Scientificbackground

Activationoftheimmunesystem
The2011NobelPrizeinPhysiologyorMedicineisawardedwithonehalfjointlytoBruceA. BeutlerandJulesA.Hoffmannfortheirdiscoveriesconcerningtheactivationofinnate immunity,andtheotherhalftoRalphM.Steinmanforhisdiscoveryofthedendriticcelland itsroleinadaptiveimmunity(1).TheworkoftheNobelLaureateshasrevolutionizedour understandingoftheimmunesystem.Asaconsequenceoftheirdiscoveries,newfieldsof researchhaveopenedupthatholdthepromisetoimprovevaccinationandtreatment againstinfection,cancerandinflammatorydiseases. Thediscoveries JulesHoffmannandcollaboratorsusedthefruitfly,Drosophilamelanogaster,asamodel systemtostudyfundamentalaspectsofhowourfirstlineofdefenseagainstmicrobes (innateimmunity)isinduced.Hoffmanndemonstratedthataparticulargene,denoted Toll,wasnecessaryforfruitfliestofightafungalinfection(2).Heconcludedthatthe productoftheTollgenewasinvolvedinsensingpathogenicmicroorganismsandtriggering hostdefenseagainstthem.BruceBeutlerandcollaboratorsusedamousemodeltosearch foragenethatactivatedinnateimmunitywhenexposedtoabacterialcomponent (lipopolysaccharide;LPS).Beutlerswork,throughtheidentificationofamutatedTolllike gene,Tlr4,inLPSresistantmice(3),unraveledthefunctionofTolllikereceptor(TLR)4asa mammaliancounterpartofDrosophilaTollinresponsetoinfection.Takentogether,these twodiscoveriesuncoveredamolecularsensorsystem(Toll/TLR),sharedbyinsectsand mammals,whichisessentialtoinducethefirstlineofdefenseagainstmicrobes. RalphSteinmanstudiedtheimmunesysteminmice,aimingtounderstandwhichcellsare importantfortheactivationoftheadaptiveimmunesystem,i.e.,thesecondlineofimmune
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defensewhereTandBlymphocytesaretriggeredtomountefficientresponsesagainst pathogenicmicrobes.Steinmandiscoveredacellwithadendriticappearanceinlymphoid organsofmice(4),andinsubsequentstudiesshowedthatthiscellhadanexceptionalability toactivateTlymphocytes(5).Hetermedthecellthedendriticcell(DC).Throughaseries ofingeniousexperimentsperformedoverseveralyears,Steinmandiscoveredafunctional systeminwhichmaturationofDCsrepresentsacrucialprocessinenablingtheactivationof Tcellresponses(6).WorkbySteinmanandotherscientistshasshownthatsignalsmediated bymoleculesoftheinnateimmunesystem,includingtheTLRs,inducethematurationofDC, thusprovidingalinkbetweeninnateimmunityandadaptiveimmunity. Earlierresearchandseminaldiscoveriesinimmunology Humansaswellasallotherspeciesaredependentonefficientdefensesystemsagainst invadingmicroorganismsfortheirsurvival.Researchontheimmunesystemhas consequentlybeenofgreatimportanceforourunderstandingofhowwecandefend ourselvesagainstmicroorganismstosurvivetheirthreat.Thisresearchhasalsoledtonovel diagnosticsandtherapies. AnumberofdiscoverieswithinthefieldofimmunologyhavebeenawardedtheNobelPrize inPhysiologyorMedicine(1).TheveryfirstNobelPrize,in1901,wasgiventovonBehring