St.John's Newfoundland ManorWUlti,.,,;,yofNewfoundland Faculty ManorW of STANFORD, .

St.John's

IMMUNOREGULATION IN MURINE EXPERIMENTAL A\ITOlMMUNETIlYROIOmS

by

MARIANNE MICHELLE STANFORD,BS<:.

AthesissubnUttedtothe Schoolof Graduate Studies

ManorWUniversityof Newfoundland inpania1fWfilmeatoftherequiremenu ofthedegreeof

Masterof Science

Facultyof Medicine ManorW Ulti,.,,;,y ofNewfoundland

September,1999

Newfoundland

Dedicated10IJwmemory of a profenorandjrlend, 0<.Garry IanMoTaggan-Cowan (1940-1997)

ACKNOWLEDGMENTS

Successfully completing. mast", dcgRc doesIIOlhappenwithoutthehelpof IJWll'important people.FU'Slly,I wouldliketothankmysupervisor, Dr.GeorgeCarayanniotisfor hisexpert guidanceandsupervision. 1could neverhavelearnedtheskillsI requiredto completethisthesis withoutyou.Icame to you astudent.andhopeto ave as aresearcher.IfIam successful,itis becauseofyou.I wouldalso liketothankthemembers of mysupervisorycommitteemeeting, Dr.Mic:hacfGrantandDr.VemonRitbardson fortheirhelpfulcommentsandcriticaldiscussion army topic.I wouldalsolike tothankKarenCarayanniotis forher tirelesstechnicalhelp,and YangDaifor his bcIpfWcommentsandmanysimulatingconversations,evenifwe didn'talways understandeachother.1would also liketothankDr.AndrejsLiepins,Dr.Ken Kao,Mr.Paul HodgsonandMs.ludy FootefortheirteduUcalhelp.Iwouldalsoliketothankthestaffof R...a.1IldGnduateSlUdies, Flallty ofMcdicinc IIldtheGSUfortheirsupponIIldhelp duringmytenureas a graduatestudentItMemorial.

Ineverwould have madeit to where I am today without the love and suppon of my family andfriends.Thisdose-knitnetworkof peoplehavebeenanint~partofmysuccess,and wordsc:annotexpressmyloveandgmitudetoaDofthem.Tomyparents..RegandBubara Stanford,I owe mypersonality,myheartandmyloveofleaming.To mygrandparents.,Harold andIda StanfordandEric:andFlorenceBrownI owe mystrength.detenninationandwillingness toDCVUgiveup.Thisbasbecomeso cleartomeillthepastyear.Despiteupsanddowns.one thinsremainstrue;theirdedication totbemseiva..eachotherandtome.Inmysiblings,Carolyn and Scott, I havefoundtrueandlifelongmends.It amazesme that even though we are so

iii

differentinour life plansandhowweaccomplishthem.we manage10stillrespectandcare for eachother so unc:onditionaIly.I am so proud ofwhat weallhaveaccomplished.I am sohappy 10 shoremy JlC'ICS with you'Tothe ....oCmyfmtiIy(can simplysay,thankyou.(loveyouall.To mybestfriendandroommale. Angda~dJe.,Ithank: you.forbeingtheblack 10 my white,the yin10myyang.Ourfriendshiphas beenmyanchor,andI will cherishitforever.Totherestof mydose:friends.newandold,Ithankyou forgivirlgme so manyperspectiveson life.Mylifeis fiIII,,1olowing _ oCyou.So10Joy,MicIIeIIe,Terri, Angie,Tanya,Angel&,Chantal.Kyle,Sue, JeremyandanyoneI mayhavemissed.thank:you.They sayittakes a village10raisea child.

Wbeolave. Iplan10take a village-sized amountofleamingandcaringwithme. FmaDy I'd liketo dcdieatcthisthesis10.maDwhogreatlyinftuenccd my decisiontopursue a postgradualedegree.Dr.GarryIan MeTaggart-eowan.Dr.Cowanwastaken fioorntheworldby cancerODJanuaryIS,1997,butDOlbefore behadthe opportunity10showagenerationof Uftiversity studentswhatitis10truly Jovewhatyou.do.He was aweDrespeacd professorin BioiOS)'who taughtmethatlcamingis athingofbcauty,andthat talentshouldnotbeWISIcd.

He believedthaieverystudentwashisequalandtaughtus accordingly.He livedCVCT)'day10its fillies!andisgrratIymissed.WellGuy,Ididit.ThanksCor everytIililg.

iv

TABLEOF CONTENTS

Acknowledgments Table of Contents ListafTable

Li$lofFigures US(ofAbbreviations

page no.

iii

xii xiii

CHAPTER I 1NTR0DUcnON

... .. 3

... . .... ... .4

... .... ....6

... .... ... .6

...6

...8

...10 2.1.1Definition ofTbsubsets

2.1.2 Thcellcross reguWion 2.2TblfTh2cellsinautoimmunity

I.ExperimenuIlUtoimmunethyroiditis

lEAn .

1.1EATISamodelohutoimmuneth)Toiddisease(AIlD) 1.2AnimalmodelsorEAT

1.2.1lnducedEAT

I.3HOSlT cdI ~

2.CD4+Thelperresponses 2.1Tbetpersubsets

2.2.1Overviewof role ofTh 1ceUsinorgan.speci5clUtoinununity 2.2.2ThlcoTb2immunedeviationin autoimmunity

2.2.3ThcellsinEAT .

3.ToeDdaenninantsinEAT 3.1TbyrogIobulinu an antigen

3.2Epitopemappingoftheth)TogIobuIinmolecule 3.2.1MappedtbyroSlobuli",pitopes

10 12

.. 15

.17

17 19 19

3.2.2Useof algorithmsinepitcpemapping 23

CllAPTER2 MATERIALSAND METIlODS

...31 ... ...32 J2 .•...29

2.1Animals .••..••.•...•...•..•...•...• 26

2.2Antigens 26

2.3 Algorithmbased searchfor Ak-binding peptidesinTg 27

2.4Culturemedia 29

29 2.5Antigenicchallengeofanimals

2.5.IIndue:tionofantigen-spccificLNe 2.S.2InduaiooofTbIandTb2 cells 2.6Enzyme-linked immuoosorbent usays

2.6.1Cytokinc~SAfor assessment of Cytokinesin cellculturesupernatant

vi

2.6.2AntibodyBJSA for assessmem ofanb"bodiesinsera 2.7Analysisofendos...eytolci1lc mRNA

2.7.1RNAextraetion 2.7.2dlNAp<epanOoo 2.7.3AmplificationbyRT-PCR 2.7.4Analysisof PeRproducts

2.8 MeasurementofpcptidcbindingtoA'or£tI

33

34 ..34 ...34 3' 36 ...36

CHAPTER 3

ATTEMPTS TO GENERATE THYROIDlTOGENlC TB2 CEUS SPEcmC FOR THYROGLOBULIN PEPTJDES

3.1 3.2Introduction 3.3ResuIu

38 39

...40 3.3.1 ThereisDO detectablemRNAfor the eytokinesIL·2.tL-4 or IFN-yinnormal

thyroid gJand... .

3.3.2TgPl-andTgP2-specificThlcyrokineseemingcellswere producedwithout the

uscof exogenouscytokines .

40

43 3.3.3 AddingexogenousrlL-4in vitrotoLNCfrom miceimmunizedwithTgPl

or TgP2 does not produce peptide-specificTh2..qtolcinesecretingcells after

10daysineulture: 47

vii

3.4 Discussion ].5FutunoclUe<:tiom

CIIAPTl:R4

AN ALGORITHM·BASE DAPPROACH FOR MAPPING A'.BINDING PATHOGENIC THYROGLOBULIN EPrroPES

49 55

4.1Abstrac:t 4.2Introduction 4.] R=Its

57 58 ...59

4.3.1 Algoritbm-basedpredictionof sevenTg peptidesthatbindtoAtmolecules.... 59 4.3.2 TherTg peptidesfound usingthe algorithmarehighlyhomologoustothe

~mTgpeptides 64

4.3.3 Thepeptide19(2597-2609)bindsSb'ODgIy to the AtrnoIeeule..whilethepeptide

rTg(2424-2437)bindsuongly10theE'molecule 64

4.3.4 Tcd!reactivity10theTg peptides_.. . ...66

4.3.5 Therewas,;gnifiamproduction ofigGanuOodyinresponsetoTg(2424-24]7) ond Tg(2597·2609)with1inI.croPreo<tM1ywith mTg. ...71 4.3.6 ThepeptideTg(2S97·2609)causes mononuclearcellinfiltrationof thyroids

inmice

4.4 Discussion 4.5Future Directions

73 ... 76 ...80

REFERENCES 81

LIST OF TABLES

Table Page

2.1 MotifsAandB withinA'-bUldingpep6des (A1nMa,uJ. .I 994) 28 3.1Comparison ofendogcoousmRNAlevelsinSJUJmousetb)Toidandspleen

uassessedbymRNADobrioo.eDNAp"J'lBlioaand RT-PCR 41

"'.1PredictionofA' binding T cellepitopcs withinr1gusing complCldyoverlapping

AandB motifs fromAltuvia ctaI.,1994 60

4.2_ ofA'bincfingpcptideswnJUDgrTgusingpanWIyoYCrlappmgAandB

motifs fromAJtuviaet11.,1994 60

4.3 mTSsequences predictedusingalgorithm fromAltuviaet.t(1994)thatcontain

ccmplCldyoverlappingAand Bmotifs . 61

4." mTgsequencespredicted usingalgorithmfromAJtuviaetaI.(1994)thatcontain paniaIIyoYCrlap¢n8 AandBmotifs .. ....•...•... ...•. 62 4.5Tg sequencesprcdiaedusing..algorithmforpcplid,lMdinglo A' from

Fremontctat.1997.... . 63

4.6 Homology comparison ofTgpcptideswithputative Ak-bindingcapacity

selectedusing ..algorithmthatpredictsA'lMdingof pcpcides 10c:orrespon<tingpcptidesof

mTg . 65

4.7R.esultsfromA'andE'competitiveinhibitionassays.,indicating relativebinding

ofelGbofthepeptides 67

4.1ImmunogenicityofpeptidesassessedinH~;:tstrainsofmice 70 4.9ComparisonofbistoJogy resultsfromdifferentH.2A'mousesuainsimmunized

withpepbdes ofTS ...•... . 74

USTOFnGIJllI:S

Fisure

Page

3.1 RT·PCRshowiDgcDNAfromSJUlthyro;dsJand 42

3.2 Schematicdrawingoftultureconditioasof LNC frommiceimmunizedwith TgPlorTgP2 .

3.3 DetcaiooofThl-typc cytolrioesintheaJkwesupernatantofUlCfrommic:e imnIwUud _ TgPlondTgP2 .

44

45 3.4 Kineticsofprolifcruion ofLNC from miceimmunizedwithTgPl 41 3.S ProductionofcytolciDesbyLNCoemia:imm.mizedwithTgPlandcultured

in\/111'0intheprescoceorabsenceofpeptide withrfL..4 48 3.6 Productionofc:ytokines by LNCtom miceimmunizedwithTgPl or TgP2,with

rlL-4in vitro,with• restingofalI cellsin the abseoceofstimulibetweens.econdaJy

Itldttrtiatyc:uJtures _... 50

4.J CompetitiveinluDition assayindicatingAlbindinginpeptideschosenusing

algorithm 68

4.2 CompetitiveinhibitionassayindicatiDg E^lbindinginpeptidesdlosenusing an algorithm that predictsAlbinding .•..••..•..•••.••..•.. 69 4.3 JgGantibodyfromseraofH-2'strainmiceimmunizedwithpcptidesselected

usingan algorithmpredictingA^lbindingpepndes 72

4.4 Histological_OIlSfromH-2'mice imnIwUud _ rTg(2597-2609) 75

LL Ab Ag AITD APe APL BB bTg BUF eDNA CFA cooA CTU CNS CPM DEPC DMEM DMSO DnI EAE EAT WSA FBS GAD GVIID IIEL lIT hTg IDDM

!FA IFN Ig n, n.-2R i.p.

