCpG only, without addition of anti-Ig, which was determined by proliferationassaysusingflowcytometry.25FurtherstudiestriedtoelucidatetheroleofTLRsignallinginmemoryBcells.Inastudyfrom2011,thedifferentiationpotentialofmemoryBcellsinresponsetoCpGandCD40Lwasinvestigated.31,32CulturingmemoryBcells inthepresenceofCpGgaveastrongactivationsignal,leadingtodifferentiationintoPCs,whichwas shown by flow cytometry and ELISPOT.31 Moreover, CpG stimulated IgMmemoryB cells to proliferate in the absence of other activation signals,whichwas assessed using flow cytometry.31,32Flow cytometry also revealed that in amemory B cell culture, CpG induced differentiation into PCs and plasmablasts(PBs)moreefficientlythanCD40L.32Inaddition,flowcytometryandexpressionprofiles indicated thatCD40L treatmentyieldedclassical circulatingCD20-PCsand PBs, whereas CpG promoted differentiation to resident CD20+ PCs andPBs.32Jungetal.focusedontheeffectofCpGDNAondifferenttypesofhumanBcells in vitro.33 They also observed that CpG (in presence of CD40L) is able topromotememoryBcelldifferentiationintoPCs.However,theyalsostudiedtheeffectofCpGonPCsincultureandfoundthatPCsproliferateinresponsetoCpG,IL2andIL10.In summary, theactivation requirements formemoryB cells are less stringentcomparedtothosefornaïveBcells,sincememoryBcellsreactstronglytoCpGby proliferating and differentiating (Box 2). This could be explained by theconstitutive expression ofmost TLRs inmemory B cells. Interestingly, CD40L-mediatedmemoryBcellactivationpromotesdifferentiationintocirculatingPCsandPBs,whereasCpGgeneratesresidentPCsandPBs.Therefore,studyingbothcirculatingandresidentPCsandPBsispossibleusingmemoryBcellcultures.

3.3Antibodyresponse

Previously,itwasshownthatmemoryBcellscanbeactivatedinvitrousingCpGand/orCD40L,whichleadstoproliferationanddifferentiationintoPCsandPBs.ThemaintaskofactivatedanddifferentiatedmemoryBcellsisthesecretionofantibodies. Therefore, it is essential to study and profile the in vitro antibodyresponse,whichcouldserveasanindicatorforBcellactivity.Using an ELISPOT assay, Bernasconi et al. established that memory B cellsrespondtoCpGbysecretingIgM,IgGandIgA.31Incontrast,naïveBcellsseemtorequire BCR stimulation in addition to CpG for secretion of IgM and IgG.However, Glaum et al. found that naïve B cells respond to a TLR7 agonist(resiquimod) by secretion of IgM and IgG.30 These opposing findings could beexplained by diverging expression of different TLRs in naïve B cells, or bycontaminationwithmemoryBcellsasdescribedpreviously.Ingeneral,memoryBcellsseemtohaveahigherantibodysecretingpotentialcomparedtonaïveBcells.SincememoryBcellsseemtoresponddifferentlytoCD40LandCpGasexplainedbefore,anotherstudyexaminedwhetherthisdifferencealsoappliestoantibodysecretion.32Here,ELISArevealedthatmemoryBcellssecretesignificantlymoreIgG,IgAandIgMinresponsetoCpGcomparedtoCD40L.Theyalsoinvestigated

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theantibodysecretionofPBsthatwheregeneratedfromCD40L-treatedmemoryBcellsandwerethereforeclassicalcirculatingCD20-PBs.CulturingthosePBsinthepresenceofCpGsignificantlyincreasedthesecretionofIgM,inducedaminorIgA increase, and a decrease in IgG secretion.32 Thus, CpG seems to inducePBdifferentiation into IgM secreting PCs rather than IgA or IgG secreting PCs.32Furthermore, these studies indicate that memory B cell activation anddifferentiationcaneasilybeassessedusingELISAtomeasureantibodysecretion.A different study investigated the effect of CpG on transitional B cells.34TransitionalBcellsaresimilartonaïveBcells,yettheyfeatureamoreimmaturephenotype. Upon CpG stimulation, transitional B cells, which express IgM andIgD, are able to differentiate into plasma cells that secrete IgM and IgG. ThesefindingsarevaluablebecauseIgMandIgDaresecretedbynon-switchedBcells,butinordertoproduceIgG,IgAorIgE,aBcellhastoundergoCSR.Thus,intheabsenceofTcellhelp,BcellsseemtobeabletoinduceCSR invitro.Therefore,the following chapter will elucidate the cellular mechanisms and signallingcascadesinvolvedinIgclassswitchinginBcellsinvitro.3.4Classswitchrecombinationandsomatichypermutation

