The Journal of Experimental Medicine

1–2 wk.Nevertheless,themechanismof immunodeficient recipientswherediabetesdevelopswithin when CD4Tcellsfromdiabeticmicearetransferredinto transgenic models(3,5,6),thediseasecanbeprecipitated Introduction it wasnotobviouswhetherthelatephaseof NOD miceexhibitedadelayedonsetofdisease(7,8),but has remainedobscure.Itbecameclearthatperforin-deficient [email protected] (617) 632-6882;Fax:632-6881; e-mail: Dana-Farber CancerInstitute,44Binney St.,,MA02115,Phone: Address correspondencetoHaraldvon Boehmer,HarvardMedicalSchool, destruction of cells causepancreaticisletcellinfiltrationwithsubsequent CD4 orCD8Tcellsspecificforantigensexpressedin been developedinwhichparticularlyhighfrequenciesof T cellreceptortransgenicmodelsoftype1diabeteshave (g7) MHCallelethatcontrolsdisease.Inaddition,several T cellsbypeptidespresentedtheparticularNODclassII model, thediseasedependsonactivationofautoimmune suitable animalmodelofhumantype1diabetes(1,2).Inthis Nonobese diabetic(NOD)micearethoughttorepresenta Fas waspresenton develop diabetes(9).Additionalanalyseshadsuggestedthat deficient lpr/lprmiceontheNODbackgrounddidnot was affected.Ontheotherhand,itshownthatFas-

cells(3,4).IntheNODandinsome

cells(9)andthatexpressionincreased

1103

Irina Apostolou, Irina e od:Fasreceptor •diabetes Key words: dispensable modeofcelldeathinthisdisease. test thishypothesis,wehavegenerateda Effective of Fas-deficient Destruction Insulin-producing genic modeloftype1autoimmunediabetesinwhichCD4 mechanism. Previousanalyseshaveshownthat

In type1diabetes,autoimmuneTcellscausedestructionofpancreatic Abstract 3 2 1 whether theFasreceptorwasanessentialmediatorof develop diabetes.However,becauseofpossiblepleiotropicfunctionsFas,itwasnotclear perforin-deficient nonobesediabetic(NOD)miceandthatFas-deficientNODdonot diabetes withslightlyacceleratedkineticsindicatingthatFas-dependentapoptosisof control oftheratinsulinpromoter.HereweshowthatFas-deficientmicedevelopautoimmune specific forinfluenzahemagglutinin(HA)arecausingdiabetesinmicethatexpressHAunder

Harvard MedicalSchool, CenterforBloodResearch, Boston, MA02115 Advanced Institute, MedicalDiscovery Toronto, M5G2C1, Ontario Canada Harvard MedicalSchool, Dana-Farber CancerInstitute, Boston, MA02115

Cellsin Type 1Diabetes

http://www.jem.org/cgi/doi/10.1084/jem.20030698 1103–1106 October6,2003 Volume 198,Number 7, J. Exp.Med.

celldestruction celldestruction  1 TheRockefeller UniversityPress•0022-1007 Zhenyue Hao, inactivation inpancreatic inactivation (unpublisheddata).ToanalyzewhetherFas flanked byloxPsites,allowingforcelltype–specificFas (fas and presentedbyclassIIE hemagglutinin (HA)expressedundercontroloftheRIP transgenic TCRrecognizespeptide111–119ofinfluenza duced intoatransgenicmodeloftype1diabetesinwhich a promoter (RIP)–controlledCretransgene(13)wereintro- Abbreviations usedinthispaper: homozygous micecarryingthe fivetransgenes(TCR-HA involved in thus leavingopenthequestionofwhetherFaswasdirectly infiltration ofpancreaticisletsbymononuclearcells(10), also revealedthatinFas-deficientNODmicetherewasno developmentofthedisease.Otherstudies,however, during Ins-HA) (5).TheonsetanddevelopmentofdiabetesinI-E Ins-HA the developmentofdiabetes,fas betic; RIP,ratinsulinpromoter. disease. ToaddressapotentialroleofFasin have aroleinpromotingpredominantlyearlystagesofthe treatment (12)wereinterpretedtoindicatethatFasmay negative conclusively, wehaveproducedaconditional 2 fl KlausRajewsky, ) inwhichthedeathdomain-encodingexon9is celldeath•autoimmunity •conditionalknockout cell–specificknockoutofthe / Fas , Fas mutation(11)orinvivoanti-FasLantibody celldeath.Morerecentstudiesusingadominant celldestructionisdelayedbutcanoccurin fl/fl , RIP-Cre celldeathintype1diabetes.Todirectly 3 andHaraldvon Boehmer HA, hemagglutinin;NOD,nonobese dia- /2003/10/1103/4 $8.00 TcellswithatransgenicTCR d cellsresultedinresistanceto MHCmolecules(TCR-HA, / mice)wascomparedwith cellsbylargelyunknown fl alleleandaratinsulin Fas geneinatrans- celldestruction Fas cellsisa allele 1 /