Lv, LNC LPS mAbs

LIST OFABBREVIAnONS

Amino Acid _body AmigCD

AutoimmuneThyroidDisease AntigenPrescmiIlgCcIJ Ahem! PeptideLigand BioBrccding BovmeTIryrogIobuIm Buffalo Compl...wy DNA Complete Freund'sAdjuvant ConcavaiinA

Cytotoxic T LymphocyteLine C-.J NOMlUS System Counts perMinute Diethypyrowbonate DuIbocco',ModifioclE&gleMedium DimotbyIsufoxide

DoIayocl Type HypersaIJitivity ExporimootIIAuloUnmunoElaphaJomyetitis ExporimootIIAutoUmounolbyroKtiti, Enzymo-liJ>kocllmmuDosorbootluKy FoulBovmeSerum

Glutamic AcidDehydrogenase GraftVcrsusHostDisease HenEggLy1Ozymo 1Iashimoto',Thyroiditis HumanTIryrogIobuIm InsutiDDopood'"llW>ol..Mdlilus Incomplete Fmmd'sAdjuvant Interferon

Immunoglobulin InterlcuJdn lotorioukio-2Roc:eptor Iotnporitoooal ImroVOOOllS LymphNodeCcIJs l.ipopoIysa=ride Mo~onalAntibodies

2.!dE MOP MHC MOO MS mTS mRNA MW NOD OS PBS PB5-T PLP PPD pTS RIP RT·PCR s.c.

sad

T, T.

TcR TIC TS TIPI

TsP2

Tbl Tb2 TNF TPO

2.Mcraptoelhanol MyelinBasic Protein Major Histocompatibility Complex MyelinOtisodendro<yte Multiple Sclerosis Mouse1byrosJobuIin M""'"8<' RNA MolecularWeisJtt Non-obesc Diabetic ObeseStniD PhosphateBufferedSaline PBS_ Tween-20 Proteolipid Protein PurifiedProteinDerivative Porcine 1byrosJobuIin IWlnsulinPromoter

ReYeneTramaipwe PolymerueChImReaaicn Subcutaneous

SevereCambincd Immunodeficiency Triiodothyronine

Thyroxine TCellReceptor ThyroidFolIicWMCells 1byrosJobuIin rTs(249S-25ll) rTs(269S-271l) T helper1 Lymphocyte Thaper 2 Lymphocyte Tumor Necrosis Factor Tb)To;dPcroxid&se

xiv

CIlAI'TER 1 lNTRODUCllON

1.EXPERIMENTAL AUTOlMMIJNE THYROmms lEA T)

1.1 EATas • •odd forAutoi • • •lllf:Tb)'f'Oid Diseut(Arm)

ExperimentaJautoimmunethyroiditis(EAT)inmiceservesas an animalmodelforthe bumanIUtoimmunethyroiddisease(AlTO), Hashimoco'sthyroiditis(HT).HTwunamedfor IIalwuHashimoto(1881. 19141 thelapanese surgcon_ "firstdescribedthediseaseinhumans (Klein.1991.p.656).InHr.organ-specmcT Iympboeytesspecificforlhyro;d"";8ensarise.

anddue to , disorderinimmunoregulation,these IUtoreac:tiveclODeSarc permitted10proliferate (reviewedinVolpe. 1990,p.78).Thisdefectinimmunoregulation maybedue to a ....rieryof W:tonsuchu;lossofSlIppressorTlymphocytefunctioo(IitalcaetII.•1988.. litalcaetII.•1988. Volpe.198 1, Volpe. 1988),defectiveclonaJ activation ofimmatuceseIf-reactiveTand B Iympboeytes(NossaJ.1983), disonl"oftheIIlli-icIiocyp;eetwcrk(BunnannetII.•198')or molc:cu.1&rminUay(Ahmann etaI.,1987,Bwmannand8&lcer.1985,lngbaretaI.,1987,Wolfet aI.•1988,WenzeletaI.,1988,Weissetal.,1983l TheautoreactiveT cells,specificfor thyroid antigenssuch as thyroglobulin(Tg)andth)Toidperoxiduc(TPO),proliferateandinteract with theantigen on thyroid follicularcetls(TFC)orintrathyroidaJantigenpresentingcellssuchas deodriticcells(KabeleaI.,1987).TheactivationofTcellsinducesfirstCD4+andwer CD8+T cdIsthatdestroy TFCIJIdevemuaIIythefollicularardlitecnueofthe myro;dsJIDd.Antibodies

dircc:led againstth)Toidantigens mayalsobeformed.andmayresuhin thedestruction ofTFCby lIltil>ody-depcodomcellula< cytotoxicity(ADCC)orbybUxtingcomplement(Volpe. \990).The thyroidgIaDdisancndoaineglandthat syntbesiushormonessuchISlhyroxine,whichare essentialforpropergrowthandmetabolism(Weir, 1997,pp.295).Subsequentto immunc desb'U<tion,thetbyro;dgWxIdocsDOtproducebonnones(orbonnoneproduction~reduced) andmetabolicprocessesare slowed.This resultsindinicaIsymptomsinthepatient.suchas sensitivityto cold,fatiguc,and obesityandsometimes.,goiter(Klein,1997,pp.656).

Thehistology of chronicEATisverysimilartothatofHT.EATwufirst desaibedby Rose and Wltcbskyin1956,whoimmunizedrabbitswithhomologousthyroidextractincompletc Fmmd',adju_(CFA)(RoseandWncbslcy,\956).Thisimmunization rcsuJt.,Hnboth 'thyroidautoantibodiesand chanctcristicdestructionofmethyroid glandarch itecture'(Roitt,p.

315,1994).InEATbothantibodiesandT edlsdirectedagainslTgdevelop,resultinginthyroid infIamnwion(Kuby.1997.pp486).

1.1 AIIimai ModdsorEAT

Animal modelsorEATaDowthe studyofmeinduction.manipulationandpossiblc immunc modification ofthcpathogenicprocess.TherearetwotypesofEATmodelscurrently underinvestigation,spontaneousEATanddirect1yindue:cdEAT.In thespontaneousmodelof EAT,autoimrrlmcdcsuuctionoflhethyroidglandoccurswithoutcxperimcotalimmune intervention.SpontaneousEAToccursinthe obesestrain(OS)chicken,theBuff'a1o(BUF)and Biobreeding(DB)ratstrains,the:ArgonneLaboratoryColonyofbeaglcdogsandinnon-obese

diabetic:(NOD)mousestram(B;gazz;,199 3).

1.1. 1 1Dd uttd EAT

EATcanalsobeexperimental1yinducedinanimals by immuniutionwiththyroidantigens suchas 18inCFATheinductionorEAT canbefurther subdivided intotwocategories. direct lDd indircainductioD. DirectEAT.whichis~in animalsimmunizedwithT8 in CFA.was pio-.dbytheRoseandWncbslcy'.experimatt.withrabbiuin1956 (Rose Illd Wrtcbslcy, 1956).Sincethen.EAThasbeeninducedusingbothhomologousandheterologous19indogs (TCfllIaoeaI.,19601mo(J....andRoitt,19611guineapigs (McMBtet ..aI.,196 1),monkeys (Kiteet&l.t1966)and

mice

(Roseet&l.t197 1).Heterologous orhomologous 19 peptideshave alsobeenusedto induceEATinmiceandrats (CarayanniotisandRao,1997).Fordirect inductionofEAT10besue<:essNI.!beproieinor peptidelllUSl beemu!sijjedwithCFA. Although theprecisem«hanismbywhichCFAenhancesthe tmmunogenicityandEAT-inducing capabilities of18isunelear,CFAbasbeendescribedas havingboth inflammatoryandantigen- depotformingproperties (WeigleClaI.,1969,Yamanakaeeal.,1992).Inadditioll toadjuVUl1,a rangeof innateandenvironmental factorsmustalsobeconsideredinEAT induction.These includethe geneticsusceptibility aCthehost.the antigendose. the immunization scheduleorthe route ofantigm(Ag)administntioD.

Inadditionto the abovemethods,EATcan be directlyinducedvia.conjugationofnormal 19 to mouse clus0 major histocompatibilitycomplex.(MHC}-specificmonoclonal antibodies (mAb)in!beobscnceof adjuvant(BaI...andCanyanniotis.19931 )and!beplacingoffresll

thyroidsUDder the renalcapsuleorintheperitonealcavity followedbyan intravenous(iv.) injectionwiththe:polyclonal BccUactivatorlipopolysaccaride (LPS)(Okayasu and Hatakeyama., 1984).

Therearcalsoseveralmethodsto indirectly induceEAT.Themostcommonindirect methodis the adoptivetnnsferof lymphoodecells(LNC)frommiceimmunizedwith Tg or Tg pcptides,.andfurtherstiIDlI&tedin vitrowith theimm.mizing Ag.Thesecellsare transferredinto naivebeatthy hostsandEAT develops2·J weekslater,A variationon thismethodis the induction of'sranukmwous'EAT usingmTg.activatedspleenceOsfrommiceprimedwithLPS.Thein vitroprimingof theseeeIls&!so0CQlJ'1ill the presmceofeitbet n...2 receptor(lL-2R)or IFN-y- specificAbsinadditiontoTg.Thisresultsinthe transferof. moresevereanddestructiveformof EAT tothenaivedonon (Braley-MullenctaI.,1991).EATisalsoinducedafter neonatal thymectomyofmicc,whichdisturbstheavailablepoolof lymphocytes., or thymectomyof adult miccinc:onjunction with imdiation,whichalten the Tcellsubsets(Rose,1998).

1.3 BOlt T ctUrapoase

ThemurineEATmodelhasprovedveryefficientindeterminingthe~ contributionofTandBcellsinEAT.Itsstrengthsincludethefactthat the murineimmunesystem basbeene:xtensivetystudied.aJongwithitsMHC,manyinbredstrainsofmice.vailabJe,aDowing for the study ofgCMticsinEAT.Using.murinemodel,VIadutiuandRose(1915)discovered thatitwas TandnotB cellsthatmnsferred Tg responsivenessin'good responder'mice.mice that wereshowntobesusceptibletoEA.TinductionusingTSod:tisinferring an effectorrolefor

T cds. Subsequentstudies inotheranimalmodelshavesupportedthisconclusion.Inthe OS chid::euspontaneousmodel,neorwaItbymeaomy alongwithin ""'"treatment withT

ceu-

speQficantibodiesresultedinprevemionof EAT(Schauenstcin,1998).In addition,EATcould be transferredtoa naive hostby Tcells derivedfrominfiltrated thyroidsofbursectomized(Bcell deprived)OS chickens.TccIlshavebeenshownto prolifente in ....1TOinresponseto mTgand adoptivelytransferEAT to normal,naiveanimals(reviewedinKongandLewis,1990).This dfectisabrogatedbytheIdditionofTby-llllliboclyinWtTO.T cell.expandedi.WtTO bythe additionofTg havealsobeen showntodestroythyroidmonolayen(Creemenet&I.,1983, SalameroetII.,1985,Simonetal.,1986).ThesestudiessuggestthatTcellsplay• more importantrolethanB ccI1s inthelnitiationof EAT.