forhisstudiesofprotectionagainstDiphtheriabyantibodytransfer(atthetimecalledserum therapy).The1908NobelPrizetoEhrlichandMechnikovwasthefirsttorecognizethe existenceoftwodifferentarmsoftheimmunesystem,i.e.,afirstlineofcellulardefense thatinvolvesmacrophages(Mechnikov)andasecondlineofhumoraldefensethat involvesantibodies(Ehrlich).Itwaslaterrealizedthattherearecellularandhumoralfactors involvedbothinthefirstlineandinthesecondlineofdefense,andthetermsinnate immunityandadaptiveimmunityforthesetwolevelsgraduallyemerged.Thatthe immunesystemhasacapacitytotolerateselfandstrikeefficientlyagainstnonselfthrough adaptiveimmunereactionswasthesubjectofanotherNobelPrize(BurnetandMedawar 1960).Genesandmoleculesofthemajorhistocompatibilitycomplex(MHC)thatcontrol immuneandtransplantreactionswerediscoveredbyBenacerraf,DaussetandSnell(Nobel Prize1980).ThesameMHCmoleculesweresubsequentlyfoundtodirecttheadaptive
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immunedefenseagainstmicrobessuchasviruses(DohertyandZinkernagel;NobelPrize 1996).Furtherdiscoveriesonthestructureandgenerationofantibodieshavetaughtushow thesemoleculescanbegeneratedwithanenormousdiversity,andhowtheycanrecognize andkillmyriadsofextracellularmicroorganisms(NobelPrizetoEdelmanandPorter1972, andtoTonegawa1987).Theclonalpropertiesofadaptiveimmuneresponsesmadepossible thegenerationofmonoclonalantibodies(Jerne,KhlerandMilstein;NobelPrize1984), whichopenedthedoorforawholenewgenerationofdiagnosticsandtherapies. Butdespitethisprogress,manyfeaturesoftheimmunesystemhaveremainedpoorly understood.FundamentalproblemsthathavebeenaddressedbythisyearsNobellaureates aretheactivationofinnateimmunityandtheroleofaccessorycellsintheactivationof adaptiveimmunity. Previousprogressandchallengesconcerningtheinnateimmunesystem Afirstlineoftheimmunedefense,alsocalledtheinnate,naturalorinbornimmune system,isimportantforsurvivalinallorganismsthatcanbeattackedbymicroorganismsin theirenvironment.Althoughsomecomponentsoftheinnateimmunesystem,including severalcellsaswellassolublemolecules,hadbeencharacterizedbefore,therewasuntilthe mid1990iesacruciallackofknowledgeregardingtheactivationofthissystem.Inparticular, itwasunclearhowitscellssenseandbecomeactivatedbymicrobialcomponents.A prevailingparadigmwasthatcellsoftheinnateimmunesystemareactivatedwithoutany specificityorstimulatingtriggers.Indeed,thisdefensewasoftenreferredtoasnon specificimmunity. Severallinesofresearchledtothediscoveryofreceptorsthattriggeractivationofinnate immunityuponmicrobialrecognition.Onelineofresearchtookoffinthe1980swhen severalscientistsinvestigatedhowthedefenseagainstmicroorganismsisaccomplishedin evolutionarilyolderorganismsthathavenotdevelopedanysecond(adaptive)lineof immunity.Animportantstepinthisprocesscamefromtherecognitionthatinsects, includingfruitflies,produceantimicrobialpeptidesthatareinvolvedintheprotection againstpathogenicmicroorganisms.ThelateHansG.Bomanandhiscolleagueswere