Inhumans,naïveBcellsthatencounteranantigenwillundergoCSRingerminalcentrestoachieveisotypeswitchingfromIgMorIgDtoIgG,IgAandIgE.10ThisprocessisusuallyperformedinthepresenceofT-cellhelp,howeverTIactivationofB cells canalso lead toCSR.10Furthermore, thesedifferentiatingB cellswillgenerateSHMinordertoproducehighaffinityantigen-specificantibodies.SHMdescribes theprocessofgeneratingmutations in the Igvariableregionson theDNA,which increases antigen-specificity and affinity.10 Both SHM and CSR arefacilitatedbyactivation-inducedcytidinedeaminase(AID),whichcausesaDNAnick. This nick is either repaired by an error-prone polymerase or leads to adouble-strand break (Fig. 4).35 Error-prone repair causes SHM, whereas adouble-strandbreakleadstoCSR(Fig.4).35Asdescribedpreviously,CpGseemstodriveclassswitchingofBcellsinordertogenerate IgG-secreting PCs.34 Therefore, class switch recombination canprobably be achieved in vitro in the absence of T cell help. The same studymeasuredmRNAlevelsofAIDusingRT-qPCR,whichisanenzymeessentialforCSR (Fig. 4). They found that transitional B cells up-regulate AID upon CpGstimulation,whichcouldexplaintheinductionofIgG-secretingPCs.Theresultsof a different study support these findings.36 Here, the authors additionallyshowedthatBcellactivatingfactor(BAFF)substantiallyincreasestransitionalBcellproliferation,differentiationandCSR.However,notonlytransitionalBcellshavebeenstudiedforthepresenceofCSRandAID expression, but also activated naïve B cells. Lanzavecchia etal. foundthatwhennaïveBcellsaretreatedwithacombinationofanti-Ig,CD40LandCpGtoinducedifferentiation,theystarttoexpressAIDfourdaysafterstimulation.27Inthisstudy,theauthorsalsorevealedthatTLRstimulationisessentialforAIDexpressioninnaïveBcellsandalsoforsuccessfulisotypeswitching.NaïveBcellstreated with all three signals were stained for surface IgG and IgA, andsubsequentflowcytometryshowedthatactivatednaïveBcellsexpressIgGandIgAinvitro.

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Interestingly,noneof thestudiesmentionedabovewasable todetect IgE in invitroclassswitchedBcells,althoughclassswitchingtoIgEisalsodependentonAIDexpression.Theanswertothis finding isprovidedbyastudyfrom2003.37The results of this publication show that CpG inhibits IgG1 and IgE classswitchingatleastinmouse-derivedpurifiedBcells.ArecentstudyspecificallyaddressedAID-mediatedCSR innaïveBcells inaTImodel with special focus on signalling pathways that are used.16 The authorsfound that BCR signalling synergizes with TLR signalling to induce AIDexpression.Incombination,BCR-TLRsignallingactivatesthenon-canonicalandcanonicalNF-κB pathways in a TImanner,which leads to AID expression andconsequentlyCSR. In thismodel,TLR1/2,TLR7orTLR9signallingsuccessfullyinducedCSR.Furthermore,LPSasaTLR4ligandinducedCSR,sinceitisabletostimulate the BCR as well as the TLR4 receptor.16 In the absence of BCRstimulation,TLRsignallingalonewasabletoinduceCSRinefficiently,whichwasshownbymeasuringsurfaceIgG.Therefore,theresultsunderlinethesynergyofBCRandTLRsignalling.Besides for CSR, AID is also responsible for SHM in B cells. However, SHM inactivated B cells in vitro has not been studied extensively yet. One studyinvestigated the ability of CpG to induce SHM during differentiation oftransitionalBcellsinvitro.38InresponsetoCpG,transitionalBcellsproliferatedanddifferentiatedasdiscussedearlier.Additionally,CpGintroducedSHMinthevariableregionsoftheheavychainofIg.SHMwasdeterminedbysequencingtheVHregionandcomparingittosequencesinthe”ImMunoGeneTics”informationsystem. The authors also compared these SHM to naturally occurring SHM inhuman peripheral blood-derived memory B cells and found that mutationfrequencyandtypearecomparable.Thus,itispossibletoactivatenaïveBcellsandtransitionalBcellsinvitrowhichleads to proliferation and differentiation. Furthermore, it is interesting to seethat in vitro activation also induces CSR and SHM in B cells, which leads toexpressionofmostIgisotypesandtothedifferentiationintoswitchedmemoryBcells,PCsandPBs.