TheTcellsthatproliferateinresponseto mTg havebeenshowntobeCD4+ (Kong, 1916,Simonet 11.,1985)andare H-2A-restricted (Simonet 11.,1985).TheproUfentiveresponse of T cellsin vitro correlateswith EAT susceptibility (Simonet11.,1985,Okayasuet11.,1981).

TherdativeroleofCD4+andCD8+TcellsinEAT has beenelucidatedby. smesof experimentsbyY..chi Kongandassociates(ReviewedinKongandLewis,1990).These experimentsindicatethatCD4+ccI1sarerequiredforthelnitiation ofEATwhilebothCD4+and CD8+Tcellsconmbute totheseverityoftbeestablished disease.

Althoughthereislittleevidenceas to the cytokineprolileof infiltratingCD4+ T cells, indirectevidenceindicatesthat theyare of the T helperI(Th1)subset.Someofthis evidence inc.ludes the hyperreactivity of peripherallymphotdc:eUs,due totheintrinsicproductionof iDttf'kukin.2(11.-2)ill theOS chicken(reviewedinWicket11.,1998)andtheexacerbationof EATbythe lbl-inducing cytokinc: fi...12(Bra.ley.Mullenetal.,1998),andtheprototypicalTbl

cytokinc IFN-y(AlimietaI.,1998).Otherindim;tevidence indudesthefinding thatantibodies to 1b1 C)1okines haveaproIu:tNeeff'minSUSoCCptiblcsninstoEAT.andthattheuscof Thelper 2(th2)cytokinessuchas 0...-10(Mignon..Qodeftoy et al.,1995)alsoconferprotectionfrom EAT.Thesestudies suppontheaurentJybcIdbeliefthatThIcdlsoftencontributetothe induction oforgan-specificautoimmunity.

1. CIU+ T HELPER SUBSETS

1.1TO.. perS...

2.1.1DdiDitiOD orT bdpcr ,ubHlI

EarlystudiesofimmuneresponsesinmofoundthatinfectiousagentShad theabilityto induce either anantibody responseoracell-medialed form of immunityandthattheseresponses

wereoftenassociatedwithdistincte:ytokiDc pro61cs(ConstantandBottomly.1997).It was Mosmannetal.(1986)who 6m:describedtwodistinctsubsetsofCD4+T helpercellpopulations asThIandTh2,based00thecytokineprofiles of mouseCD4+T cellclones{Mosmannetal, 1986}.Ithassinc:ebeat

disco_

thatbot!lrats(East<on ..aI.,1990)andhumans(SoonetaI., 1988)alsoexhibitthesetwodistinct. subsets.

Thecytokine profile ofthesc twosubsets has beenwelleharactcrizcd.Thlcellssecrete thecy10IcinesIL-2,IF!o!:r,lympI>otmcinandTNF-jl.TheybavealsobeatimpUwcd...myriadof immune responses through activation ofmacrcpbeges,initiation of delaycd.-type hypersensitivity

(OTH)reactionsandIgG2aandIgG) antibodyproduction.These: includeprotectionagainst intraceiJularpan.sites.organ--spec:ifie autoimrrwnity,aUogrUlrejection, Crohn' sdiseaseand uoexplaiDed ...1bortions(1lomagllllli, 1997).Th2cellsproduce thecyIokilleIL-4,lL-S, lL-IO and lL-1l.TheyarcUlVoIv<dinIntibody_ suchIShelpingB cell.produce Ig.'d.

IgOl , IgAand19B,attractionof eosinophilsandinhibitionof macrophages(Druet etaI.,1996).

1b2respoasesare oftenimplicated inprotectionapinstmetazoanparasites,successfuJ pregnancy.vernalconjunctMtis,1I0picandaUergicdisorders,chronicGVHD(graftversushost diseue).syslemicsclerosis andprogression10AIDSin HI\'infcaion(Romagnani.1997).

Th1andTb2 cellsarebelievedto developfromthe samematurenaiveCD4+Tcell,often describedas ThO(ConstantandBottomly,1997).Initiationofei1herTblor 1b2 responseslikely depends onthecdJu1arenviromnemItthetimeofamigenstinIJlIlion.ThepresenceoCfi.. 12, producedbyldMled maaopbagcs anddendriticecIIs,induoesI1b Iresponsel!voughinitWion ofthetranscriptionfactor 5tat-4.Knockingoutfi..12(MagrametaI.,1996)or5tat-4 (Thierl"e1deret 11.,1996,Kaplanet11.,1996)rcsuluinmultcdlyreduced1b I respoeses.lFN-y alsobasan inducingeffectonTbl ceUs,partlybyenhatJciDgIL--12secredcnbymaaopbages (Thindlieri etaI.1995)and panIybymainuiningfimctionofn.·12 receptors000>4+TcdI.s (GuIereta1,1996).Tb2responsesare inducedthrough DA,producedbybasopbils,mastcells andT cells(Thomson, 1994)signaledthroughthetranscriptionfactorStat~(HeuetaI.,1994) Similarly'0 lL-12,knockingoutIL-4(KuhnetaI.,1991,Kopf" aI.,1993)and SOI,-6(I<oplm"

11.,1996,Takeda" 11.,1996,SbimodaetaI.,1996)rcsoIuindeficieotTh2.!"'PO"'C' Previous studies havefoundthatonly eytolcinesecretionandsubscquc:n1immune responsescoulddifferentiate betweenTh1andTb2 cens.Recently,however,severalstudies have

implicatedseveralchemokincreceptors u possiblemukers forThlandTh2cells.Thechernok:ine r«oplorsCCRS (Zingonietal.,1998)andCCR' (1loneccIUetaI.,1998)an:differentially expressedOD aaivaJ:edTh2cells,whileCCR.S andCXCRJ are differartiaUyapre:ssedon Thl ce11s(Boneechiet&1.,1998,Sivekeet&1.,1998).Thesestudieshaveopenedup a largearea of researdlin thisfidd, sirlceDOWTh1andTb2 tellsCIftbecbaraaeriudItthe singlecelllevel based onsurfacemarkers,insteadofddinio gapopulation ofcellsbasedthe cylolrinesseemed iDloal1turesupernatant.

2.1.1

n

uUcross nplatioa

ODeofthemost studiedaspectsofT helpersubsetsisthefactthatThl andTh2 ceUsare crossreguiuory.TbeC)'lokiDcsproducedbytheThlsubsethavetheabilityto downregWaiea Th2 responseandvicevena(reviewedinCor1SW1tandBottomJy, 1997).Thisfindinghashad a greatimpact instudiesofimmmoreguJationandillareasofresearchwhereITh1 or Th2 typeof responseis unfavorable tothe host,such as orpn-spccificautoimmunity(TbI) orallergy(Th2)

Tb14ypeC)1okinesrespoDsibleforthedownregulationof Tb2-type responsesinclude IFN-yand JL.12.IFN-yuw"bitstheproliferation oCTh2clones<Gajew>lOetaI.,1988)whilen.- 12 can beused to reverseanestablishedTh2 response(Romaniet11.,1997).The abilityof n.-12 to reverseanestablishedTh2 responsewudemonstratedinexperirnentaI~shmaniasis(Nabonct 11.,1995).n.-12 wasusedinconjunctionwith .Ieishmaniacidaldrug duringlate phase infection, andwushown10switchthedominantTb2mpoosetoa Tb1 response,resultinginbca1ingand resistancetoreinfectionwithLmajor.

CytokinesproducedbyTb2cellsknownto intu"bit Tb1ceUs andpromotelh2

deYd_

^indudc1L-4,ll.-IOIIId ll.-13(RomagnIni.1991).lblcelJsgrown ...:u1_

withoutexogenouse:ytokines., sueh.as eeDs spcc:i6eforpurifiedproteinderivative(PPD),can becomeTh2·likcinculturebytheaddition of n.-4earlyinbulk culture(parronchietaI.,1992).

TceIJsfromhoststJwareinfo<led withLmajor,poWiud10thelblpopulltio.bavebeen modi5edto produee Th2 C)1okinesbyreculturein

w tro

withn.-4,fi...2andAPe(Moeciand Coffman.1995).fi...IOhassimilareffects,suppressinglb 1 cells whiledecnasing n.-2andIFN-y production(Yo...et al.,1991, Top et 01.,1992).

OneofthemostimerestingdebatesofthelblfTh2paradigmiswhether1111or Th2 cytokiDe seeretingecIlsan aetuaDy'switch'to producethe opposingsubsetcytokines.TheIcveI ofeommitmcntfor cytosineproductionatthe singlecell levelisunclear(KongandLewis,1990).

Itisalsounclear wbetberduringa'switch'fromlbltoTb2or viceversainbulk c:uhures, the cytolcines presentin thecnvironmemIClU&IJycauseaswrtebinthe e:ytokine profileofindividual cells,or alloweontaminatingeeUsoftheoppositephenotypetoproliferate.oracton uneommined ThOtellsinthe cultureandallowthem to proliferate.MurphyctaI.(1996) foundthatfbi clones andeeJls thatW'CrCc:uJturedunderchronieTb1polarizins^eooditionsresisteda S'Nitcbto1b2,and a reductionofthcIFN-yproducingcellswasobserved.Also,Th2ccUsare oftenresistantto phenotypereversal,possibly due10aloss of responsivenessto0..-12(SzaboctaI.,1995).

Tbcrcare sevmJ other faetorstIw:affectthebalancebetweenThlandTb2 responses Antigendosein1Juenc:c:s the Tc:cUresponseandgeneraIty,lowdosesfavortbl responses,whiJe higherdoses favorTh2 (BretschcretaI.,1992,Hoskenet el.,1995).Co-stimulatory signalsinT edl aetivatiooalso playarole.Insome systemsthetypeofB7 moleculethatbindsto C028has

10

aneffect.with B7·1inducingThldevelopmentand 87-2favoringTh2(XuchrooetaI.,1995).

Othersrudicsindicatethathighlevelsofeostimulation in gencnl£avor•Th2response (DiMolfettoetaI.,1998).Lowerc:ostimu1ationmayrauhinTblresponsebecauseTh!cdlsmay belessdependentonthe levelofTcellactivation astheygettheirstimulatorycytokinell.·t 2

&omAPe(Murphyet11.,1994) .Someotherfacton mcJucle thegenetic baclcg10undof the animal.,as demonstratedbyLmajor studies where susceptibilitytoL.majorwasdependanton thestrainof mouse.,aodtherouteof antigeuadministratioll.