particularlyactiveinthisfield;theyunraveledaninducibleantibacterialdefensein Drosophila(7)anddiscoveredthececropins(8),nowknownasoneamongseveralclassesof solubleantimicrobialpeptidesthatcanbindtoandlysebacteria.Today,weknowthatsuch peptidescompriseanessentialpartoftheinnateimmunedefense,notonlyininsectsbut alsoinmammals,includinghumans(9).Anumberofscientistsmadeimportantdiscoveries relatingtoothereffectormoleculesininnateimmunityandthegeneticelementsthat regulatetheirproductionininsects(10,11),butitremainedunclearhowtheseresponses wereinducedbymicrobesormicrobialproducts.Astomammals,theprevailingviewwas thatthespecifictriggeringstimuliinimmuneresponsesexclusivelyrelatedtoadaptive immunity.However,itwasknownfrommanystudiesthatanumberofdifferent,moreor lesswelldefined,stimuliincludingbacterialsubstancescouldenhancetheeffectsof vaccines,likelyviaactivationofcellsoftheinnateimmunesystem.This,andotherinsights, ledthelateCharlesA.Janewaytopostulatetheexistenceofarecognitionsystemfor microbialcomponents(patternrecognitionreceptors)(12),differentfromtheantigen specificreceptorsofTandBlymphocytes.However,therewerenocluesregardingthe molecularnatureofthesensingmechanisms. Discoveryofreceptorsthattriggeractivationofinnateimmunity JulesHoffmanandhiscolleaguesstudiedmechanismsthatactivateinnateimmunityin Drosophila.Amajortoolinthiswork,andtheonethatenabledthemajorbreakthrough,was theavailabilityofmutantfruitfliesthathadoriginallybeendevelopedinChristianeNsslein Volhardslaboratoryforstudiesofembryonicdevelopment(13).Herworkwasawardedthe NobelPrizein1995.OneofthesepreviouslygeneratedmutantswascalledToll;thelegend tellsthatthisnamewascoinedbyChristianeNssleinVolhardafterseeingaverypeculiar typeoffruitflyinthemicroscopeandexclaimingdaswartoll(thislooksgreator peculiar).Thepeculiarappearanceofthismutantflywascausedbyadefectinthe dorsoventralpatterningduringdevelopment. Studiesinseverallaboratoriesintheearlytomid1990s,includingHoffmanns,had suggestedsimilaritiesbetweensignalingcascadesinvolvedindorsoventralpatterningand activationofimmunegenesinDrosophila(10,11).Atthetime,thisfieldofresearchhad
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developedsignificantlywithkeycontributionsbyseveralinvestigators(1425).Findingsin thisareainspiredHoffmannandcollaboratorstoaddressthepossibleroleoftheTollgenein sensinginfectionleadingtoinductionofinnateimmunity.WhenHoffmanandhiscolleagues infectedDrosophilathatcarriedatemperaturesensitivemutationintheTollgenewitha fungus(Aspergillusfumigatus),allofthefliesdiedduetoimpaireddefense(2).Itisnow knownthattheTollreceptorbindsanothermolecule(Sptzle)thatinturnrecognizes structurespresentonthefungusandthatformationoftheToll/Sptzlecomplexisessential foractivationofinnateimmunityagainstfungalinfectioninthefly(2628).Aclassicalpicture ofafruitflywithamutatedTollgenesuccumbingtoafungalinfection(Figure1)provided thecoverofthejournalissueinwhichthispaperwaspublished(2).Thisdiscoverywasa breakthrough,asitidentifiedaspecificreceptorcomplexinvolvedinactivationofinnate immunityuponinfectionwithapathogenicmicroorganism.

