OralToleranceStateofimmunologicalunresponsivenesstoantigeninducedbyfeeding.Itisafeatureofthecommonmucosalimmunesystem.OralToleranceStateofimmunol1ThemucosalimmunesystemConsistsofthegastro-intestinaltract,respiratorysystem,genito-urinarysystem,liver.CommonlymphoidcirculationEpithelialcellslinethemucosaLargestareaexposedtotheexternalenvironmentHeaviestantigenicloadThemucosalimmunesystemConsi2Featuresofmucosaltolerance?NormalimmunefunctionTolerancecanbelocalorsystemicItrequiresafunctionalimmunesystemSymbiosis-intheabsenceofcommensals,apoorimmuneresponsedevelopsandoraltolerancecannotbeinducedFeaturesofmucosaltolerance?3Generalpropertiesofmucosaltolerance:Antigenspecific.Oftenpartial(eg.antibodiesinhibited,butTcellresponsesmayremain).Notcomplete(eg.maybeaquantitativereductioninantibodylevels).Waneswithtime.Generalpropertiesofmucosal4Generalpropertiesofmucosaltolerancecont’dEasiertoabrogatearesponsethanreduceandestablishedresponse.Goodimmunogensarebetteratinducingtolerance!(adjuvant…)Doseandroutedependent.Generalpropertiesofmucosal5BreakdownoforaltoleranceImmuneresponsestofoodleadstofoodintoleranceegcoeliacdiseaseImmuneresponsestocommensalbacterialeadstoinflammatoryboweldisease(IBD)egcrohn’sdisease,ulcerativecolitisBreakdownoforaltoleranceImm6BalanceRespond Don’trespond

fight anderadicate IgnorePATHOGENS SELF FOODBalanceRespond Don’trespon7Mechanism?Centraltolerance

deletionofself-reactiveTcellsinthethymusPeripheraltolerance

anareaofveryactiveresearch!

1.deletion

2.immunedeviation

3.anergy

4.suppression/regulationMechanism?Centraltolerance8Regulationofselftolerance?CentraltoleranceisincompleteTCRbindatlowaffinityandcanpotentiallyrecogniseanumberofMHC/peptideAuto-reactiveTcellsexistathighfrequencyintheperipheryAuto-immunity-isitaresultofdefectiveTcellregulation?Regulationofselftolerance?C91.DeletionMechanismof‘central’tolerance(negativeselectioninthethymus)ApoptosisofspecificTlymphocytes(egfas-fasL)Showntoplayarolein‘peripheral’toleranceinsitesofimmuneprivilege(egstromalcellsinthetestesexpressfasL)1.DeletionMechanismof‘centr10Peripheraldeletionofantigen-reactiveTcellsinoraltoleranceREF:Nature1995Jul13;376(6536):177-80ChenY,InobeJ,MarksR,GonnellaP,KuchrooVK,WeinerHLPeripheraldeletionofantigen11oralantigencandeleteantigen-reactiveTcellsinPeyer'spatches,inmicetransgenicfortheovalbumin-specificT-cellreceptorgenes.Thedeletionwasmediatedbyapoptosis,andwasdependentondosageandfrequencyoffeeding.Atlowerdosesdeletionwasnotobserved;insteadtherewasinductionofantigen-specificcellsthatproducedtransforminggrowthfactor(TGF)-betaandinterleukin(IL)-4andIL-10cytokines.Athigherdoses,bothTh1andTh2cellsweredeletedfollowingtheirinitialactivation,whereascellswhichsecreteTGF-betawereresistanttodeletion.Thesefindingsdemonstratethatorallyadministeredantigencaninducetolerancenotonlybyactivesuppressionandclonalanergybutbyextrathymicdeletionofantigen-reactiveTh1andTh2cellsoralantigencandeleteantige12DeletionsummaryGenerallyobservedathighdosesoffedantigen:Activationinducedcelldeath(AICD)mediatedbyfas/fasLinteractionsGrowthfactordeprivalDeletionsummaryGenerallyobse132.ImmuneDeviation

CD4+Tlymphocytesareactivatedbyantigenpresentingcells(APC)Th1cells-importantininflammatoryresponses(egdelayedtypehypersensitivity)Th2cells-importantinhelpingantibodyresponses.SuppressTh1cells(IL-4,IL-10).