d ,

Downloaded from from Downloaded jem.rupress.org on August 13, 2017 13, August on The Journal of Experimental Medicine the deletionofexon9.(B)PCRanalysis onDNAfromliver,heart,,andpurifiedisletsofoneFas amplified productindicatedthepresenceofaWT DNA. Primersequenceswere5 School. GenotypingoftransgeneswasdeterminedbyPCRontail guidelines oftheCommitteeonAnimalsHarvardMedical were maintainedinapathogen-freefacilityaccordancewiththe mixed 129sv,C57Bl/6,andDBA-2background(13).Allanimals exon 9hasbeenflankedbyloxPsites.RIP-Cremicewereona Cre heterozygousmouse. TCACCCG-3 CTAAGAGG-3 gene, amplified productindicatedthepresenceofadeletedexon9. product indicatedthepresenceofintactexon9,whereasa260-bp mediated Harvard MedicalSchoolIsletCoreRodentIsolation.RIP-Cre– prepared fromthethymus,liver,heart,andisletsisolatedat gous recombinationusingaconditional Materials andMethods CTG-3 velopment ofspontaneousdiabeteswasassessedbymeasuring floxed whereas a470-bpamplifiedproductindicatedthepresenceof with thesequence 5 with ground. Fas bred asheterozygoustransgenicmiceandareontheBALB/cback- Fas Figure 1. (P3) locatedupstreamofthe5 TCR-HA mice) andtherebyexpressingFasin transgene (TCR-HA that inmicewithonlyfourtransgeneslackingtheRIP-cre located downstreamofthe3 CAGGA-3 A). HomozygosityforI-E 5 GTCT-3 the antisenseone3 blood leukocytes. -CATCGCTCGACCAGTTTAGT-3 Diabetes MonitoringandCyclophosphamideAdministration. RIP-Cre–mediated FasRecombinationinIsletDNA. Mice andGenotyping. , andrecombinedfloxed and 5 Fas forthe transgene,5 allele,sincethesenseprimerwaslocatedinexon9and (P1)and5 Fas Successful eliminationofthefloxed fl and5 micederivedfromC57Bl/6werecreatedbyhomolo- -TGCAGTTGCTGAGATGAACCATTTTCTCT- recombinationwasassessedusingasenseprimer and5 (P2)forWTandfloxed HA -ACAGTCAGTCTGGTTCCTGA-3 totheloxPsitedownstreamofexon9(Fig.1 -GTCCTCTATTATCCTCATCATGAG-3 transgene,5 -CGATGCAACGAGTGATGAGG-3 -GGCTTTGGAAAGGAATTTCCTC- -CTCCGTCAGCCATAGCAAATTT- / Fas TCR-HA miceandIns-HAwere d wasdeterminedbystainingperipheral , Ins-HA 1104 alleles,andlocationoftheprimers used inPCRanalyses.Cre-mediatedrecombinationofthefloxed loxPsite(P2).A1.7-kbamplified loxPsiteandtheantisenseprimer -GGCTACCATGCGAACAAT- -ACAAGGTGGCAGTAA- Fas-deficient / Fas , Fas cells. forthe