Figure1.GerminatinghyphesofA.fumingatusonadeadfruitflyillustratingtheinabilityofTolldeficientfruit fliestodefendthemselvesagainstfungalinfections(fromref.2).ReproducedwithpermissionfromCellPress.

Weresuchreceptors,withabilitytosensemicrobialcomponents,conservedinmammals? Tolllikegeneshadbeenidentifiedinthehumangenome19941997(2932).Someofthem hadbeenisolatedandcharacterizedascDNAcloneswithunknownfunction(29). Furthermore,in1997thegeneforaTolllikereceptor(TLR)calledTLR4hadbeenclonedand engineeredtodemonstratethatitcouldactivateNFBtranslocationtothenucleus,a typicalimmuneactivationpathway(30).Thiswasanimportantfinding,butitremained unclearwhetherthisreceptorhadaroleinpathogensensing.

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BruceBeutlersolvedthisquestion.Hehadsinceseveralyearsstudiedthemechanisms involvedintheinductionofsepticshockbyGramnegativebacteria.Thisphenomenonhad beenknownsince1892(33).Itinvolvesasystemicresponse,foundlatertobeelicitedby LPS,andcanbelethalinanindividualexposedtohighdosesofLPSorbacteria(34).Atalow infectiondose,theresponsecaninsteadcontributetoprotectionofthehost(33).Amost importanttoolintheresearchonsepticshockwasdiscoveredinthe1960s,namelythatthe C3H/HeJmousestrainhasaseverelyimpairedLPSresponse(35).Thenotionofadefective receptorasamechanismbehindthedefectiveLPSresponseinC3H/HeJmouseemerged graduallythroughworkbyseveralscientistsincludingBeutler,eachprovidingpiecesintoa stillhighlymysteriouspuzzle.Thecontributionsincludedthefindingthatasinglelocusis responsibleforthedefect,thatthesamelocusalsocontrolscertainBcellresponses,that macrophagesarecrucialfortheresponse,andthatTNFisamajorsolublemediatorofthe response,aswellasidentificationofother(nonsignaltransducing)componentsofthe receptorcomplexsuchasCD14(3650). Withthesepiecesofthepuzzleathand,Beutlerlaunchedalongtermsearchaimedat identifyingthereceptorinthemousethatcouldrecognizebacterialLPSandactivatethe innateimmunesystem(3).Heusedgeneticstrategiesinasearchthatwouldtakeseveral years(Figure2),endingwithadiscoverythatcouldnothavebeenexpectedattheinitiation oftheproject.

Figure2.Diagramofasmallportionofthe2.6MbcontigspanningthecriticalregionoftheLpsgene.Thisgene mappingpavedthewayfortheidentificationofTlr4astheLPSreceptor(fromref.3).Reproducedwith permissionfromtheAAAS.

 Thus,histeamfinallyidentifiedthesignaltransducingLPSreceptor,andrevealedbyDNA sequencingthatitwashomologoustotheTollgeneinDrosophila(3).Thegeneidentifiedby BeutlerwasmutatedintheC3H/HeJstrainaswellasinanotherLPSresistantmousestrain. Beutlersseminaldiscoverygalvanizedthefieldbyprovidinganinfectionrelated,non redundant,invivofunctionforTLRinmammals,aswellasthefirstpathogenderivedligand foramammalianTLR.Hisdiscoverywassoonconfirmedbyothersinthefieldworkingon similarlinesofresearch(51). Together,thediscoveriesofHoffmannandBeutlerdemonstratedthatverysimilarreceptors areinvolvedinsensingthepresenceofinvadingmicroorganismsandactivatinginnate immunityinawaythatultimatelymaydeterminelifeordeathofananimal.Severalother scientistsweresoonabletoconfirmandextendtheirfindingsinotherspeciesincluding man.Takentogether,thisresearchhasprovideduswithacompletelynewviewofhow patternrecognitionreceptors(12,52),amongthemthefamilyofTolllikereceptors,sense andactivateinnateimmunityagainstdifferentmicroorganisms.Tolllikereceptorshavealso beensuggestedtobeinvolvedinrecognitionofendogenousTLRligandsfromwithinthe body,forexampleaftertrauma(53). FollowingthefindingsbyHoffmannandBeutler,thefieldvirtuallyexploded.Tenandtwelve TLRshavebeenidentifiedinhumansandmice,respectively,andallaretransmembrane proteinswithextracellularleucinrichrepeatsandanintracellularToll/interleukin1receptor (TIR)domain.Someoftheseareexpressedontheoutercellmembrane,otherinintracellular vesicles.Somereceptorsrecognizebacterialglycoconjugatesorproteins,otherbindto aberrantnucleicacidpatternstypicalofbacterialorviralinfections(54).Additionalreceptor familieswithsimilarfunctionhavebeenidentified.Detailedinsightsintosignaltransduction andgeneregulationhavebeenobtained,andeffectorfunctionshavebeencharacterizedin largedetail.Stimulationofdifferentcombinationsofreceptorshasbeenshowntoelicit distinctabilitiesofDCstostimulateTlymphocytesandthusgenerateappropriateresponses fromtheadaptiveimmunesystem(55,56).