ThereforeTh1immuneresponsesmaybeinhibitedifTh2cellsarestimulatedinstead.2.ImmuneDeviationCD4+Tly14

Inhibitorycytokines

Transforminggrowthfactorbeta(TGF

)non-specificallyinhibitsthegrowthoflymphocytes(Th3)SpecificimmuneresponsescanbeinhibitedbyTh2cytokines(IL-4andIL-10)SomepopulationsofTlymphocytes(bothCD4andCD8)canconsumeIL-2,theTcellgrowthfactor.SurroundingcellsthereforefailtogrowInhibitorycytokinesTransfor15Oneexampleofmany

Feedingoralinsulintomicepreventsvirusinducedinsulin-dependentdiabetesinamousemodel.IL-4andIL-10weregeneratedwhichinhibitedaspecificimmuneresponse.REF:VonHerrathetal.,JClinInvest98,1324.1996OneexampleofmanyFeedingo16口服免疫和口服疫苗課件17BystandersuppressionAntigen-specificsuppressionisinducedbyfeedingSuppressionistriggeredbyre-encounterofantigenReleaseofinhibitorycytokineswillnon-specificallyinhibitothercellsBystandersuppressionAntigen-s183.Anergy3.Anergy19

Non-productiveantigenpresentation

TcellsareactivatedbyantigenpresentingcellsNon-productiveantigenprese203signalsarerequiredtoactivateaTcellSpecificrecognition-TCR‘sees’therightMHC-peptidecomplex….signal1Costimulation-CD28bindsB7…signal2Cytokines-localmicro-environmentwillinstructthekindofTcellneeded…signal33signalsarerequiredtoacti21Responsevsnon-response