Box2.InvitroBcellactivation:AnoverviewNaïveBcellactivationNaïveBcellscanbeactivated invitroeitherinpresenceorabsenceofBCRstimuli.Inabsence of BCR activation, CD40L and TLR9 agonists will induce naïve B cells tosecretecytokinessuchasIL6.28,29AfterBCRstimulation,naïveBcellswillup-regulateTLRs and respond strongly to CD40L and TLR agonists by proliferating anddifferentiating.27 Furthermore, in vitro activation of naïve B cellswill cause them toundergoCSRandSHM.16MemoryBcellactivationIncontrasttonaïveBcells,memoryBcellshavelessstringentactivationrequirements.MemoryBcellswillproliferateanddifferentiatetoPCsorPBsinthepresenceofeitherCD40LorTLRagonists,oracombinationofboth.25MemoryBcellsthatundergoCSRduringdifferentiationareabletosecreteIgGandIgA.31 Interestingly, treatmentwithCD40LorTLRagonistsyieldsdifferenttypesofPCsandPBs:classicalcirculatingCD20-orresidentCD20+PCsandPBs,respectively.32

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Figure4.Mechanismsofclassswitchrecombinationandsomatichypermutation.Duringtranscription,activation-inducedcytidinedeaminase(AID)convertscytosinetouracil on single-stranded DNA. Uracil is recognised as a wrong base and excised byUracilN-glycosylase(UNG),leadingtoasingle-strandnick.Thenickcanberepairedbyerror-pronemismatchrepair,whichresults insomatichypermutation(SHM).Thenickcan also cause double-strand DNA break, which is repaired through non-homologousend-joining (NHEJ), leading to class switch recombination (CSR). AID = Activation-inducedcytidinedeaminase,UNG=UracilN-glycosylase,SHM=somatichypermutation,NHEJ=non-homologousend-joining,CSR=Classswitchrecombination.

4TowardsevaluatingvaccineefficacyusingBcellculturesTo answer the question whether evaluating vaccine efficacy by using B cellculturesisavalidapproach,itisessentialtoconsiderseveralaspects.TheabilityofinvitroBcellactivationtomimictheeffectofvaccinationinhumansmustbeexamined. Furthermore, it is essential to discuss possible readouts anddetermineavalidmodelofaBcellculturesystem.Itisimportanttonotethatto

