Ezrin, Maspin, Peroxiredoxin 2, and Heat Shock Protein 27: Potential Targets of a Streptococcal-Induced Autoimmune Response in Psoriasis This information is current as of April 6, 2019. Petra Besgen, Paul Trommler, Sigrid Vollmer and Joerg Christoph Prinz J Immunol 2010; 184:5392-5402; Prepublished online 2 April 2010; doi: 10.4049/jimmunol.0903520 http://www.jimmunol.org/content/184/9/5392 DD oo ww nn lolo aa dd ee dd References hTthtpis: /a/wrtiwclwe .cjiimtems 5u5n oalr.otircgl/ecso,n 1t6en ot/f1 w84h/i9ch/5 y3o9u2 .cfualnl #arcecfe-lsiss tf-o1r free at: from from h h ttpttp ://w://w Why The JI? Submit online. ww ww • Rapid Reviews! 30 days* from submission to initial decision .jim.jim mm uu nn • No Triage! Every submission reviewed by practicing scientists oo l.ol.o rgrg • Fast Publication! 4 weeks from acceptance to publication b/ b/ y gy g *average ueue ss t ot o nn Subscription Information about subscribing to The Journal of Immunology is online at: A A pp http://jimmunol.org/subscription ril 6ril 6 Permissions Submit copyright permission requests at: , 20, 20 11 http://www.aai.org/About/Publications/JI/copyright.html 99 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The JournalofImmunology Ezrin, Maspin, Peroxiredoxin 2, and Heat Shock Protein 27: Potential Targets of a Streptococcal-Induced Autoimmune Response in Psoriasis Petra Besgen, Paul Trommler, Sigrid Vollmer, and Joerg Christoph Prinz PsoriasisisanHLA-Cw6–associatedTcell-mediatedautoimmunediseaseoftheskinthatisoftentriggeredbystreptococcalangina. Toidentifykeratinocyteproteins,whichmaybecomepsoriaticautoantigensastheresultofanimmuneresponseagainststreptococci, rabbitswereimmunizedwithheat-killedStreptococcuspyogenes.StreptococcalimmunizationinducedAbformationagainstvarious human keratinocyte proteins. Sera from psoriasis patients reacted against several of these proteins as well. Common serologic reactivitiesofrabbitsandpatientsincludedtheproteinsezrin,maspin,peroxiredoxin2(PRDX2),heatshockprotein(hsp)27,and keratin6.Whenusedforstimulationofbloodlymphocytes,ezrin,maspin,PRDX2,andhsp27inducedincreasedTcellactivationin psoriasispatients,whichwasparticularlyevidentforHLA-Cw6+individuals.Ag-specificTcelllinesgeneratedwiththeseproteins consistedpredominantlyofCD8+TcellsandusedTCRb-chainrearrangements,whichwerehighlyhomologoustothoseexpanded D o withinthecorrespondingskinlesion.Severalimmunodominantepitopesonthedifferentproteinscouldbedefinedaccordingto w n sequencealignmentswiththewholegenomeofS.pyogenes.Ourdataindicatethatmaspin,ezrin,PRDX2,hsp27,andpotentially lo a keratin6couldactasautoantigensofastreptococcal-inducedautoimmuneresponseandrepresenttargetsoftheexaggeratedTcell de d response in psoriasis. Additionally, ezrin and hsp27 might constitute antigenic links between psoriasis and inflammatory bowel fro disease,uveitis,orarteriosclerosis,whichareclinicallyassociated. TheJournalofImmunology,2010,184:5392–5402. m h ttp P ://w soriasis vulgaris is a common chronic inflammatory skin psoriatic skin lesions over years and reappeared in psoriasis re- w diseaseaffecting2–3%ofthewhitepopulation.Itischar- lapses, while being absent from uninvolved skin (6–8). Together w acterizedbyscaly,erythematousplaquesthatresultfroman withaconservedaminoacid motifinthethird complementarity- .jim inflammatory hyperproliferation of the epidermis and may cover determining region (CDR3) of clonally expanded lesional TCR m u large areas of the body (1). Psoriasis represents a multifactorial b-chain rearrangements (9), these findings emphasize that the no disorderwithapolygenicpredisposition,whichincludesastrong psoriatic T cell response is directed against dominant psoriatic l.o rg association with HLA-Cw6 and