Previousprogressandchallengesconcerningtheadaptiveimmunesystem TherehasbeengreatprogressovertherecentdecadesinourunderstandingofhowBandT cells(lymphocytes)oftheadaptiveimmunesystemrecognizetheirtargetscalledantigens. WhenRalphSteinmanenteredthefieldintheearly70s,itwasknownthatantigenscould bythemselvesnottriggerlymphocytes.Accessorycellswereneeded,whichcouldbe removedfromcellculturesbyadherencetoplasticsurfaces.Itwas,however,notknown whetherallofthesemacrophagelikecellshadthesameabilitytopresentantigenstoTcells orwhethersomecellsweremoreimportantthanothers.Intheprevailingparadigm,therole oftheaccessorycellswasmainlyconsideredintermsofmerelydeliveringtheantigen,while Tcellswereconsideredtotakeallthenecessarydecisionsrequiredtoorchestratethe ensuingresponse. Acellwithanexceptionalcapacitytoactivatetheadaptiveimmunesystem Steinmanworkedfromthebeginningofthe1970ieswithanaimtoidentifycellsand mechanismsthatcouldactivateTcellsoftheadaptiveimmunesystemtocarryouttheir manyfunctionsindirectingspecificimmunereactionstowardsmicroorganismsaswellas againstotherantigens.Inaseminalpublicationfrom1973(4),heidentifiedacellwitha dendritic(treelike;fromtheGreekworddendron)appearanceinthespleenofmice (Figure3).Helatershowedthatthiscell,whichhetermedthedendriticcell,hadan exceptionalcapacitytoactivateTlymphocytes(5).Hisfindingswerenotimmediately acceptedbythescientificcommunity,asitwaswellknownatthetimethatothercellssuch asmacrophageswereabletoactivateTcellsagainstspecificantigens.Throughaseriesof ingeniousexperimentsoverseveralyears,Steinmanshowed,however,thattheDChad uniqueproperties,distinctfromothercellsincludingmacrophages,inbeingabletoactivate naveTcellstospecificantigens.


Figure3.Phasecontrastmicrographofadendriticcellasfirstobservedinthemousespleen(fromref.4). ReproducedwithpermissionfromRockefellerUniversityPress.