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date,nostudieshavebeenpublishedthatattemptedestablishingaBcellinvitromodelforvaccinecandidatetesting.Vaccineefficacyinhumanscanbeassessedgenerallyintwodifferentways.First,adoubleblind,randomized,clinicalcontrolledtrialcanassessvariablessuchasinfection incidence and hospitalisation. Evaluating the disease attack rate ofvaccinatedcompared tounvaccinatedsubjects consequently reflects theabilityof a vaccine to confer immunity against an infection. Naturally, this approachcannot be used in B cell cultures. Second, a serological evaluation of vaccineefficacy offers a method that can be applied to in vitro systems as well.Serologicalassaysincludemeasuringantigen-specificantibodytitressuchasIgG,IgMandIgAdirectly.3,20Moreover,HAIassayscanbeused,whichdeterminetheamountofHA-specificantibodies inafunctionalassay.24,39Therefore,assessingvaccine efficacy using antibody responses offers a valid form of testingimmunogenicityinvitro.Recently, a different strategy of anticipating vaccine efficacy has beendescribed.24Theauthorsusedtwoexistingmicroarraydatasets,whichmeasuredgeneexpressionprofilesofPBMCsfollowingvaccination.Here,theyfoundthatIgandproliferationgenesetsareup-regulatedinPBMCs7daysaftervaccination.Furthermore,theyfoundastrongassociationofexpressionofthesegenesetsinB cells with HAI titres in vaccinated subjects. Up-regulation of Ig andproliferationgenesetsinBcellswasthereforepredictiveforhighHAItitresandvaccineefficacy.24Thus, inadditiontoserologicalassays,expressionprofilesofsingleBcellscouldbeusedtodeterminevaccineefficacy,whichisapplicabletoBcellculturesaswell.HavingdiscussedthepossiblereadoutsandassaysthatcoulddeterminevaccineefficacyinBcellcultures,itisimportanttoexaminepotentialmodelsofinvitroBcell activation. First of all, when establishing a B cell culture, primary B cellsderived fromhumanperipheralblood shouldbeusedpreferentially.ObtainingprimaryBcellsfromvariousindividualswouldallowtestingvaccinesinahighlyheterogeneouspopulationwithdistinctgeneticandenvironmentalbackground.An optimal approach would be to immortalise these cells to form cell lines.Primarycellsonlydivideadefinitenumberoftimesafterwhichtheywillgointosenescence. By introducing mutations or viral genes (e.g. Epstein-Barr Virus),primaryBcellscanbeimmortalised.Thismeansthatthatthesecellscandivideinfinitely without entering senescence.40 Of course, this would make vaccinetesting easier, since the same donors could be used for each test and B cellswouldnothavetobeisolatedfrombloodrepeatedly.However,itisknownthatimmortalisation of cells will change their genetic composition and thereforebehaviour. Thus, immortalised B cell lines should be extensively evaluated fortheiruseinvaccinetesting.Second,asuitable typeofBcellhas tobechosen,whichcouldbenaïveBcellsand/ormemoryBcells.UsingnaïveBcellswouldbeadvantageous in termsofantigen-specific activation, since BCR stimulation is required for activation.Therefore,naïveBcellscouldbestimulatedinvitrousinganantigen(orvaccine)and would proliferate and differentiate following CD40L and TLR agonisttreatment.27However,studieshaveonlyusedanti-IgtostimulatetheBCRsofarandnotantigens,whichiswhyantigen-specificactivationofnaïveBcellswouldhavetobeinvestigatedbeforehand.DuetotheabilityofBcellstoundergoCSRandSHMinvitro,itisverylikelythatuponantigenstimulationnaïveBcellswill

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differentiate into antigen-specific memory B cells. The presence of antigen-specific B cells could easily be determined by measuring antigen-specificantibody titres or surface antibodies using ELISA and/or flow cytometry.However, antigen-mediated naïve B cell activation would probably be muchweaker compared to anti-Ig activation, since only a small fraction of antigen-specificBcellswouldrespond.ActivationofnaïveBcellscouldbeassessedusingvariousdifferentapproaches.For example, proliferation assays using flow cytometry and ELISA tomeasuresecretion of IL6 or TNFα could be employed to determine naïve B cellactivation.28,29SincenaïveBcellssecreteIgMandIgGfollowingactivation,ELISAcouldalsobeusedtodetecttheseantibodiesasareadout.30Additionally,naïveBcells express TLRs only after BCR stimulation, so that up-regulation of TLRswould be an indicator for antigen-specific B cell activation. It has also beenshown previously that in vitro activation of naïve B cells leads to CSR andSHM.16,27Therefore,theexpressionofAIDcouldbemeasuredusingRT-qPCRasareadoutfornaïveBcellactivation,CSRandSHM.Another suitable approach would be the use of memory B cells. As explainedearlier,uponvaccinationasmallfractionofpre-existingantigen-specificmemoryB cells proliferates and differentiates in response to the antigen in humans.21Isolating the whole population of memory B cells from blood of subjects andstimulating thesememoryB cells invitrowould lead to an extensive responseincluding antigen-specific antibody secretion.Readouts for this response couldbetheuseofproliferationassaysandflowcytometrytodeterminethedegreeofdifferentiation into PCs and PBs. Furthermore, the amount and antigen-specificityofantibodiescouldbemeasuredtoexaminetheefficacyofvaccines.ApossibilitywouldalsobetousebothnaïveBcellsandmemoryBcellsfromonedonorandaccessvaccineefficacy separately inboth.This approach couldgiveinformationaboutthemodeofactionofthevaccine.Forexample,ifnaïveBcellsdo not react strongly to a vaccine, whereas memory B cells do, this could beevidenceforastrongTLR-activatingcomponentofavaccine.Interestingly,ahuman-derived invitromodelthatshouldenableresearcherstotest vaccine efficacy was introduced recently.41 VaxDesign, a biotechnologycompany, established a technology called Modular IMmune In vitro Construct(MIMIC).Inthisapproach,DCsandTcellsareusedinaco-culturetogetherwithBcells.Theculturesystemisoughttoresembleagerminalcentreinthelymphnode.Specifically, theuse for influenzavaccine testinghasbeenexamined inacase study clinical trial. In this case study, antibody titres in response to thevaccine invitroalmost resembled findings inhumans.41This system illustratesthatinvitromodellingofthehumanimmunesystemispossibletosomeextent.ItwillbeinterestingtoexaminethepotentialuseofprimaryBcellsonly,sincetheantibodyresponsetoavaccineshouldbeevaluablewithouttheuseofDCsandTcells.Basedonthisdiscussion,theuseofprimaryBcellstoevaluatevaccineefficacycouldbeavalidapproach.However,establishingsuchasmodelwillbedifficult,sincemanyvariableshavetoberegarded.