1.2Thlll'h2 cdb ia autoimmua iry

2.1.1Overviewof tbe role of Thl cdls ia OrplHpcdrlC autoimmu.ity

Manyorgan-spec:ific autoimmunediseases.,such as Wulin-dependentdiabetes mellitus (IDDM).multiplesdetOsis(MS)andItTan:mediatodbyTbI ceD.(Ublauet11.,1995).The cressregulation oftheThelpersubsetshasfocusedonthePOSSlbilityofdeviatingthe Tcell responseinthesediseases fromI'patho genic'Tbl responseto.'protective' Th2responseinthe animalmodelsforMSand100M(LiblauClaI.,1995, O'Garra etaI.,1997.DruetetaI_.1996).

E.xperlmenJal...hdotmmunreEncqJhaJomytlitis(EAE):

Experimentalautoimmune encephalomyelitis(EAE)isa modelofcertainaspectsorMSin taunans.Itisinducedinrodentsbyinjection of mydin antigenssuchasmyelinbasicprotein (MBP).proteolipidprotein (pLP)andmyelinoligodendrocyte protein (MOO)inCFA(reviewed

11

inO'Gam dat.1997).ThepathogenesisofEAE involvesCD4+TceUsbutnot CDS+T cells T'bereare~linesof evidencethatindicatethatTbIcellsareiDvoIvcd intheindUdionof EAE (reviewedbyDrudetaI.,1996,Ltblauelal.,1995).FntIy,theintJammatoryresponsein thecentralnervoussystcm(CNS) resembles.

om

reaction.whichis known tobeTal- mediated.Tberc:is •strongcorrelationbetweenDTHresponsesto myelinantigens andthe development ofEAE.Immunohistochemicalstudieshaverevealed thepresenceorTbleytokines, fL.2,TNF-llon<!IFN-yinCNS tissues IIthe heigblof diseases,on<!theabscuocofTb2 cytokines suchasn....-4.EAECIDbetransf"errcdpassivelyinto naive bostsbyTbIceil clonesspecific::for onc:cphalit<>gali<pcptides,derivedfromenc:cphalit08aU<lesioos.butnotbyTb2<ellclones (Barondol.•1993).

/1UJI/inlkf¥ndem D;abetnM,llitllS (lDDM):

Inthecaseof IDDM, studiesintheB8ratandNOD mouseindicatethatIDOMisT-«U mcdWcd(O'Gam.etaI.,1997). Thereisevidencethat bothCD4+andCDS+-T cells are involvedinthepathogenesis oCIDOM,throughstudiesusing depletingantibodies againsteither subset(Kikutanid II.,1992).Howewr.thereareotherstudies thatshow CD4+Tcellsalone caninduceIDOM(Katzetal.,1995).IIItheIDOM modeithespecificpathogenicAgUsnot boondetmnincd, yetth.lkeUproteingIutanU• .ad d_ xyluc(GAD) ... likely candi<iate(Druct et al.,1996).TheideathatmDMis. Tbt-mediateddisease:issupponcdby the observationthatT cdls fromNODmiceprodu.celargeamountsofIFN.'Yinresponseto GAD (Kaufinandel.,1993,Ttsebelal.,199 3).Also.theincreasedexpression ofIFN-yunderthe controloftberatinsulinpromoter(RIP)resultedillinflammation of isletsanddiabetesin

12

tnnsgenic:mice(Sarventni ctCIaI.,1990.StewartdaI.,199).BothIFN-y(CampbellCIaI.•

1991)andD..-J2(Tranbleauetat. 1995)havebeenshownto promoteIDOMinmousemodels.

1Jladdition, asintheEAEmodel.Tcellclonesspecific:forGAD.thathadthe abilityto accelerate mOMinNODmice.produoc:dfbitypecytokinesafterrestirrKllationinvitro(Bergmane aI.•

1994).

1.1.2n ltoTh2immuDe dniltio. i.luloimmg.ify

Manystudies have focusedon switchingthe 'proinflammatory'Tb1 responseinorgan- specific:autoimmunityto.'protecti ve 'Th2response(reviewedinO'GametaI.,1997).

However,the effectivenessofTh2 cdJsin havingaprotectiveand/or therapeuticeffectmorgan- specificautoimmunityhasyet tobedefinitivelyshown.Insome cases such uin imrnuoocomprimihosts.lb2cellshavebeenshowntobepathogenicintheirownright (Lafaillee..I.,1997,PabIaet~.•1991).

In theEAEmodel.thefirstanemptstoamelioratethediseasewere made using monoclonalantibodiestotheproin1lammatory C)1okines Lt2mdIFN-y.Administration of a amibodies toL12inmicefollowingadoptivetrans1'er ofPLP-primedLNC(LeonardetaI., 1995)or immunizationwithPLPinCFA(GijbclsClaI.,1997)inhibitedthedevelopment ofEAE.

InCOntt'Ut,the use ofIFN-y specific: mAbshadDOefl'ec;t on EAE(DoungetaI.,1992)orwas showntoenhmteitsdevelopment(BilliauetaI.,1988).OtherSlUCfiesinhunw\sindicatedthat

13

treatmentwith lFN.yeu<erl>alcdMS 'l""ptoms(panitch "II.,1987).

Theusc ofTh2cytokinestoskewtheinftammatoryresponsehasalsobeenstUdiedinthis modd.InEAE.uin thehumancondition.thereareoften stagesofrdapseandremission,and during the stage ofreccvery,IFN-yRNAandproteinlevels arelowwhileIL-4,IL-10andTGF-~

levdsan:high,imp!iatinS'Tb2responsewithroc:ovuy(K!lowy"II.,1992,KennedyetII., 1992).IntheEAEmodel.adminisuaiooofD..-4.IL-IOand ll..-13hushownbenefiWJeffectson the progress ofthe disease(RIckeC1aI.•1994,RonetaI.,1994.CashetaI.,1994).Yet there have beensome disctepanciesbetweenthesefindingsandthe studiesofothers.pantybecauseof differencesinanimalmodelsandtimeof cytokineintroduetioa..Forexample. earlytreatmentwith fi....4amelioratesEAEinan adoptive transfermodel(RackeCIaI.,1994),yetn..-4administration at the time of diseaseinductionor onset exacerbatedEAE(GijbelsetaI.,1997).Inthesame study,the weorIL·IOIwllittleelf"".Enc:ephalitos"'"'Teeuh}bridonwuansduccdwith.

retrcviralgenetoexpressfi....4delaytheonseI:ofEA£:whenadoptivelytransferredinto mice immunizedwith MBP(Shaw etaI.,1997),andsimilariyPLP-specific memorycellstransduced withIL-IOeDNAwerealso showntoinhabitEAE(Mathisenet al.,1997).Finally,theuse of PLP-derMdalteredpeptideligands(APL)which _ Tb2ccU,ronfencdprotet:tionfrom EAE inducedbytheadministrationofPLPinCFAwhenadoptivelytransferredintomice (Nicholson et al.,1995).

Inodditioototheabovestu<fi...Khoruts..II.(1995)foundIIwPLP(139-151)-spcci1i<:

Tb2 cells<OUktIlOlsuppressEAEinducedbyTbIccUs

LaWll."

II.(1997) foundIIwin immunodeficienthosts.,

Jh2

cellsClWsed •fonn ofEAEwithallergy-likepathology.The above datasuggestthat the ideathatThIceUsare pathogenicandTb2cellsare protectivemaybetoo

I'

simplisticIDddwtheremaybesomeothertypeof regulatory medwtismromingintoplaythan theThltrb2balance.

[DVM,

InthemDMmodd.,antibodiesto

n...

12RandIFN-yhavehadbeneficialeffectsonthe disease(Liblau etaI.,1995).Also.administrationofII....4prevents diabetesinNOD femalemice (IlapoponttaI.,1993).AdminiSlBlionoeLIO amdiom:cs diseaseinNODmice(pennline et aI.,1994)yetmousepc;dbwith 0..-10 expressedby. transgcnearedestroyedmore rapidly (WogcnscnetaI.,1994).This destruction maybe dueto .Iocalizcdhighconcentrationof the cytokine.Rccemliodinp inthismoddhavebeeDsimilartodatiobtainedin theEAE modd.in that Th2cellsthemselvesarecapableofinduc:ing diabetesinimmunocompromiscdNOD-scid mice(pakalaetaI.,1997).Theseresults also suggestthat anothertypeof regulatory Tcell,such as TregulatoryedI1(1rI)or Th3 maybeimportantinthedevdopmentofIUtoimmunediseases (pabla .ul.,1997).

NovelapproachestothestudyaCtbe ThlfIb2baJanceinautoimmunestates include modelssuchasH.-t OandIL-4uansgc:rocanddeficientmice(Bettdli etaI.,1998),TcR antagonists(AndertonetaI.,1998),genetic imrmmization\Vimeytolrinegenes(Ramshaw etaI.•

1997)andthe usc ofandrogens toaffecttheThlfI'h2 shift.The resultsin theseexperiments supponthe ideathatother!Klanbesides theThlfI'h2b&lance.suchasregulatoryTcells,are involvedinorgan-speeificIUloimmWlity,andthai anypotentialimmuno~pymust takethese factorsinto account.

IS 2.2.3Tb uIIIiaEAT

The role of'Ihland Tb2ce1lsininununorcguJation orEAThasnotbeenas well tban.cterized.EATinducedbyimmuniutionofmieewithTginCFA.is consideredtobe mediated byCD4+Tcells(KongetaI.,1990)andthesecellsarebelievedtobeoftbeTbltype (DruetetaI.,1996).11basalsobeenshownthat theCD4+Tce1lsinfi1tn.tingthe thyroidsof patientswithHTare oCtheThlrype(Miossec.1997).Thishas notbeendefinitively showninthe mousemodel.although,as described below,muchirJdirectevidenceindicatesthat they are Thl.

n.-12has been showntoexacerbatethegranuJomatousformof EAT.resultinginan extremdysevereanddesuuaiveformorEATinrecipieatmice(Braley-MullenetaI.,1998).

IntrathyroidalinjectionofIFN-yalonebasabobeenableto induce EAT (Remyetal.,1987) MAbs toIFN-yduringthedevelopmentorEAT significantly attenuatedthe diseaseinmice(Tang etaI.,1993).Using

mice

thatweredeficientintheIF'N.yreceptor gene,AIimietal.(1998)found thaialthoughIFN-ywasoot requiredfortheinductionof EAT.itwas required for progressionto tunblowndisease.Contradicting thisfinding,injectioaofmAbs to IFN-yintomiceimmunized with19hasresultedinan~ledformof gnnuIonwousEATinnaivehosts,whithis inducedusing rnTg-aaivatcdspleenceIlso(theimmunizedmiceculturedin the presence

or

anti- D..-2-R(TangetaI.•1998).lmrnunizationof IFN-y-deficient micewith 18hasalsoresultedin this cucerb&led.formofgnnulonwous EATin thenaivehosts(Stulletal.,1992).Theabove dataseemtocomradia:theassumedroleofTbl eeusISinitiators orEAT.