Tlymphocyteactivationrequires2signalsSignal

Tcellproliferation+Signal

(IL-2&IL-2r)Signal

alone

NoproliferationResponsevsnon-responseTly22Signal2absence/blockadeSomeepithelialcellsinthegutandlungnormallyexpressclassIIMHC,butnotcostimulatorymoleculesandthereforecannotprovidesignal2Reagents(egCTLA4Ig)havebeendevelopedtoblocktheinteractionofCD28withB7onAPCandthereforeblocksignal2Signal2absence/blockadeSom23AnergyResultsinaspecifichypresponsivenessAnergiccellsdonotrespondtospecificMHC+peptidepluscostimulationAnergiccellsmaythenblockAPC-andinhibitimmuneresponsesAnergiccellsmayconsumeIL-2Anergiccellsaremoresusceptibletoprogrammedcelldeath(apoptosis)AnergyResultsinaspecifichy24口服免疫和口服疫苗課件253.RegulationTherehasbeenagreatdealofdiscussionof'suppressorcells’(especiallyinthe1980s)SuppressorcellshaveproveddifficulttocloneandphenotypeManycellsexertasuppressiveeffectArangeof‘regulatoryTcells(Treg)’havenowbeendescribed3.RegulationTherehasbeena26RegulatoryTcellsApopulationofCD4+TcellshasbeenimplicatedinthesuppressionofinflammatoryimmuneresponsesAntigenspecificTurnoffspecificinflammatoryimmuneresponsesMechanismunclear…RegulatoryTcellsApopulation27TreginmurineinflammatoryboweldiseasePathogenic‘Tcells’(Tpath)-cantransferthediseaseto‘na?ve’recipientsRegulatory‘Tcells’(Treg)-inhibitdisease&TpathTregareasubsetofhelperTcells(CD4)whichexpressCD25MajorareaofinvestigationinImmunologyResearchTreginmurineinflammatoryb28ModelsoforaltoleranceEatsolubleantigenInjectantigenMeasureimmuneresponseTcellproliferationantibodyproductioncytokineprofileModelsoforaltoleranceEatso29Multiplemodelsoforaltolerancehavebeenproposed(Weiner,1997)AnimalmodelsHumanmodelsClinicaltrialsMultiplemodelsoforaltolera30Amurinemodel-GarsideMurinemodelinwhichOVA-specificTcellscouldbetrackedwithaspecificmonoclonalantibodyFedOVAWatchimmuneresponsebytrackingOVA-specificTcellsSmithKM,McAskillF,GarsideP.OrallytolerizedTcellsareonlyabletoenterBcellfolliclesfollowingchallengewithantigeninadjuvant,buttheyremainunabletoprovideBcellhelp.JImmunol2002May1;168(9):4318-25Amurinemodel-GarsideMurine31ResultsPRIMING-Ovainjectionresultedin:specificantibodyproductionproliferationofOVAspecificTcellsDTHresponseTOLERANCE-FeedingOvaabrogatedtheseresponsesdemonstratedthatprimingandtolerancecouldbeinducedinthismodel.ResultsPRIMING-Ovainjection32Wheredidtheresponsestakeplace?PRIMINGd3peakofOVAspecificTcellsinperipherallymphnodeTOLERANCEd3peakofOVAspecificTcellsinperipherallymphnodeWheredidtheresponsestakep33TcellproliferationPRIMINGTcelldivisioninperipherallymphnodes(pln),mesentericlymphnodes(mln)andpeyerspatches(pp)at2daysTOLERANCETcelldivisioninperipherallymphnodes(pln),mesentericlymphnodes(mln)andpeyerspatches(pp)at2daysTcellproliferationPRIMINGTOL34TcellphenotypePRIMINGOvaspecificTcellsdevelopa‘memory’phenotype.Changesdetectedasearlyas6hafterfeeding.TOLERANCEOvaspecificTcellsdevelopa‘memory’phenotype.Changesdetectedasearlyas6hafterfeeding.TcellphenotypePRIMINGTOLERAN35Differences...EarlysystemicandlocalimmuneresponseinprimingandtolerancewasverysimilarHowever,laterimmuneresponseswereverydifferent(immunityvstolerance)TolerantTcellsdidnotmoveintoBcellareaandstimulatetheirexpansionDifferences...Earlysystemica36PotentialCanoraltolerancebeusedtherapeutically?Doinbredanimalmodelsrelatetooutbredhumanpopulations?Canmechanismsofregulationbegeneratedexvivoorinvivoforclinicaltreatment?PotentialCanoraltolerancebe37ClinicaltrialsAnumberofclinicaltrialsforauto-immunediseaseareinprogress:Disease Antigen MultipleSclerosis(MS) MyelinBasic Protein(MPB) RheumatoidArthritis(RA) TypeIIcollagen TypeIDiabetes Insulin Uveitis S-antigen TransplantRejection MHCmolecules ClinicaltrialsAnumberofcli38HumanMStrial1ydoubleblindstudy6/15MSpatientsfedMBPhadattacks15/15controlMSpatientsfedplacebohadattacksThoseindividualsfedmyelinhadahigherfrequencyofTGF

producingcells(Fukuaraetal.,1996.JClinInvest98,70).HumanMStrial1ydoubleblind39HumanRAtrialsSeveralstudieshaveinvestigatedtheeffectoffeedingtypeIIcollagentoRApatientsStudyNoofcentresDose(mg) Time ResultsGermany 5 0,1,10 12weeksnd USA 6 0.02-2.5 12weeks 1+ InvestigatorsarefindingthatdoseandfrequencyareimportantfactorsHumanRAtrialsSeveralstudies40DiabetesTypeIinsulindependentdiabetesmellitus(IDDM)Organspecificauto-immunediseaseTcellmediateddestructionofpancreaticbetacellsAnti-insulinantibodiesdevelopSusceptibilitycontrolledbyenvironmental&geneticfactors(particularlyMHCgenes)DiabetesTypeIinsulindepende41DiabetesPreventionTrial

(DPT-1)

Multi-centre,randomized,controlled,clinicaltrialdesignedtodetermineifitispossibletopreventordelaytheonsetoftypeIdiabetes(1994-2002)>50%riskinjectedinsulin25-50%risk