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5ConclusionTheinitialquestionwaswhetherevaluatingvaccineefficacyinvitrousingBcellscouldbeavalidapproach.On thebasisof theprecedingdiscussion, suchan invitro B cell system could probably be established to allow vaccine efficacytesting.Establishing a primary B cell in vitro system to evaluate vaccine efficacy ofcandidates can have several advantages. Apparently, the use of invivomodelscould potentially be reduced, which is beneficial as animal experiments arelaborious and expensive. Moreover, translation from animals to humans hasalwaysbeenastruggle.Thus,manyvaccinecandidates(oradjuvants)thatweresuccessful inanimalstudiesturnedout tobe inapplicabletohumans inclinicaltrials.Therefore, theuseofahuman-based invitrosystemmightsmoothenthestepfromanimalexperimentstohumantrials.AnotheradvantagewouldbethatBcellsfrommanydifferentsubjectscouldbeused.Consequently,vaccinescouldbe tested in a highly heterogeneous population with distinct genetic andenvironmentalbackground.Furthermore,invitrosystemsallowrapidscreeningofmanydifferentvaccinecandidatesandadjuvants,whichisdifficulttoaddressinanimalmodels.Ofcourse,testingvaccineefficacyinBcellcultureswouldhavelimitations.Thebiggestlimitationwouldbethatinvitrosystemsneverreflectinvivoconditionsperfectly.Thehumanimmunesystemishighlyversatileandischaracterisedbythe interaction of a variety of immune cells. Therefore, an in vitro system isprobably an oversimplification and thus cannot replace in vivo models.Additionally, it will be difficult to establish a valid in vitro system, especiallybecause there is a lack of studies, which investigated an antigen-specificactivationofhumanBcellsinculture.Albeit, an in vitro B cell model should not be regarded as a replacement foranimalmodelsorclinical trials.More likely, thisapproachhasthecapability tosmoothenthestepbetweenanimaltestingandhumanclinicaltrials.Inconclusion,thepotentialuseofBcells invitro totestvaccineefficacyshouldbe pursued, since it offers great advantages and will also shed light on themechanismsunderlyingBcellactivationandfunctionality.6AcknowledgementsIwouldliketotakethisopportunitytooffermanythankstoAnkeHuckriedeandGabrielaTapia,whoseguidanceandsupportwasessentialforwritingthisessay.

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7References

1.BeigelJH.Concisedefinitivereview:Influenza.CritCareMed.2008;36(9):2660-2666.

2.LofanoG,KumarA,FincoO,DelGiudiceG,BertholetS.Bcellsandfunctionalantibodyresponsestocombatinfluenza.FrontImmunol.2015;6:336.

3.LiGM,ChiuC,WrammertJ,etal.PandemicH1N1influenzavaccineinducesarecallresponseinhumansthatfavorsbroadlycross-reactivememoryBcells.ProcNatlAcadSciUSA.2012;109(23):9047-9052.