The use ofTb2C)1okines

n....

andfL.10has alsoyielded interestingresultsinEAT studies.Mignon-GodefroyetaJ.(1995)studiedthe effea ofinvivoadministrationof recombinant

16

bumaD IL-IO(rftlL.IO)onthedevelopmentofEATfoDowingdirectimmunization with mTgin CFAor adoptivetransferofmTs-specific: Tcellsinto naivehosts.They found thatadministration ofrfill..IOwas~c:f6cicntinbothpreventingandtreatingEAT.Theycontributed thisfinding inpan to rhll..-IO'senhancementof T-cell dwh.Inthe'transfer EAT'model, spleen cells from CBA/1doDOrspreviouslyprimedinvivowithmr g wereeulturcdwith orwithout rII..4 or rn.·I O.

L IOwas shown to decrease mTg.speci6eproliferativeandcytot oxicTcdlresponses.whilen.,.

4hadlittleeffea (MignoJKJodefroyetat,I99S).Anothergroup.de laVega eraI.(1998) found that LIDmRNAexpressioninhuman th)TOid canbeincreasedinthyroidautoimmunitybut only inusesof severeinfiltration.Thepresenceof albl·inhibitingc:ytolcineinlesionsthatcontainT cellsof the Thlcytokincprofilewas not explained.Incontrastto thisfinding.the application of a plasmid containingIL-IOontothethyroid.inlIiwJhad.curative drec:lonEAT(BaneuxClal., 1999).Theuseofthe plasmidwas shown to inducefastandlong lastingexpressionorn.·IDin mouseTFC.Itresultedin•loweringof the mononuclearcellinfiIuationof thyroidglands anda diminishedanti-18Tcellprolifention.Italsoinduced• trendtowardtheTh2response. indicated by. decreasedproductionofIFN..yandincreasein theratio oflgGllIgG2a.In the gmwlonwousEATmodd, theadditionofrll..-4ill\IItrotoo.1Ituresofspleencellsfromdonor miceimmunized with18badlittJetonoeffecton the dcvdopment orEAlinthenaiverecipient mice(Tang etaI.,1998).

Theseresulu indicatethatforEAT.themostpowerfulThl·inhibfting factoris

n..•

O,Vrr'ith Q...4playingljttleroleindecreasingthe severityofEAl.Theyalsoshowthatnovel tedmiques.

such asthewe ofplumids encodingcytokines toinduc:e cytokiDeexpressionbyTFC (Baneuxet:

aI.•19(9).maybeSU«eSSfu1inboththeprevention andUeatmenlofthyroidautoimmunitylothe

17

3.T CELL DETERMINANTS IN EAT

3.1DJ1"'OCIobulilllS . . alltizm

Thyroglobulinis•large,highlyconservedglycoproteinwithIhomodimeric:molecular weightofapproximately660kDa.It istmISCribcdfromIgenefoundon chromosome8in lIJm&nsandon chromosomeISinmice.TIissyntbesiudin theendoplasmic:reticulumof tbyrocytel,modifiedpost-transWionaDyinthe Golgi,andthenseemedintothefollicular lumen (VanHcrle eta1.•1979andEkholm andBjorlanan,1990).Itis expressed onlyin thethyroid gland,whereitis themainconstituentofthcc:oUoid oftbctb)Toidfollicles(CaturegiietaI.•

1997).llSmaUlfunction~inthesynthesisoflhyro;dhormones,throughiodinationof n.tyrOSine$

(Catureglietal.•1997)10formT.(thyroxine)andT1(triiodothyronine),yetit hasbeenrecently shown topossess TGF-tlactivity(Huang eeaJ.•1998).

Tghasbeenthemajo<...cantigenDnptiatcdinthepathogenesisofEAT.In1fT.bolhTg andTPO are believedtobemajorIUCoantigens.In1956,Roitt et11.showedthatpatients with fITdevelopedTg-specificantibodieswhileRoseandWitebslcy (1956)foundthatimmunization of rabbitswithTginCFAresultedinthyroidinfiltration.Sincethattime.nwnerow:sud ieshave confirmedthe pathogenic roleofT ginautoimmune: tb)Toiddisease,andTg-specificT cellsin both the inductionandeffector phases orEAT (reviewedbyKong,1994).

T8isalso ahighly conservedmolecule.'ThreespeciesofTghavebeenclonedandfully

18

"'lUencedIttheeDNA 1M.TheseIn:humanTg (hTg)(MaItlliayClI1.,1937),bovineTg (hTg) (MerkCllClI1.,1985)andreccntIymou.. Tg(mTg) (Caturegli ClI1.,1997).TheC·

terminusoCm thyroglobulin(r1g)hasalsobeen sequenced(DiLauroeeaI.,1985).Amongthese spec:ies thereis •>70%identity intheLa.sequencewith,lUghdegreeof conservative substitutions(Can.yanniotisandRao.1997,CatureglietaI.,1997).ThishighdegreeoChomology iDdic:atesthaIpathogenicepitopesof 18 mayalsobehighlyeoeservedacross species.Tgalso has

• highdegreeoftmemalbomology(MaItlliayeeaI.,1987,Merken ctaI.,1985)wheremorethan 75'1,aCthe sequenceiscomposedof3typeS.or domainsof repetitive sequences(Can yanniotis andbo. 1997).These domainsare;DomainA with 10tyrosine-andcysteiJle.richrepeatsof SO La.betweenpositions29-1196.DomainB whichbasJrepeats of 14-17highlycooserved residuesbetweenposition1436-1483,DomainCwith SrepeatSbetweenposition 1583·2109.The last Domain(0) lacksintema1homologyandisat theC-tc:nnina1endofthemolecule.

There are 11leasttwofeaturesof 18 synthesisthatmayaffeclgc:nention ofits path~cepitopes.FtrSlly,19isnot. sequesteredantigen,butit iscontinuouslyreleasedinto thecirculation,alongwithIsandT. (CarayanniorisandRao,1997).Itwasbelievedtobea sequesteredantigenuntilthedevelopmentofsensitivetedmique$ tomea.sureTgin circulation (SdmcidcrandIkekubo.1979).IthasDOtbeen determinediftlUs released18isintactorrdcased insmallerfragments.ProcessingofTg fragmentsbyAPein

"'\10

maydifferfromtheprocessing ofintact18inIIItro,resultingin thegeneratioa of pathogeniccpitopc:s. Secondly,iodinationof T8mayinfluenceitspatbogalicity.TheTgmolecule,consistingoftwoidenticalsubunits.ismore stablewhen iodinated(Championet&1.,1987).Championet&1.(1987) also foundthat poorly iodinatedT8failedtodicitEATinmice,indicating thatdominantpathogenic epitcpes maybe

19

iodinated.Also,epidemiological evidencesuggeststhat geographical areas ofhighiodine eotrdatcwiththeincidenceof thyroidIUtoimnwnity(BouJcisetal,1983,SzaboIcsetaI.,1997).

3.2 Epitope MlppiaJortb~TbyroglobaliamolKu~

3.2.1 Mlppcd tlI)TOIIobulia epitopa

Identificationof pathogenic TcellepitopesofTgisvitalto studyofthyroiditis.Itisalso erucial tothedevelopmentofpocentiaJ strItegies forimmunetherapy.Due tothemolecularsize afTs. epitope mappingtechniquesbased on overlappingpeptidesare both impracticalandcostly.

nw..

other meansoffindingepitopesIWStbe employed.takingintoIICCOWlt CUrrentkDowlcdge ofbow peptides;"'endwithT <dis.TheseothermethodsincludealgorithmstIw predict pcptidesthatmaybindto MHC molecules,andthus are candidatesforTcell.eti....tion.Others includestudyingthehomology ofTgwithotherthyroidal antigens suchas TPO.the exploration ofpept.idcscontainingthe bormonogcnic:sites cfTg,andthe useof hybridomas derivedfrom lhyroid Jcsions.

To date, there are five mapped T cell epitopesinT8.Thelargestoftbeseis.40a.a peptide [Tg(1672-171 I)], discoveredbyTexieret al.(l992).Thisgroupused a cytotoxicT

un

bybridoma (KIO),as amappingtool.HTC2hadtheabilitytolyseinvitro,macrophagcspulsed witheitherintaaporcinetbyrogIobulin(pTg)or asmalltrypsinfragmentafpTg.Theyalsofound thatthesmallerfngmcntoepTghadthe abilitytoinduceEATinH-2'strainmice.Pre- immunization of these micewithHTC2conferredprotectionfromEAT.Thisindicatedthatthe

20

peptidefngmentwasindeedpal!log";c.Theypurifiedand sequencedthe pTg-peptid. fngmenl andfowldthatitbad70%bomoIogy withtheknownbTg eDNA sequence.Asthe sequenceof pTgwasunknown,they syrrtbesiud. peptidefromthehumanT8 sequence homologousto tJUs region.Theyimmuniz.ed mice with hTg(1672.1711)anddiscoveredthat EAT was induced They alsofoundthatautomtibodiesinthe sen ofthese

eeee

weredireaed onlytothe peptide, bTg(1672-I7II)and DOlintactbTg

Therestof the four mappedT ceOepitopesclusterattheC-terminalendofthyroglobulin,

• eysteme..poorregion homologousto acetylcholinesteraseandother esterases (MaltJUeryand Lissitzky.1987,TakagietaI.,1991).Romandcolleaguesstudied a sequencecontaining.

thyroxinesite u. possiblepathogeniccpitope.Inhumans.T)andT. residuesmaybefanned fromtyrosinesat positionsS.2553,2567,and2746(HutchingsetaI,1992).Theybelievedthat iodinationatthesepositions mayaffea~.Previous studiesbythisgroup indicated that twomurineTgautoreac:tivc bybridomasrecognizedan epitcpecontainingT.acposition 2553.The peptideepitopeTg(2549a2S60) lostitsimmunogcnicityifthe T. at position2553 was substituted with any otherI.a.ThepathogenicpotentialofthispeptidewastestedinCOAlJ(Ii- 2')miceanditwasfoundthat thispeptide _ EATbyadoptMcnnsf"butDOlbydUect cballeage into recipientmice.ThispeptidealsoinducedT.eellproliferativeresponsesand antibodyresponsesinmiceimmunized \IrIith18(2549.2560).

HoshiobetaI.(1993)investigatedthetwokno9rr'n thyroidantigens.TgandTPOandthey sc:arched(orcommonsequencesthat mayaa uT-epitopes. TheyscannedthehumanTgand TPOsequencesandf~fourS a.Lresiduesequencesthatwerecommoninboth.Theythen testedtheTgaDdTPO pcptidcscontainingtheseidenticalresiduesfor their immunogenicityin

21

mice.TbeyfoundthatTS<273~274])wasantigenic.aswaitsTPOcoumClpUt.TPO(I1S.131) (Hoshioka eaI.,1993)iDthatthey irxfl.adEATin mice.yetonlythroughanindirectmelhod.

Theyfoundthatwhensplenocyte!frommiceimmunizedwithmTSwere stimulatedin vitrowith thesepqmdcs.asweDas with mTg,theytnmferredthyroiditisintonaiverecipients.Thefaa WI thyroiditisc:ouldnotbedircafyinducedin animalschallengedwiththis peptideinCfAwas•

majordnvro.ekofthisstudy.