oralinsulinDiabetesPreventionTrial

(DPT42ResultstodateInjectedinsulinfailedtopreventordelaytheonsetofdiabetes.SkylerJSetal.,(2002)Effectsofinsulininrelativesofpatientswithtype1diabetesmellitusNewEnglandJournalofMedicine346(22):1685-1691ResultstodateInjectedinsuli43ResultstofollowTheOralInsulinTrialcompletedenrollmentonOctober31,2002.Studyinvestigatorsarenowcollectingfinaldatatodetermineiforalinsulincandelaytheonsetoftype1diabetes.TheyexpecttoannouncetrialresultsinJune2003.ResultstofollowTheOralInsu44SummaryofhumanclinicaltrialsAgentsthatenhanceclinicaltoleranceareunderinvestigation,egothercytokines,adjuvants(cholera-toxin).HoweverclinicaltrialshavebeendisappointingPoorstudydesign/suboptimaldose/typeofantigen/route???/index.htmlSummaryofhumanclinicaltria45DiffuseMALTLaminaproprialymphocytes(primarilyBcells)(LPmajorsiteofIgsynthesis)Laminapropria:thelayerofconnectivetissueunderlyingtheepitheliumofamucousmembraneDerivedfromO-MALTandrepresenteffectorandmemorycellsfromcellsstimulatedbyantigenIntraepitheliallymphocytes(IELs)PlasmacellsproducingdimericIgAAntigen-presentingcells(macrophagesanddendriticcells)DiffuseMALTLaminaproprialym46ModesofAntigenSamplingStratified,non-keratinizedorparakaratinizedepithelia(oralcavity,pharynx,esophagus,urethra,vagina)AntigensamplingdependsonDendriticcellsLangerhanscells,phagocytic,antigen-presentingmotile“scouts”)Dendriticcellsmaythentransportantigentolocalandregionallymphoidfollicles.Simpleepithelia(bronchiole,intestine,bronchi)AntigensamplingdependsonMcellsandTransepithelialtransportDendriticcellsmayalsoparticipateinantigentransportModesofAntigenSamplingStrat47DendriticcellsCaptureantigenintissuesTransporttosecondarylymphoidorgansProcessandpresenttoTcellsAnessentiallinkbetweeninnateandadaptiveimmunityMayalsorepresentthe“Achille’sHeel”ofthehost?(Cutleretal.2001)DendriticcellsCaptureantigen48MaturationofDendriticCellsLossofendocyticandphagocyticreceptorsIncreasedexpressionofMHCUp-regulationofco-stimulatorymolecules(CD80andCD86)requiredforT-cellstimulationUp-regulationofCD40andadhesionmoleculesICAM-1andLFA-3Fcreceptors(endocytosis)decreaseThis"DC-precursor"foundinTDLlooksverymuchlikealymphocytewithseveralimportantexceptions.Mitochondriawerefarmorenumerousinthecytoplasm,andtheDCnucleuswasconvolutedwith

moredelicatelydistributedheterochromatinandlightereuchromatinthanisnormallyfoundinlymphocytes.(FromA.Anderson)MaturationofDendriticCellsL49AntigenSamplingacrossSimpleEpitheliaMucosalsurfacesgenerallylinedbyasinglelayerofepithelialcellsBarriersealedbytightjunctionsthatexcludepeptidesandmacromoleculesUptakeofantigenrequiresactivetransepithelialtransport(M-cellsorDendriticcells)Samplingisblockedbymechanismssuchaslocalsecretions,sIgA,mucins,etc.AntigenSamplingacrossSimple50OrganizationofO-MALTM-CellFollicle-associatedepitheliumDomeregionGerminalCenterParafollicularregionLUMENLymphoidFollicleOrganizationofO-MALTM-CellFo51AntigenAdherencetoM-CellsAdherencefavorsendocytosisandtranscytosisAdherentmaterialstendtoevokestrongimmuneresponsesWidevarietyofpathogensadheretoM-cellsMechanismofadherenceisunclearManycommensalmicroorganismsavoidadherencetoM-cellsAntigenAdherencetoM-CellsAd52M-CellsMayServeasEntrysitesforPathogenicMicroorganismsBacteriaVibriocholeraeEscherichiacoliSalmonellatyphiSalmonellatyphimuriumShigellaflexneriYersiniaenterocoliticaYersiniapseudotuberculosisCampylobacterjejuniVirusesReoviruspoliovirusHIVM-CellsMayServeasEntrysit53AntigenRecognitionAntigentransportiseffectedbyM-CellswhichoccuroverOrganizedMucosa-AssociatedLymphoidTissue(O-MALT)Afterantigenstimulation,effectorB-lymphocytesleaveO-MALTandmigratetodistantmucosalorglandularsitesAntigenRecognitionAntigentra54MigrationandHomingofLymphocytesDistributionofHomingSpecificitiesinMucosalTissues