4.PolandGA,JacobsonRM,OvsyannikovaIG.Influenzavirusresistancetoantiviralagents:Apleaforrationaluse.Clinicalinfectiousdiseases:anofficialpublicationoftheInfectiousDiseasesSocietyofAmerica.2009;48(9):1254-1256.

5.WongS,WebbyRJ.Traditionalandnewinfluenzavaccines.ClinMicrobiolRev.2013;26(3):476-492.

6.HeW,MullarkeyCE,MillerMS.MeasuringtheneutralizationpotencyofinfluenzaAvirushemagglutininstalk/stem-bindingantibodiesinpolyclonalpreparationsbymicroneutralizationassay.Methods.2015;90:95-100.

7.KurosakiT.B-lymphocytebiology.ImmunolRev.2010;237(1):5-9.

8.RothaeuslerK,BaumgarthN.B-cellfatedecisionsfollowinginfluenzavirusinfection.EurJImmunol.2010;40(2):366-377.

9.WaffarnEE,BaumgarthN.ProtectiveBcellresponsestoflu--nofluke!JImmunol.2011;186(7):3823-3829.

10.LaffleurB,Denis-LagacheN,PeronS,SiracC,MoreauJ,CogneM.AID-inducedremodelingofimmunoglobulingenesandBcellfate.Oncotarget.2014;5(5):1118-1131.

11.SchroederHW,Jr,CavaciniL.Structureandfunctionofimmunoglobulins.JAllergyClinImmunol.2010;125(2Suppl2):S41-52.

12.ParamithiotisE,CooperM.MemoryBlymphocytesmigratetobonemarrowin humans.ProceedingsoftheNationalAcademyofSciences.1997;94(1):208-212.

13.MakIWY,EvaniewN,GhertM.Lostintranslation:Animalmodelsandclinicaltrialsincancertreatment.AmericanJournalofTranslationalResearch.2013;6(2):114-118.

14.HannounC.Theevolvinghistoryofinfluenzavirusesandinfluenzavaccines.ExpertRevVaccines.2013;12(9):1085-1094.

15.GriffinDO,HolodickNE,RothsteinTL.HumanB1cellsinumbilicalcordandadultperipheralbloodexpressthenovelphenotypeCD20+CD27+CD43+CD70-.TheJournalofExperimentalMedicine.2011;208(1):67-80.

16.PoneEJ,ZhangJ,MaiT,etal.BCR-signallingsynergizeswithTLR-signallingforinductionofAIDandimmunoglobulinclass-switchingthroughthenon-canonicalNF-kappaBpathway.NatCommun.2012;3:767.

19

17.WileyDC,SkehelJJ.Thestructureandfunctionofthehemagglutininmembraneglycoproteinofinfluenzavirus.AnnuRevBiochem.1987;56:365-394.

18.HuangR,ZhangL,GuQ,etal.ProfilesofacutecytokineandantibodyresponsesinpatientsinfectedwithavianinfluenzaAH7N9.PLoSOne.2014;9(7):e101788.

19.VazquezMI,Catalan-DibeneJ,ZlotnikA.Bcellsresponsesandcytokineproductionareregulatedbytheirimmunemicroenvironment.Cytokine.2015;74(2):318-326.

20.SasakiS,JaimesMC,HolmesTH,etal.Comparisonoftheinfluenzavirus-specificeffectorandmemoryB-cellresponsestoimmunizationofchildrenandadultswithliveattenuatedorinactivatedinfluenzavirusvaccines.JVirol.2007;81(1):215-228.

21.WrammertJ,SmithK,MillerJ,etal.Rapidcloningofhigh-affinityhumanmonoclonalantibodiesagainstinfluenzavirus.Nature.2008;453(7195):667-671.

22.PinnaD,CortiD,JarrossayD,SallustoF,LanzavecchiaA.ClonaldissectionofthehumanmemoryB-cellrepertoirefollowinginfectionandvaccination.EurJImmunol.2009;39(5):1260-1270.

23.HoekKL,SamirP,HowardLM,etal.Acell-basedsystemsbiologyassessmentofhumanbloodtomonitorimmuneresponsesafterinfluenzavaccination.PLoSOne.2015;10(2):e0118528.