Carayanniotis andcoDeagueshave mappedtwopathogenicTcellepitopesattheC- terminalendafTS·Thetim,r1g(2495-2511 ).designated TgPl.wasfoundusing lhecomputer algorittuns•AMPHI'and'teeaee-motif '(Chronopoulouand Carayanniotis,t992).Thistr-mer peptidewtlich_ _in!belIvyoidsofEATsusceptiblesuainsof mice(SJUJ,

em

&tidBIO.BR)butnotEAT resistantarains(BALBIc and BIO).Itwaslaterfoundthatthispeptide alsoind~EATinrats(Balasa et 11.,1993).TgPlWISidentifiedas non-immunodomiJwlt,as TgPl-primedTcellsdidDOtresponsetointactmTgortTSin

w U'O

and.convcne/y,mTS-and rTg- primedTcellsfailedto respondto TgPl. Serologically,hTg-primedmiceshowedstrong mTg-specificJgGresponsebutnoresponseto TgPl. However,19Pt-primedmicewereshown toelicitstrongIgGresponsestoTgPlthataossreactwithmTg,rTg, bTg.bTgandpTg, indicating•stronglyimmunogenic:peptide.Thisgroupbassincedelineateda9-mcrpeptide of TgPl,(2496-2504)

IS.

minimalpathogenicepitope ofTg (RaoetaI., 1994),

Anotherpeptide.rTg(269S-2713)wasalsofoundusingthesametompUteralgorithms.

andwasdesigrwedasTgP2.Thispeptidehada unique genetic patternof~u<:tionofEAT.It causedEATinSJlJJmice(H.2")micebutnotinH_2^lmice(C3H,B10.DR),whichare typically 'goodresponders'toEAT(Canyanniotisetai, 1994)_EATinSJlJ)micewasinducedboth

22

dinctJyandviaadoptivetransfer,andtheTeeDsofthesemiceproliferated;n vitro toTgP2.All strainstestedexJubited TgP2.specificIsO (Can.yanniotisetaI.,1994).

OfthefiwTedlepitopestNthavebeendisco....cl, nonehasbeenshowntobe immunodominant.'Ibisisnotparticularlysurprisingconsidering thesizeoftheT8molecule. However,recentdevdopmcnuEnthefield. suehISthedetermination of the sequence for mTs (Catureglietal.,1991)andthe aystaJSU'\IClUTeofthemouseA' molecule(Fremontct11.,1998) mayleadtothediscoveryofotherTcellepitopes.Due tothehighhomologyacross many speciesof Ts.thesenew epitopesermTgVriIllike/ybesharedaaoss other speciesof Tg.

3.1.2

n e

^UK

.r

^alcorithms18 epitope mappiDC

Vladutiuand Rose(1971) cliseovered that miceo(H·2'or H·2"haplotypeSare'good respond",',highlysus, . ptibletoTg-induced EAT.Miceof theH-2'andH.2' haplotype ee d&ssi.fiedas'poor responders''WithlowerTceDresponsesandIirtlethyroidtnfiltntio n (Vladutiu andRose,1971).Susceptibility to19·induced EATiJcomroUed bygenesinthe H·2A subr<gK>ns,whilethe H2-DandH-2Ksubregionsha", _ control(BeiselandRose,1983).

Forthisreason.algorithmsscreening the T8 sequencefor the existenceofpeptidcsbindingtoH·

2AandH·2Einsusceptible

H·r

miceareusedto map pathogenicT cell epitopes (A1tuviaeta1., 1994).

There are othermethodsused topredict T ceUepitcpes.Antigen-specific T cell hybridomasdaived frominflammatorylesions havebeenused to predict Tcellepitopesof myosin inmyocarditis(Donenneyerelal.,1995)andTSinthyroiditis(Texier etal., 1992).Forsmaller

2J

antigens,suchasmyelinproteolipidprotein(p1.P).anantigenimplicau:dinthepathogenesisof EAE,overlappingpeptidescoveringthe whole moleculeweretestedfOrtheirabilitytobindto A moIccu1es,anduItimaIdycauseEAE(GRor" 01.,1996).Thisoverlapping-peptideteehnique wouldbeverycostlyandtimeconsumingifperfonnedontheTg molecule.

Earlyalgorithmswere developedempiriea1tyandwere basedoncommonfeaturesfrom peptidcsbindingtoAorEmolecules.AbrcaJcthroughin thisareac;amefromADenetal.,1987, whofirstdemonstratedthat certainfeatures ofapeptide assoc;:iatedwiththe A'molecule(inthis case HEL(S2-61)a dominantepitopefromheneggIyzozymc) wereresponsibleforit!

imrmmogemcity.Theywere abletoshowthrougha.a substitutionanalysis thatwithinapeptide.

certain I.a.residues make contactwith the A'moleculeandothers make contactwiththeTcR.

ThesediscoverieslaidthefoundarioaforstUdiesthat lookedatmWtiple lmmunogenic peptides andascertainedconunon"I.sequencefeaturessharedbyaDpeptidcsthatboundtotheA~.They establishedthatthepresenceor absenceofcertIin"I.11given positionswithinIpeptidebasI directimpacton itsimmunogcnicity.Thestudyof manypeptidesthatbindto moleculessuchas A^kand the commonfeaturesofthcse peptidcshave resultedinthe formationof algorithms

BothA'andF moleculeshavebeenstudiedcxtensi\'Clyandseveralalgorithmspublished, estimatingpcptidesthatshouldbind toit(reviewedinNelsonetaI.,1996).AlTuviaetal.(1994) developed•computerizedmethodfor recognitionof peptidcsbindingtoA' or E' usingacobon ofpc:ptidesthaiwere

mown

tobindtotbc:semoleculesandelicitITccDresponse.Asacontrol theyused another cohort ofpeptidesthatfailedto bindto A'orE~.Motifs wereconstruct edthat consistedofbothpbysical-cherrUcalandstrudUn1propertiesoftbeaminoacidsthatcould occupy apartiQ.llarpositionintheprospectivepeptide(AlnMaetaI.,1994).These motifsspecifya.aat

24

certainpositionsIScriticalforthebindingofpeptidc10

MMe.

AstudybyKelneret aI.,1992 supported.theideathatA'andE'-binding peptideshavecritieaJanchorresidues.Thestudy demonstraledthat.singleLa.substitutionin theAk-rcstriaedpeptideMteyt (93-1 04)(mouse testicularcytochromec)atposition 96 froma.a.lysinetoalaninechanged therestrictionofthe peptidetoE'.'IbisiDdicatedthatthisLa.wummebowaltic:&Jfortheisotypespec:ilicity.Several other studies(NelsonetaI.,1996andItohetaI.,1996)havealsodemonstratedthatcertainLa areessentialforIpeptidetobindtotheA'molec:uJe

AnA'- peptide bindingmotiflw~beenpublished. stemmingfromthediscovery of theaystaJstructureofA^kincomplexwiththedominantepitopeHEL(S0-62)(FremontetaI., 1998 ).Thisdiscoverygives.visualrepresentationandastJUawalbasisfor an accun.teA^k peptide-bindingmotif.ThecrystalresolutionindicatedthatIpeptidebindstothe A' molecule by the'burial'of5 ....sidechainsinto pocketsintheAgroove.TbcsepocketSarefoundatposition 1.4,6,, and 9 (FreemontetaI.,1998).Ofa11thesepockets,PI edUbits the most exclusive binding,withtheLa asparticacid •nearperfectfit.Position P6basthenextmostexclusive fit, acceptins titherglutamineor glutamicacid.Position9typicallyac.ceptssmallresiduessuch as serine, but will accepc:largerresiduessuchISthreonine.Positions4 and7willaccepta larger numberof substitutions,yetforP4mediwn-sizehy(ltophobic:residuesarepreferredandP7can acceptanyaminoacids except thosewitha positive charge.Inadditio n.the crystal structure also lrnp6QtessevenJpositionsaspossibleTcRcontldresidues,duetotheirhigh exposureto solvent.Theseoccurat positions3, S,8andII.Thisstudyconfirmsmmy oftheassumptions of previousmotifsanditprovidesclearrulesthatdi<:we peptide--binding10 A~.

NOTE TO USERS

Pagels) miss ing in number only ; text follows. Pagels) were microfilmed as received .

25

This reproduction is the best copy available .

UMl

26

CllAYTER2 MATERIALS AND METHODS

2.1ANIMALS

Female Sllll.011. COM.AKR.mdmal. Sllll. ...werepurclw<dfromtheJackson Laboratories,Bar Rubor,ME,USA.Thefemalemicewere used for immunizationsbetween4- 10wceIcsof age.ThemaleSJlll.. micewereusedfor extractionofthyroid,spleenandlivertissue onlyandwere\lSCIdat2()"2Sweeksofage.

2.2ANTIGENS

peptides designatedTgPl [rTg(249S.2SI I)JmdTgP2[rTg(2695-2713))weresynthesizedII

>95% purity at the AlbertaPeptide Institute(Calgary,Alberta.Canada).They were synthesized Oftan AppliedBtosystCllU(Foster City,CAl 430Asynthesizerusing.generalprocedurefor soUd-phasesynthesisnuilincdbyEriksonmdMerrifield(1976)withmodiJialions byHodgeset at (1988).Thisprocedureinvolved hydrogenfluoridedcavagc ofpeptideresinat·S-Cforone hourinhydrogenflouride:anisole:dimethytsulfoxide(DMSO):p-thiocresol:peptide resin (IOmI:lmI:O.SmI:O.2ml:lg).peptide puritywuossessedbyIlPLCmdmassspearoscopicanoIy·

sU.

Peptides of rTg used toimmunizemicewere chosenusing overlappingAandB motifsfrom

27

Altum ..

aI.(I994~u outIin<dinsection:UThese ...Ts(1827-183~Tg(2()29.2040~

Tg(2108-2125), Tg(2424-2437)ond Tg(2597-2609).ThesepeptideeweresynthesizedbySynpep (Dublin, CA, USA).

:uALGORJTIIM-BASED SEARCB FOR A'-BINDING PEPTIDES IN TG

'Ibepaper entitled'SequenceFeaturesthat CorrelatewithMHCRestriction'byAltuviaetII (1994) desaibed analgorithmforpredictionofpeptides that maybindtoA~orF-molecules. From this paper.weusedtwomotifsto screenfor peptidesthai:maybindtotheA~molecule (Table 1.1).The pcGENEsoftwarewasusedtoSCIIIthe961a.a.on the C·tenninalendof the rTg moiea1lc(DiLauro et&1.,1985).andlaterthecompletemTSsequence(Caturegliet&1., 1997).We initiaDyusedrTgbceausethe completesequencefor mTghadnot been publishcd Usingthisprogram.we fOWld sequenceswithinthisportionofrTg that fit motifAandmotifB, sepanteIy.

w.