Fas Fas

alleleinwhichthe

–exon 9byhomologousrecombination inpancreatic fl/fl

alleles.A430-bp

, RIP-Cre

CellsSusceptible toImmuneDestruction

Cre DNA was

Fas

trans- forthe

allele,

and

De-

/

and -nonexpressingFas from purifiedisletsandvarioustissuesCre-expressing RIP-Cre (Fig.1B).Amuchfainter470-bpbandwasseen spective ofRIP-Creexpressionandinisletsmicelacking single 470-bpbandinnonpancreatictissuesofmiceirre- Results andDiscussion uct indicativeofthesolepresence pancreatic originyieldedasingle1.7-kbP2/P3PCRprod- bined butnottorecombined Ins-HA 1 B sorted 6.5 were monitoreddaily. mg/kg. Bloodglucoselevelsofcyclophosphamide-treatedmice inPBSwasinjectedintraperitoneallyatadoseof200 Aldrich) (Accu-chek Advantage;Roche).Cyclophosphamide(Sigma- blood glucosetwiceaweekwithanautomaticglucometer tion of notype and and thepositionofprimersusedtoassesslittermatege- TCR-HA) andanti-CD4(GK1.5;BDBiosciences)mAbs.10 Fas tested daily. of RAG-2 cells werepurifiedbysortinglymphocytesfromspleenandLN Even tracesofrecombined a schematicrepresentationofmutatedandWT using theP1/P2primersthathybridizeto Cre-nonexpressing mice.PCRanalysisofthesamesamples whereas asingle1.7-kbbandwasdetectedfromtheisletsof uct thatwasobtainedfromisletsofCre-expressingmice, of exon9camefromthesingle260-bpP2/P3PCRprod- fragment. Evidenceforislet-specificCre-mediateddeletion cause ofitsshortsizewouldbefavoredoverthe1.7-kb detected bythepresenceofa260-bpfragment,whichbe-

The efficacyandtissuespecificityofCre-mediateddele- Adoptive TransferofDiabetes. fl/fl

shows that,

, Rip-cre

Fas

/

mice.Bloodglucoselevelsoftherecipientswere CD4 –exon 9wasassessedbyPCRonDNAprepared

/

, TCR-HA cell-specificlossoffunctional

/

cellswerei.v.injectedintoeitherRAG-2

regardless ofCreexpression,DNAnon- , Ins-HA

fl

homozygousandoneFas

fl

cells.(A)RepresentationofWT homozygousmice.Fig.1Adepicts

/

/

micestainedwiththe6.5(anti–

or RAG-2

Fas

Fas

–exon 9wouldhavebeen CD4

alleles (Fig.1A)yieldeda

Fas

TCR-HA–expressing

/

–exon 9(Fig.1A).

, Fas

fl

WT nonrecom- homozygous,RIP-

Fas

Fas fl/fl

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allele.Fig.

Fas

Fas

alleles

, floxed

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5 , ,

Downloaded from from Downloaded jem.rupress.org on August 13, 2017 13, August on The Journal of Experimental Medicine blood glucosemeasurements over aperiodof32wk.Micewereconsidereddiabeticaftertwosuccessive HA of diabetesinthelatterquadruple transgeniclittermates recombined bytheCrerecombinase (TCR-HA recombined assess theimpactofFasexpressionby tween RIP-Cre–expressingand–nonexpressingmice.To RIP-Cre transgeneinpotentialphenotypicaldifferencesbe- among theirlitter.Thus,wecouldexcludearoleforthe opment, fertility,andfrequencyoftheexpectedgenotypes HA doubletransgenicmicewithrespecttodiabetesdevel- HA, Ins-HAmicebehavedidenticallytoTCR-HA,Ins- study togeneratetheFas-competentand-deficientTCR- HA, Ins-HA,RIP-Cretransgenicbreedersusedinthis Rip-Cre Fas in thepresentstudycontaincellsotherthan likely toreflectthefactthatwholeisletssuchasthoseused in isletsofCre-expressingmice(Fig.1B). of Fas Fas-competent TCR-HA,Ins-HAtransgenicmice.Bloodglucoselevels Figure 2. termates lackingCreexpression (TCR-HA HA transgenicmodelinwhich thefloxed spontaneous autoimmunediabetesintheTCR-HA,Ins- mediated destruction,wecomparedthedevelopment of e.g., non– tional Fasinthevastmajority recombination resultedintissue-specificdeletionoffunc- isletsstronglysuggestthat expressing the diminutionof470-bpfragmentintensityinCre- atic tissuesanditsdetectiononlyinCre-expressingislets vored overthe1.7-kbP2/P3PCRproduct)innonpancre- tion ofa260-bpP2/P3PCRproduct(whichwillbefa- etc., lackingCreexpression.Thus,theabsenceofamplifica- mice (13,14).Furthermore,theFas function, ortheglucosetoleranceofRIP-Cretransgenic dence foratoxiceffectofthetransgeneto line identicaltothatofthepresentstudycouldnotfindevi- diabetes. PreviousworkusingaRIP-Cretransgenicmouse in theseparticularmice,andthusbeinsufficienttoresult Fas-dependent destructionof tial roleofFasinthedevelopmentdiabetes,sinceany conditional knockoutwassuitabletorevealapossibleessen- dl requiresdestructionofwellover50%all ment ofdiabeteswithbloodsugarlevelsexceeding200mg/ fl/fl fl/fl / , RIP-Cre , Rip-Cre , Fas / , TCR-HA Effective destructionof endocrinecells,macrophages,endothelial fl/fl , RIP-Cre / / , TCR-HA mice)(Fig.2).Theincidence andonset / , Ins-HA 1105 300 mg/dl. / mice)tothatoftransgeniclit- /