Epithelialcellsliningpostcapillaryvenules(HEV’s)displayorgan-specificrecognitionsitescalled“vascularaddressins”Recognizedbycelladhesionmolecules“homingreceptors”MigrationandHomingofLympho55HighEndothelialVenules(HEV)Containspecializedendothelialcellsliningpostcapillaryvenules.Displayorgan-specificrecognitionsitescalled“vascularaddressins”thatarerecognizedbyspecificcelladhesionmoleculesonlymphocytes.HEVcellsarecharacterizedby:ElongatedshapeandprominentglycocalyxonluminalsurfacePolarized,withadomedluminalsurfaceseparatedfromthebasolateralsurfacebyadherentjunctions,butnottightjunctionsCellsrestonabasallaminathatconstitutestherate-limitingbarriertomigratinglymphocytesHighEndothelialVenules(HEV)56HEV(continued)InO-MALT,HEV’sarepresentinT-cellareasbetweenBcellfolliclesInD-MALT,venuleshaveflatendothelialcellsthatsharemanyfeatureswithHEV’sHEV’sproducesulfatedglycolipidsandglycoproteinsintothevascularlumen(notknownwhethertheseproductsplayaroleinhomingorextravasation)HEV(continued)InO-MALT,HEV’57AdhesionmoleculesclonedsofarbelongtofourmainproteinfamiliesIntegrinsSelectinsCAMs(celladhesionmolecules)Proteoglycan-link.coreproteinsAdhesionmoleculesclonedsof58ModulationofHomingSpecificitiesNaivelymphocytespriortoantigenicstimulationdemonstratenomigrationpreferenceFollowingantigenicstimulation,lymphocytesacquirehomingspecificitiesModulationofHomingSpecifici59LymphocytesinHEVLymphocytesadheringtoluminalsurfacesofHEVendothelialcells.Notemicrovillionsurfaceoflymphocytes.Cross-sectionofHEVLymphocytesinHEVLymphocytes60TransepithelialTransportinMucosalImmunitySamplingSiteEnvironmentEffectorSiteDiffuseMALTOrganizedMALTMucosalorGlandularTissueTransepithelialTransportinM61TransepithelialTransportofIgAAntibodiesPolymericimmunoglobulinreceptoranditsintracellulartraffickingpoly-IgreceptorBindingofIgAtopolymericimmunoglobulinreceptorTransepithelialTransportofI62TransportandDistributionofIgAAntibodiesTransportandDistributionof63EffectorFunctionsofMucosalAntibodiesIgAantibodiesarenotgoodmediatorsofinflammatoryreactionscomplementactivationneutrophilchemotaxisphagocytosisImmuneExclusion/Serve“escort"functionBeneficialnottoinduceinflammationIntra-epithelialvirusneutralizationbyIgAExcretoryfunctionforIgAEffectorFunctionsofMucosal64RelationshipbetweenSystemicandMucosalImmunityOraltolerance(anergy)Oraladministrationofantigensuppressessystemicimmunity“MucosalInternet”Epithelialcell-ImmuneCellInteractionsMaybecriticalforinductionofadaptiveresponseDangertheoryRelationshipbetweenSystemic65EpithelialCellResponsetoPathogensEpithelialCellResponsetoPa66RequirementsofProtectiveVaccinesBlockadherenceofmicroorganismtohostFacilitateclearancefromhostNeutralizetoxinMustrecognize“virulence”epitopesMustbeimmunogenicMustnotinduceautoimmunediseaseShouldinducelong-lastingimmunityMustinducethetypeofresponsethatiseffectivetoeliminatepathogen(eg.TH1orTH2)RequirementsofProtectiveVac67RationalStrategiesforMucosalImmunizationRequirementsSafetakenorallyLong-lastingduetocontinuedmaintenanceofmemorySurviveingastricandintestinalenvironmentsMustescapenormalclearancemechanismsMustcompeteforinclusionwithinM-CelltransportMustarriveintacttoantigen-processingcellsMustinducedimericsIgAreactivewithcellsurfaceRationalStrategiesforMucosa68RationalStrategiesforMucosalImmunization(continued)StrategiesforDeliveryofVaccineIntoO-MALTInertparticulatecarriersBiodegradablecopolymersImmune-stimulatingcomplexes(ISCOMs)HydroxyapatitecrystalsLivevaccinevectors(recombinant)Vacciniavirus