^thenebcse peptid.. inwhichmotifAandmotifBccmpIetelyoverIappecIwitllin the peptidesequence.Wethenalignedtheponion aCmerTg sequencewiththeknown mTg molec:ule(withoutthepresenceoftheleadc:rsequence)togetthepeptideco-ordinatesof our candidate peptides

28

T. bIt 1.1Motif.Alad Bwitbit.A'·bia di acptptida(AltllYiattai.1994) Position "

MOTIF A I

MOTIF 8 I

Characteristicof La.

hydrogenacceptor.non-bydropbobic.

notsmall.alipbaric any a.a.exceptamides anyu.

medium-Sud. oIiphalic, hydrophobic nocharge,cannotbeatomaticoramide anyu

anya.a.except uomatic hydrophobic,DOcIIarg. andnotbe Anamide

hydrogen acceptor,notsmall.polar, non-aliphatic

anyu.

medium-Sud.oIipllllic.hydrophobic no charge,cannotbearomaticor amide hydrogenacceptor,oon-hydrophobic., notsmoll,oIipllllic

IncludclExcludea.a.

iadudt:S:asparticacid., glutamicacid.,histidine.

asparagine.glutamine e-d uda:asparagine., g1uwnine iad udaalla.l.

iad udes:isoleucine.

leucine,threonine.valine bldudesall •.•.

cldudes:phenylalanine, histidine.tryptophane iDdudes:alanine, cysteine.

phenyialanine.isoleucine.

leuc:ine,methionine, proline.threonine.valine. ttyplOPhanc. ".,...,.

bld ades:CYUcine.

asparticacid., glutamic acid,histidine,asparagine, glutamine ud uda:a.spartkac:i.d., glutamic acid I.dudaanyLa.

iDd udcs:isoleucine.

leucine.threonine, valine ia d udes:asparticacid, gluwnica.cid.,histidine.

gJuwnine,

ospuog;n.

29 U CULTURE MEDIA

All ....!"weeperformed inDu!bcc<o',modifiedEagle(DMEM) medium(Gibco, BorIington,Onurio,Canada)suppl...edwith10'"fetalbovineserum[(FBS)(BKlprodueufor Scieoce,lndianapoIis, lN,USAL20mM IlEPES buffer, 2mML-GIuwnine,100unitslmI pencicillin,loo ..w mlSUeplomy<in(aDfrom Gibco)IIldS.10"'M2-...ptoetbanol (MEXSigmaCbenUcaIs, SI.Louis,MO,USA).

1.5ANTIGENICCHALLENGEOF ANIMALS

1.5.1 bdactioaoraatip_specificLNC

MicewereinummizedwithS0-200~ofTgPlorTgP2,or 100nmolofeitherofthesix peptides[Tg(1827· 1837),Tg(2029-2040),Tg(2108-212S1Tg(2424-24l 7),orTg(2S97-2609)) ina1:1emulsionmComplete FreundsAdjuvant (CFA)(withMycohot:tm llJJlIntt)riaun. Difco Labontoriesln<.Detroit,MI,USA) They __inunwUzcdsubcutaneously (underether anaesthesia)at3sitesalongtheback.After9-11days.somemicewereeuthanisedwith etherand

theirdraininginguinal.auxiliary andbrachiallymphnodescollectedaseptically.A singleeeu suspensionwas prepared by passingtheIympb BOdeuS5LIethrough•sterilestainless seedwire

mesh(Si- . StLouis,MO,USA111ldwashedincompletecuItun:mcdUlPMEM+10'"

FBS)

we

wereculturedin96wellplates11aconcentmion of 4:II:10'celbIwellfor 4

days

inthe

30

praeooeoftitratedamountsof the appropriateIDtigatin200~microeultures.Eighteenboun priorto harvesting.1IolCiof'[H]-thymidinewasaddedtoeachculturein25IJ,Iofculture medium.Theoellswere harvested18hourlater. usinga semi-automated~harvester(Skatro n, Sterling. VA)andincorporatedthymidinewas countedinaliquidseintillationcounter (LS3801;

_lnslJumcnts.PaloMo.CA,USA) StimuIaliooindex~clefincdIS(CPMinthe presenceof antigenICPMin abseDc:eofantigea).

To determineifthe peptidcswere thyroiditogenic.somemicewere boosted at 3weekswith SOnmoIofpeptideinIncomplete Freund'sAdjuvant(lFA).Aftertwo weeks,thesemice were bloc!, Uldtheresultant .... usedinantibodyWSA(see%.602).TheUthyro;dglandswerethen removeden blocwiththetracheaandfixedinbufferedformalin.Thelobeswere then dissected fromthe tracheaandembeddedinmethaaylatc.Approximately40 sectionsat3.0J.11nintervals.

wereobuinedlbroughouteadlgland.Theywerefixed

'0

gla>sslid..Uldstainedwith haematoxylin andeosin.Scoringwaspafonncdasfollows:0 '"'00infiltration;l-im mtitial accumulationofinflammatorycells;2'"one or morefociof int1&mmatory cellsat least thesizeof one follicle;)-extmsivcinfiltration,10-40%ofthe loWarea.4-extensiveinfiltration,40-800/0of the totalarea;,S&CXtensiveinfiltration,>80".4 ofthe'toWarea.Thehighestinfiltrationscore observedperglandwasassignedtoeachmouse.

1.S.2IDduC1ioa

.rnt

IDdT1a1cdb

Antigen-primedLNC generatedas describedin2.5.1wereculturedinvitrointwo separate selUP'.Theywere placedinculnuescontainingeither 10' ceIlsIml,10>'S'm1peptideUld30og/m1

31

rn....4 (Gibc:o), or a culturewith5x 10' ceUslml,10~Wm1peptideandno exogenousC)'tokines.

Thisfollowedthe protocoloutlinedinKhotutsetaI.,1997.Cellswerethenc:ulturc:dfor 4to 10 days.LNCare resuspended fromcultureandwashed incompletemedia.1·5 x10'eeDsarcput into 2mlofmedia.and3m1sterileFicoU-Hypaque (phannaciaBiotechInc.,Baied'Urfe.

Quebec.Canada)layeredbeneathit.Thetubewu thenspunina centrifuge (BeclonanGPR Centrifuge. Becbnan, PaloAha.CA, USA)&12000 rpm for 1Sminuteswith no brake.TheeeUs werethenremovedfromthemedit/F'1COIlinter&ceandwashed ineompletemediaandcounted using0.02%uypanblue(Gibcc,Burlington, Ontario,Canada).ViableLNCwerecultured at a c:onccntTation of Irf' cellsfmlandrtStimuIated in thepresenceof 10 I!IVmI of peptideandSx 10' cellsfmIofmitomycinC·treatedsplenoc::ytes.SpleenswerecoUecledaseptieallyfromeaivebcalthy SJUL miceandsingleeell suspensionswere preparedbypassingthespleentissuethroughsterile stIinIesssteelmesh(Sigma.St.Louis.MO. USA).The resultingcell suspension was pipened vigorouslytoobtainan uniformsuspension of cells.TheccUswerewashed3 timesincomplete medium.adjustedtoSx10'c:elIsimIandincubatedIt3rtwith 100IJ8of MitomycinC(Sigma.

51.Louis.,MO,USA)for 20 minutes.avoidingexposureto light.The cells are then washed three timesincompletemediaandusedasAPe.Inaddition, viable LNC werealsousedinLNC proliferation assaysISoutlinedin1.5.1.After48boon inculture.theeeIlsupernatantwas collectedfor determination of cytokinelevelsviaC)1okineELISA

32 1.6ENZYME-LINKEDIMMUNOSORBENTASSAVS

1.6.1CytOkiD~ELISAforusasm~Dtof cytokinaincdl culturt'IlIptmataat

Theenzyme: linkedimmwIosorbcmassay(ELISA)wasusedfordetectionofq10kinesas follows.Disposable po/yWIyIchloride

96-_

ELISAplates(DynalechUbonlori...CbantiUy.

VA, USA)were coatedwithpurifiedanti-cytokine capturemonoclonal antibodiesata concentrationof2J!&,ml.ThesemAbs were:purifiedratanti-mouse lL-2 and lL-IO (PhatMiDgen.Mississauga,Ontario.Canada),andthepurifiedmAbsfromthesupernatant from thecelllineMOl70IDd11811(AmericanTypeCuItun:CeDection(ATCC).Rockville.MD.

USA).forIFN-rand

n.-4

respectively.Plateswereincubatedovernightat4-candwuhed with PBS buffer.Theywerethenblocked overnightwith1%eSAinPBS.AfterwuhingwithPBS containingTween-20(pBS.T),standardsofeachcytckiee were added.inaddition to thesample supem.atanlS.to generateastandardcurve.usingrecombinantll.-2.ll.·IO(bothfrom PbarMingeaMissi....ga,Onurio.CuwIa),IL-4(1l&DSystems,MinneapoU~MN.USA). IDd lfN·y (Gibec,llurUngton,Omario,CuwIa).Theplateswerethenincubaledovern;g!Ill1

."C.

andwashedagainwithPBS-T.Theywerethenincubatedwithbiotinylatedmanti-mouseanti-n..- 2,ll.-IO,IL-4andIFN-'Ydetecting mADs (pharMingen.Mississauga,Ontario,Canada)atO.S Jl.glml.Theplateswere inalbatedatroom temperaturefor45minutes..washed3timeswithPBS·

T. thenstreptavidUHIkaphospbawe(SigmaChemicals,St.Louis,MO.USA)odded.After a one-boutU>ad>I1K>nIDdwashing,100'" .fsubsttlle[1m&,m1~phospbale(Sigma Chemicals.StLouis..MO, USA)in10'1,diet.hanolaminc(Fisher Scientific. Napcan.Onwio,

33

Canada)]wasadded.Afteranboucincubationatroomtemperature,theplatewasreadon a micrcplolc'- (MolecularDeYk<s.SunnydIIe.CA. USA)at40Som.

:z.U Aatibody £USA (or UHSIme.t of aatibodyill~ra

Thepresenceof spec;ificserumIgGantibodywasdeterminedbyELISA.Microwe1lsof

polyvinylchlorideplate(DynateehLaboratories,Chantilly,VA. USA) wen:COaledovermg!ttwrth 10IJ8ofmTg orthespecificpeptide(describedabove)dissolvedincarbonate bu.fl'er.pH9.6.

Theplates wue then b&octed

ovemisht

withPBS+0.1%BSASen.samples or theTa-specific:

mAb55MB(.kindgiftofDr.P.Lymbcri,HellenicPasteur Instinnc,Athens..Greece)u •ccnrrct, were addedtotheplatesforone hourat roomtemperatureandtheplateswerethenwashedthree

timeswithPBS-ToThen thesecond antibody,anaIkaJine..phosphaweconjugated goatanti- mouseIgG(Sigma., St.Louis.MO.USA)wasaddedtoeachwell.AfteranhourItroom

_ !heplateswen:again wubedthreetimeswrthPBS-Tandp-nitrophenylphosplwe subslmesoIutioo(Sign>&, SI. Louis,MO, USA) wasadded(I mWm!p- ndtophenylphosphale"

10'''cfiethanoiamUle,100,.JlwdI)andincubatedfor 30 minutes Absorbanceof!hep- nitrOphenylalc product wasmeasuredat405omusinganautomatedmiaoplatcreader (MolCOJlar Devices,Suooydale,CA. USA).

34

2.7 ANALYSISOF ENDOGENOUSTHYROID CYTOKINE .. RNA LEVELS

1.7.1RNAntnttioa

Mi<:e wereeuthanisedusingether.thethyroidglandremoved~nblocstillattachedto the tnchea,and!henthethyroidlobes teased&omthe _ A piecerouglllysimilarinsize10the thyroidglandwasremoved fromtheliverandspleenofthesameanimals. Thetissuewas homogenized,!henplacedin0.5mIofTRlzoI(Mol«:uJarRosoarchCentreInc, Cio<innori.OR, USA).Thetissuewas then separatedinto(WOphases using0.1mIchloroformandcentrifugedat 4-C at 1200rpm.TheRNAremains

m.

theaqueousphase.ThisphaseWISplacedin.freshtube and precipitated using0.25mlof isopropanol.andagaincentrifuged.Theresultant pelletwas then washedwith0.5mlof 75%ethanol andair dried.ThepelletwasthenresuspendedinaJJlof cfiethypyrocart>on.(DEPC)treated_or.ThepurifiedRNAabsorbancewas!henreadin•

spectrophotometer atthe00of26Oand 280nmto determineRNAyieldandpurity.