24.TanY,TamayoP,NakayaH,PulendranB,MesirovJP,HainingWN.GenesignaturesrelatedtoB-cellproliferationpredictinfluenzavaccine-inducedantibodyresponse.EurJImmunol.2014;44(1):285-295.

25.BernasconiNL,OnaiN,LanzavecchiaA.Arolefortoll-likereceptorsinacquiredimmunity:Up-regulationofTLR9byBCRtriggeringinnaiveBcellsandconstitutiveexpressioninmemoryBcells.Blood.2003;101(11):4500-4504.

26.LaneP,BrockerT,HubeleS,PadovanE,LanzavecchiaA,McConnellF.SolubleCD40ligandcanreplacethenormalTcell-derivedCD40ligandsignaltoBcellsinTcell-dependentactivation.JExpMed.1993;177(4):1209-1213.

27.RuprechtCR,LanzavecchiaA.Toll-likereceptorstimulationasathirdsignalrequiredforactivationofhumannaiveBcells.EurJImmunol.2006;36(4):810-816.

28.CognasseF,Hamzeh-CognasseH,LafargeS,etal.IdentificationoftwosubpopulationsofpurifiedhumanbloodBcells,CD27-CD23+andCD27highCD80+,thatstronglyexpresscellsurfacetoll-likereceptor9andsecretehighlevelsofinterleukin-6.Immunology.2008;125(3):430-437.

29.WagnerM,PoeckH,JahrsdoerferB,etal.IL-12p70-dependentTh1inductionbyhumanBcellsrequirescombinedactivationwithCD40ligandandCpGDNA.JImmunol.2004;172(2):954-963.

30.GlaumMC,NarulaS,SongD,etal.Toll-likereceptor7-inducednaivehumanB-celldifferentiationandimmunoglobulinproduction.JAllergyClinImmunol.2009;123(1):224-230.e4.

20

31.BernasconiNL,TraggiaiE,LanzavecchiaA.MaintenanceofserologicalmemorybypolyclonalactivationofhumanmemoryBcells.Science.2002;298(5601):2199-2202.

32.Geffroy-LuseauA,ChironD,DescampsG,JegoG,AmiotM,Pellat-DeceunynckC.TLR9ligandinducesthegenerationofCD20+plasmablastsandplasmacellsfromCD27+memoryB-cells.FrontImmunol.2011;2:83.

33.JungJ,YiAK,ZhangX,ChoeJ,LiL,ChoiYS.DistinctresponseofhumanBcellsubpopulationsinrecognitionofaninnateimmunesignal,CpGDNA.JImmunol.2002;169(5):2368-2373.

34.CapolunghiF,CascioliS,GiordaE,etal.CpGdriveshumantransitionalBcellstoterminaldifferentiationandproductionofnaturalantibodies.JImmunol.2008;180(2):800-808.

35.deVillartayJP,FischerA,DurandyA.Themechanismsofimmunediversificationandtheirdisorders.NatRevImmunol.2003;3(12):962-972.

36.UedaY,LiaoD,YangK,PatelA,KelsoeG.T-independentactivation-inducedcytidinedeaminaseexpression,class-switchrecombination,andantibodyproductionbyimmature/transitional1Bcells.JImmunol.2007;178(6):3593-3601.

37.LiuN,OhnishiN,NiL,AkiraS,BaconKB.CpGdirectlyinducesT-betexpressionandinhibitsIgG1andIgEswitchinginBcells.NatImmunol.2003;4(7):687-693.

38.AranburuA,CeccarelliS,GiordaE,LasorellaR,BallatoreG,CarsettiR.TLRligationtriggerssomatichypermutationintransitionalBcellsinducingthegenerationofIgMmemoryBcells.JImmunol.2010;185(12):7293-7301.

39.HirotaY,KajiM,IdeS,etal.Antibodyefficacyasakeenindextoevaluateinfluenzavaccineeffectiveness.Vaccine.1997;15(9):962-967.

40.TosatoG,CohenJI.Generationofepstein-barrvirus(EBV)-immortalizedBcelllines.CurrProtocImmunol.2007;Chapter7:Unit7.22.

41.DrakeIIIDR,SinghI,NguyenMN,etal.Invitrobiomimeticmodelofthehumanimmunesystemforpredictivevaccineassessments.DisruptiveScienceandTechnology.2012;1(1):28-40.