, Ins-HA of / cellscouldnotexceed50% -islet cellsfromFas-deficientand ( Apostolou etal. cells.Sincethedevelop- ,

the RIP-Cre–mediated fl n heterozygous,TCR- / cellsintheirTcell– ( 63) micewereassessed

This faintbandis , n / Fas cells( , Ins-HA 46) andFas allelesare cells,their cells,the / 30%), , Ins-

was / fl/fl , , of Ins-HA micetransgenicwitha tes, wasreachedwhendiabetesanalyzedinTCR-HA, namely thatFasdeficiencydidnotdelaytheonsetofdiabe- of theonsetdiabetes(Fig.2).Asimilarconclusion, did notproduceadelayintheonsetbutsomeacceleration toimmunity. Theeliminationof glucose levelseveryday. 200 mg/kgofcyclophosphamideonday0andmonitoredfortheirblood sorted, and10 Ins-HA Figure 3. ( RAG-2 Figure 4. Ins-HA genic cells.6.5 caused byautoimmuneTcells(Fig.3).Finally,itwastested tions Fasdidnotplayadiscerniblerolein ment withthedrug,indicatingthatalsounderthesecondi- micecamedownwithdiabetes4–5daftertreat- expressing that controlthedevelopmentofdisease.TheRIP-Cre– perhaps byinterferingwithregulatory(suppressive)Tcells NOD andTCR-HA,Ins-HAmicewithsimilarkinetics, mide. Thistreatmentisknowntoprecipitatediabetesin the FasfloxedandRIP-Cretransgenicmice Fas genic mice,indicatingthatneithertheintroduced not differentfromthatinTCR-HA,Ins-HAdoubletrans- , Fas allelesnorthemixedgeneticbackgroundbroughtby n thatweretreatedwith200mg/kgcyclosphospha- 4)ondayzero.Bloodglucoselevels weremonitoreddaily. / / / ( , Ins-HA , Fas , Development ofdiabetesinFas-deficient andFas-competent 5 Cyclophosphamide-induced diabetesinTCR-HA cellsweretransferredintoRAG-2 , fl/fl n CD4 , RIP-Cre 4) andRAG-2 / cellsfromRAG-2 recipientsinjectedwithCD4 / mice.Mice( / , Fas Fas / fl/fl n , TCR-HA byCrerecombinase cell–specificdeletion , Rip-cre 10)wereinjectedwith / , Fas