First strand eDNAsynthesisWISperformed using. kit fromPhaJmacj. (Uppsala,Sweden) Briefly, 1-5~gofRNA(made uptoIvolwnc0£20JJlwithDEPC-treatedwater)wasplacedin an Eppendorfrubeanddenaturedin.Pedcin-Elmer^lWthcrmoc:yeIer&I6S-cfor 10minutes.,then chilledon ice.The RNA wasthenaddedto •pre-mixedsolution of11JJlBulkmix, 1JJl dithiothreitol solution, IJJlNot 1-d(T)18,aUprovidedbythe FU'SlStrand eDNAsynthesiskit.

35

Theresultant mixturewasthen heatedin thethermocyclcr(perkin Elmer DNATbennocycler, Cetus.,Norwalk, CT.USA)at37"( for I hour.andterminatedbybeatingthe tube at6SoC for 10 minutes.TheeDNAwasstored at-700c.

2.7.3 Amplificatiod byRT~PCR

PeRamplificationwascarriedout using reagents from thePROMEGATWPCRkit (Promege BiologicalResearchProducts,Madison,WI,USA).Briefly,1~of eDNAwasadded to 2~of eachoftheforwardandreverseprimers,purdw.sed fromUniversity Core DNAservices, UnivenityofCalgary(Calgary.AIbcru, Canada).0.4mMdNTPnUx,4mMMgCl,.2.5unitsof Taq DNApolymcrue.TheprimersusedwereforgIycen1dehyde.3-phosphatc dehydrogenate (GAPDH)[S' CCATCACCATCTICCAGGAG. 3' TTGAGATGATGCTTTGACAI. Thyroglobulin[5'CGGGATCCACCATGGGCCCTTTCCACTACTGGGG,3' GGTTGTCGATGTCGTTTACTCCTAGGGClandthe cytolcine, lL-2[ 5' AACAGCGCACCCACTICAA 3' TTGAGATGATGCTTTGACAl.lL-4 [5' TAGTTGCATCCTGCTCTI. 3' CTACGAGTAATCCATIGCI andJFN-y

[S'AACGCTACACACTGCATCT,3' TGCTCATTGTAATGCTTGGj. Theresultantmixture wasthen layeredwith SOJJ1ofmineraloil and placedinthe thermocycler(perkinElmerDNA Thermocycler,Cetus, Norwalk, CT, USA). A 3()..cycle step program(95"Cfor I minute.56"Cfor 1 minuteand7:zoefor I minute)was precededby.5 minutedenatuntionstepat 94"Candwas followedbya7minute~ensionstepat7re.

36 1.7.4AaaJrsil.rPCR Produeu

TheamplifiedPCR produetsweresubjected to electrophoresis on .1.5%agarosegel containingO.S1l&'m1ethidiumbromide,inan electrophoresisclwnberforapproximately I hour at lOOv.Thegdwasviewedand pbotographedusing.Cbcmilmager4000computersystem from AlpbalDDoteeh Corpontion(SanLeandro.CA.USA).andsavedondiskfor laterquantitation andanaJym.

1.1 MEASUREMENT OF PEMlDE BINDING TO A'ORE'

Determination of peptidebindingto A' aCE' molecules wasachievedusing. competitive iahib;,;""&SKy.This ....yusedtheTcellhybridomu,3,47(A' -reslrict od,Daiet01.,1999)and 8F9 (E'-mtri<tod, 0';.. 01., 1999)RCOgniDng the peptidesT.(2SSl )(akindgiftfromDr.Vi Chi Kong)andTgPl(RaoetaI.•1994),respeet ively.InIflat-bottommicrcwellplate,10' hybridomacellswere incubatedwith.constantamountoftbeir respectiveligand(O.OSI!Wmlof T~2SS3)and0.04,.gImi.fTgPl~andscriaIdilutions.ftheUlIubit",peptide(lUghestdilution 100I!W'mI)and10'TAJcellsasAPe.TAJexpress H_2A'IH_2A^kandH-~IH-2E ~(AllenetaI., 1985).whichwerea IciDdgiftfromL.H.GlimcheratHarvard MedicalSchool,andwere couneoustyprovidedbyr.Wans&ltheUaivenityofToromo.ThecellswereculturedinatoW or2 00J,d culture mediumperwcD.After14houn.100J,LIofsu pemawu wucollcetcdfromeach weU.truslerredto a newplate.andkept frozenat·200c.Therelativen.·2contentoftheculture supernatantfromthe~bindingassaywasdeterminedbythawing themicrotitreplates

37

ondoddiDa10' lL-2 depcnclem CTI.L-2[(Gillis.tll.,19n)(ATCC,Rockville,MD,USA)]per wcIJ.AllerIIbounincuIturc'{IIJthym;dinc(I. CliwclJ)wasodded.The_ werebanested6 hourslaterandthelnccrporal:ed radioactivity measuredasoutlined insection3.4.2.CTLL-2tells weremaintainedinculturemedium supplementedwith100ftsupernatant fromeoncavalin·A activatedratspIcnoeytcsIS •sourteofL2.

38 CllAPTER3

A1TEMPTS TO GENERAn:THYROWITOGENIC TID CEllS SPECD1C FOR THYROGLOBULIN PEPTIDES

3.1 ABSTRACI

Cross regulation cr lblandlb2 cdlsbythe cytolcinesofmeopposingTh subsethasbeen usedinimImmotherapyofTbl-mediated.,orpn.specific diseases.Therole ofTg.peptide.specific tb2 cellsinmurineEAT basyet:tobeassessedinvivo.Thisstudyat1emptsto'switch'Tg- peptide.specific1111 populationsofLNCbyculturingtheminvitrowithrll...4.a prototypicalTh2 cytckine.Italso examinesanyendogenousC)'tolcinc productionbythethyroidglandinnormal miceusingRT-PCR.Both the originalTblcellpopulationsandthe Th2cellpopulationproduced weretobeadoptivelytransferredinto naivemiceandtheireffecton EATassessedbothby hi~oIogyo(andcytokincmRNAexpressioninthe thyroidglands.ThenonnaImurinethyroid glandb.tsno endogenouscytokincmRNAexpressionHo_ .Tg-peptide-speeifieTh2cells couldDOtbeproducedusingrn. -4In

"'tro.

ThepropmyoenAISan autocrinegrowthfactor mayhaveaffea:editsabilitytoswitchthe e:ytokine profileer n1cells.andretaintheir antigen specificity.The inabilitytogenerateTg-peptide-spec:ific:1b2 cellsby thismethodpreventedus fromexploringthe biologicalfi.maionoCTh2cellsinmurineEAT.

39 3.1INTRODUcnON

ExperimentalautoimmunethyroiditiJ

CEAn

isinducedinsusceptiblestrainsof mice(H-2^k IDdH-2")CoDowingcllallenge _ TgioCF A.TheTgpeptidesTgPl (rT g(24 95-2SIO)andTgP2 (rTg(2695-2 7 13)]

mo

havetheobility"'ioduceEAT,andactu _ epitopes (ChronopoulouandCarayamUotis,1993,!laoet II.,1994).Manyorgan-specifi<outoUnmune diseases,such u thyroiddisease.multiplesclerosisandinsulirHSependenl diabetesmellitusare thoughltobemediated byCD4+Tcells,andThlcdlsinparticular.CD4+Tcellsdifferentiate intotwodislinctsubsets,withThlcdlsproducing n.,.2andIFN-yandUM>Ivedprimarilyin..u mediatedresponses,whileTb2cellsproducethec:ytokiDesn...n.·I Oand0...-13andare typicallyinvolvedinbumonJraponses (O'Gan.andHoskm, 1997),

1111andTb2cdlsare cross-regulatory,meaningthat e:ytokines producedfromonesubset havetheabilityto downregulate theresponse of tile opposingsubset.Theobjectivecrmy l"e$CUChwastoinduceanTg-peptide-specificTh2populationbythedeviationof anestablished ThlcellpopulationusingtheTh2cytokinell..-4,andtoex.aminctheeffectoftileTb2 subseton theinductionandprogressionof EATinSJUJmice.Tbc melhodIusedwasbasedonthework byKborutsetaI.(1995).IntheirexperimentsintheEAEanima!modelforMS,theyimmunized micewithan enc:cpWtogeNCpeptidePLP(139.1SI)inCFAandafter 9·11 days invitro, removedtheLNC.whichwere oftbelblphenotype.Theythenproceededto'switch'thesecells from.!hItoaTb2 pancmofcytokinesecraioobytheadditionof rll..-4invitro.Theythen exominedthedUea>e-U1duciDgpotentialoCTh londTh2cdlsiotheEAEacimaImodel,by adoptivetransfer intosyngeneicSn.JJfcmalemice.Weattempt edtoreproduce thisprotocolin

40

the EATmodel.usinstheth)TOiditOlcnic pcptidesTgPlandTgP2 u antigens.

3.3RESULTS

3.3.I1bt~isDOdetectable IIlRNA ror the cytokincsIL-l.fL.4or IFN-y in normal thyroid ped.

Themeasumncnt oftbcimnthyroid&J cylokinemRNAaimedItestablishing.baseline such thatinlater experiments., the mRNAexpressionofe:ytolcinesinthe thyroidglandsofmitc c:ouldbeusedto indicatea 1b I or lb2typeofresponse.Ten normalSJlJ1miceWU'etestedfor thePRSCllCCofimrolhyro;dalmRNAfo<the cy1OIriDe$JL.2,IL-Iand1fN..,.ThemRNAwas extractedfromtheth)ToidRJandofS maleandS femalemiee,alongwith •similarsizepiece of liverandspleen.ThemRNAwasusedto produceeDNAwhichwasllllpliliedusingRT-PCR.and viewedon an agaroscgel.~SCCIIinTable 3.1,of the ten SJLIJmicetested,noneshowed the preseeceofJL.2,

n...

or1fN..,mRNA.y<tall showedthepreseeceof thelXXlSlitutivcenzyme GAPDHandthyrogJobulin(FIpFe3.1).As. conuol mRNAwucxnaed frombothspleen andliver.OfthcmRNAextractedfrom the spleen,mostmice(6 of9tested)showedthepresence:

oen.... ,

wtill.e 2 of9 showed thepresenceofn...2.None exhibitedIFN-yexpression.Screening oelivermRNAshowedsimiJ&rresults (datanocshown).iDdieatingthatthereisexpressionof cytolcinegeocsinsome tissuessuch asIivcrandspleen. butDOneillnormalthyroidtissue.This lndicalesthatthelevelof cylokinc: mRNAexpressionc:oWd beusedto detecttheTb subset presentin glandsthat have beeninfiltratedafter adopti'Vc transferofTg-peptide-specificThland