Importantly,healsoshowedthattheDCisaverydynamiccell,whichupondifferentiation fromanimmaturetoamatureDC,inducedbyimmunecytokines,acquiresanexceptional capacitytoactivateanddirectTcellstowardsspecificeffectorfunctions(6).Thiswasakey discovery,asitconceptuallytiedtogetherSteinmansresearchontheDCandanumberof observationsonsocalledLangerhanscellsoftheskin(seereviewin(57)),originally describedbyLangerhansin1868(58).ParalleltotheworkofSteinman,severalgroupshad reportedonthemorphologyandpossiblefunctionofLangerhanscells(seereviewin(58)), implicatingfunctionsrelatingtotheimmunesystem:thesecellswereoftenobservedclose tolymphocytesintissuesandlymphaticvessels;theyexpressedMHCclassIImolecules;they appearedtobindtocontactallergens,andtheycouldstimulateTcellsinmixedlymphocyte reactions(MLR)(5962).Steinmandemonstratedthatthelattercapacitywasweak,butthat LangerhanscellsacquiredallthepropertiesofDCsuponmaturationinducedbycytokines, includinganexceptionalcapacitytoactivateanddirectTcellstowardsspecificeffector functions(6).Thiswasakeydiscovery,asitconceptuallytiedtogetherSteinmansresearch ontheDCandanumberofobservationsonLangerhanscells.Thispavedthewayforthe currentparadigm,whichstatesthatimmatureDC(whereLangerhanscellsareconsidered onespecializedsubsetofDC)patrolperipheraltissuestotakeupantigen,canbeinducedby
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avarietyofstimulitomigratetolymphoidorgans,andconcurrentlydevelopaphenotype withallthecellsurfacemoleculesrequiredfortriggeringnaveTcells.DC,withtheir distributioninalmostallorgansofthebody,thusprovidethefirstcellularconnectiontocells oftheadaptiveimmunesystem,directingmuchofthesubsequentTandBcellmediated immunity. FurtherworkbySteinmanandothersonthedynamicsoftheDCdemonstratedthatthiscell notonlyhadanexceptionalcapacitytoactivateadaptiveimmunity,butthatitcouldalso inducesubsetsofTcellsthatcanpreventordownregulateimmunity(63,64).Thislatter findinghasprofoundimplicationsfortheunderstandingofhowtheadaptiveimmune systemisregulatedtoattackforeignintruders,whiletoleratingnormalautologouscells. ThediscoveryoftheDCanditsfunctionsbySteinmanhasopenedupalargefieldof research.MethodologicaldevelopmentintheSteinmangrouphasbeencriticalforthis progress,astheyhavetaughtushowDCscanbeisolated,culturedandpropagatedinlarge numbers,andhowtheycanbedirectedtowardsperformingseveraldifferentfunctions (65,66).Interestingly,thediscoveriesconcerningtheroleofTolllikereceptorsininnate immunityrapidlyledtotherecognitionbyseveralgroupsthatsignalsmediatedviathese receptorscandeterminepreciselywhichfunctionswillbeexecutedbytheDC.Thus,theDC isacellthatcreatesalinkbetweentheinnateandadaptiveimmunesystems(56). ThediscoveriesconcerningthecrucialrolesoftheDCindirectingadaptiveimmunityhave alreadyresultedinaseriesofclinicaldevelopments(6668).Theseinsightsareusedto createbettervaccinestowardsinfections.Theyhavealsobeenusedtoconstructvarious formsoftherapeuticvaccinesagainstcancer.ThefactthattheDCcanalsobeturnedintoa cellthatdirectsdownregulationofspecificimmunityprovidesabasisforseveralongoing studiesinmiceaswellasinman,aimedatspecificallydownregulateimmunereactionsthat contributetochronicinflammatorydiseases. PotentialfutureeffectsoftheNobelPrizeawardeddiscoveriesinclinicalmedicine Thediscoveriesawardedthe2011NobelPrizeinPhysiologyorMedicinehasprovidedanew understandingofhowboththefirst(innate)andsecond(adaptive)linesofimmunedefense
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areactivated.Expectedclinicaloutcomesinclude,amongotherthings,improvedvaccines. Ongoingworkinthisfieldisexpectedtoimprovenotonlyvaccinationagainstmicrobesbut alsovaccinationsaimedattriggeringandenhancingimmunereactionsagainsttumors (66,67).Progressinthisareadependsbothonabetteruseofadjuvantstoenhanceinnate immunitythoughstimulationofTolllikereceptorsandothersimilarreceptors,andon methodsforturningDCsintoefficientantigenpresentingcells(66).Additionalclinical progressishopedforwithintheareaofautoimmuneandinflammatorydiseases.Here, promisingresultshavebeenachievedinanimalmodelsofinflammatorydiseasesbothby interferingwithinnateimmunitythroughTLRblockade(69),andbydownregulating diseaseassociatedadaptiveimmuneresponsesthroughDCmanipulation(66). Takentogether,thediscoveriesofBruceA.Beutler,JulesA.HoffmannandRalphM. Steinmanhavebroughtusclosertothegoaloftreatingandpreventinginfections,cancer andinflammatorydiseasesbymobilizingandregulatinginnateandadaptiveimmunity. HansGustafLjunggren ProfessorofInfectiousDiseases,KarolinskaInstitutet,Stockholm MemberoftheNobelAssembly AnnikaScheynius ProfessorofClinicalAllergyResearch,KarolinskaInstitutet,Stockholm MemberoftheNobelAssembly LarsKlareskog ProfessorofRheumatology,KarolinskaInstitutet,Stockholm ChairmanoftheNobelAssembly

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