did perturbau- TCR-HAtrans- fl/fl / / celldeath , Rip-cre , Ins-HA micewere

floxed / /

/

, ,

Downloaded from from Downloaded jem.rupress.org on August 13, 2017 13, August on The Journal of Experimental Medicine TCR-HA transgenicRAG-2 short timeperiodafterinjectionof10 4, diabetesdevelopedinbothtypesofrecipientswithina ground lackingorexpressingfason References Accepted: 25August2003 Revised: 21July2003 Submitted: 28April2003 Research Foundation. Prize forEuropeanSciencein1997andbytheJuvenileDiabetes ber Foundation,,,intheformofKoerber Boston, MA)forprovidingtheRIP-Cremice. We thankR.N.KulkarniandC.R.Kahn(JoslinDiabetesCenter, consistent withthenotionthat rect cellcontact.Ourfindingsrefutethishypothesisandare stroyed press Fasligand,itwasconceivablethatsuchTcellsde- whether purifiedCD4 them intoIns-HAtransgenicmiceontheRAG-2 destruction intheabsenceofCD8 few activatedCD4 mice thatthemselvesdonothaveanyTcells.Sinceonlya ferred withpurifiedCD4Tcellsintoimmunodeficient genic models(3,5,6),theautoimmunediseasecanbetrans- nism asthatof tion ofthetransplanted though itisnotclearinthelatterscenariowhetherdestruc- fectively destroyedbydiabetogenicTcells(14–16)even mice thatweretransplantedintoFas-competentareef- with thefindingthatadominantnegative cells and cell deaththatdonotrequiredirectcontactbetweenT sults shouldshiftthefocustoothermediatorsofapoptotic sential mediatorof into x-irradiatedtransgenicnondiabeticrecipients(11). when splenocytesfromdiabeticanimalsweretransferred NOD micehadlittleeffectonthedevelopmentofdiabetes II MHCon out thatthereisapparentlynosignificantexpressionofclass cells isnotrequiredfortheTcell–mediateddestructionof sively thatantigen-specificcontactbetweenTcellsand role in struction needstofocusonmoleculesthatplayanessential diators of and differfromFas.Theidentificationofsuchessentialme- and firstsymptomsaredetectable. with thediseaseonceprocessofdestructionhasbegun .Bach,J.F.,andD.Mathis.1997.TheNODmouse. 1. cells(17).Thus,thesearchformediatorsof H. vonBoehmerandK.RajewskyweresupportedbytheKoer- In boththeNODmodel(2)andinseveralTCRtrans- By eliminatingtheFasL–Fasapoptoticsystemases- munol. celldestructionintype1autoimmunediabetes 148:285–286. cellsbyaFasL–Fasinteractionthatrequireddi- cells.Withregardtothisnotion,wemaypoint celldeathwouldperhapspermitinterference isletcells(1)andperhapsevenmoreconclu- cellsinsitu.Ourresultsarealsoconsistent Tcellsexpressperforinbutmostex- celldeathintype1diabetes,ourre- TCR-HA–expressingTcellsfrom 1106 cellsoccursbythesamemecha- / micecouldcause Fas-deficient cellsfromFas-deficient Tcellsbytransferring cells.AsshowninFig. 5 CD4 Fas Tcells. CellsSusceptible toImmuneDestruction transgenein cellde- / Res. Im- back- cell 5 Kim,Y.H.,S.K.A.H.Yagita,N. Kayagaki, 15. Allison,J.,andA.Strasser.1998.Mechanisms ofbetacell 14. Kulkarni,R.N.,J.C.Bruning,J.N.Winnay,C.Postic,M.A. 13. Nakayama,M.,M.Nagata,H.Yasuda,K.Arisawa,R.Ko- 12. Savinov,A.Y.,A.Tcherepanov,E.A.Green,R.A.Flavell, 11. Itoh,N.,A.Imagawa,T.Hanafusa,M.Waguri,K.Yama- 10. 7 Sarukhan,A., O.Lechner,andH.vonBoehmer.1999.Au- 17. 6 Pakala,S.V.,M.Chivetta,C.B.Kelly,andJ.D. Katz.1999. 16. .Amrani,A.,J.Verdaguer,B.Anderson,T.Utsugi,S.Bou, 8. Kagi,D.,B.Odermatt,P.Seiler,R.M.Zinkernagel,T.W. 7. .Chervonsky,A.V.,Y.Wang,F.S.Wong,I.Visintin,R.A. 9. .Sarukhan,A.,A.Lanoue,Franzke,N.Brousse,J.Buer, 5. Scott,B.,R.Liblau,S.Degermann,L.A.Marconi,L.Ogata, 4. Degermann,S.,C.Reilly,B.Scott,L.Ogata,H.vonBoeh- 3. 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