SalmonellaMycobacteriumbovisRationalStrategiesforMucosa69RationalStrategiesforMucosalImmunization(continued)StrategiesforEnhancingMucosalImmuneResponseCo-deliverywithcytokinesCo-immunogens(Choleratoxin)PeptidespresentedwithpotentT-cellepitopesRationalStrategiesforMucosa70OralVaccinesITHACA,N.Y.--TheBoyceThompsonInstituteforPlantResearchInc.(BTI),anaffiliateofCornellUniversity,announcedthatclinicaltrialswillbegintoday(July7)atRoswellParkCancerInstitute(RPCI)inBuffalo,N.Y.,totestthesafetyandimmunogenicityoftheworld'sfirstpotentialoralvaccineagainstthehepatitisBvirus.Thevaccinewillbedeliveredsimplybyeatingpotatoesgeneticallydesignedtocontainthevaccine.OralVaccineProtectsInfantsfromSevereRotavirusDiarrheaFirstSuccessinaDevelopingCountryAnoralvaccineagainstrotavirus--themostimportantcauseoflife-threateningdiarrheainchildrenunderage2--reducedseverediarrhealillnessby88percentinastudyofmorethan2,000infantsinVenezuela.Thisisthelargestandmostsuccessfultrialtodateofarotavirusvaccineamongchildreninadevelopingcountry.OralVaccinesITHACA,N.Y.--T71OralVaccines(cont’d)VaccineNowAvailableasanOralSeriesoraSingleDoseInjection

TyphoidfeverimmunizationisrecommendedforalltravelerstolessordevelopedcountriesespeciallythoseinCentralandSouthAmerica,Africa,SoutheastAsia,andTheIndianSubcontinent.ThehighestriskcountriesarePeru,India,Pakistan,andChile.However,abouthalfofallcasesoftyphoidfeverreportedinAmericantouristsareacquiredfromtraveltoMexicoeventhoughtheriskofdiseaseislowerthere.Typhoidfeverisgenerallyspreadpersontopersonespeciallybyfoodhandlerswhodonotwashtheirhandsadequatelyafterbowelmovements.Visitorswhostrayoffthebeatenpathandeatmealspreparedatfoodstandsorbystreetvendorsareathighestrisk.Carefullyselectingrestaurantswithcloseattentiontotheirsanitationstandardscanreducetherisk.Therenowisanoraltyphoidvaccineandanewsingledoseinjectablevaccinethatproducesfewersideeffectsthattheoldertwodoseinjectablevaccine.Bothvaccinesareequallyeffectiveandoffer65-75%protectionagainstthedisease.Alzheimer'svaccinelookspromising&BraindeteriorationslowedbynosedropsMedicalresearchershavesuccessfullytreatedAlzheimer’sdiseaseinmicebyputtingdropsofvaccineintheirnoses.Theythinkitwillultimatelybepossibletodothesamewithpeople."Weplantobeginhumantrialsnextyear,"saysHowardWeiner,aneurologistatHarvardMedicalSchoolwhohaspioneeredtheuseoforalandnasalvaccines.

OralVaccines(cont’d)Vaccine72OralVaccines(Cont’d)AVANTRECEIVESPATENTLICENSEONORALTYPHOIDFEVERVACCINENEEDHAM,MA(August22,2000):

AVANTImmunotherapeutics,Inc.(Nasdaq:AVAN)announcedtodaythesigningofacross-licensingagreementwithMeganHealthInc.forexclusiverightstoapatentportfoliosupportiveofAVANT’ssingle-dose,oralvaccinecandidateagainsttyphoidfever,calledTy800.

TheagreementallowsAVANTtofurtheritsclinicaldevelopmentofTy800inexpandedPhaseIIstudies,whileMeganHealthgainsnon-exclusiverightstouseAVANT'shigh-levelexpressionsystemforhumanand