polymorphisms invarious genes autoantigensthatarepreservedwithinindividualpatientsandmay b/ relevantforinflammationandimmuneresponses,suchasIL-23Ror bepublictopsoriasisingeneral.Accordingly,identificationofthe y g theIL-12p40subunit(2–4). psoriatic autoantigens represents an essential clue to the patho- u e Selective immunosuppressive therapies targeting T cells or genesisofpsoriasis.Itmaydefinethetargetsmaintainingdisease st o cytokinessuggestthatthehyperproliferativepsoriaticinflammation activity as the result of persistent T cell activation and identify n A mayresultfromaTcell-mediatedimmuneresponse(3,5).Although novel,causallydeterminedtherapeuticapproaches. p the T cell-derived psoriatic cytokine network has become in- Psoriasis onset or relapses are seen in strong association with ril 6 creasingly clear, the precise mechanisms leading to activation of a prior tonsil infection with group A b-hemolytic streptococci , 2 T cells in the skin of psoriasis patients are less well understood. (GAS/Streptococcus pyogenes) (10–14), and HLA-Cw6+ patients 01 9 Extensive analysis of the lesional TCR usage revealed dominant areparticularlysensitivetothestreptococcaltrigger(12,13).The TcellclonesinthepsoriaticTcellinfiltratethatpersistedwithin simultaneous presence of the same T cell clones in skin lesions andtonsilsinpatientswithstreptococcal-drivenpsoriasissupport apathogeneticlinkbetweenstreptococcalanginaandthelesional DepartmentofDermatology,Ludwig-Maximilians-UniversityofMunich, Munich, psoriatic immuneresponse(15). Germany TheseobservationssuggestthatGASinfectionofthetonsilscould ReceivedforpublicationOctober27,2009.AcceptedforpublicationFebruary18, induceaTcell-mediatedautoimmuneresponseagainstskinproteins, 2010. whichmaypreferentiallybepresentedbyHLA-Cw6(16–18).After ThisworkwassupportedbySonderforschungsbereich571oftheGermanResearch an initial priming phase, which might also involve costimulatory SocietyandbyagrantoftheWilhelm-Sander-Stiftung. signalsby streptococcalsuperantigens(19),thisGAS-inducedim- AddresscorrespondenceandreprintrequeststoDr.JoergC.Prinz,Departmentof mune response could extend to the skin by molecular mimicry in Dermatology, Ludwig-Maximilians-University of Munich, Frauenlobstr. 9-11, D-80337Munich,Germany.E-mailaddress:[email protected] asimilarfashionasthecross-reactivestreptococci-specificautoim- Abbreviationsusedinthispaper:BJ,joiningregiongene;BV,Vregiongene;CDR3, mune responsesinotherpoststreptococcalsequelae,suchasacute thirdcomplementarity-determiningregion;CK,cytokeratin;GAS,groupAb-hemolytic rheumaticfeverorpoststreptococcalglomerulonephritis(20). streptococci/Streptococcuspyogenes;H,healthycontrol;hsp,heatshockprotein;K6-N, To identify potential psoriatic autoantigens resulting from an N-terminalproteinpartofkeratin6;K6-C,C-terminalproteinpartofkeratin6;Kc, keratinocyte;ND,notdetermined;N-D-N,rearrangementsite;PRDX2,peroxiredoxin antistreptococcal immune response, we immunized rabbits with 2;PV,psoriasispatient;SFC,spot-formingcell;TCL,Tcelllineraisedagainstthe GAS.GASimmunizationinducedAbsagainstseveralclearlydefined indicatedprotein;TCRBV,TCRb-chainvariable;TT,tetanustoxoid. proteins expressed in keratinocytes. According to their particular Copyright(cid:1)2010byTheAmericanAssociationofImmunologists,Inc.0022-1767/10/$16.00 humoralandcellularimmunogenicityforpsoriasispatients,someof www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903520 TheJournal ofImmunology 5393 theseproteinscouldrepresentantigenictargetsofapolyspecific(21) scribed(27).Resultswereexpressedasspot-formingcells(SFCs)per106 streptococcal-induced T cell-mediated autoimmune response and PBMCs.For[3H]thymidineincorporation, triplicatesamplesof 2 3 105 cells were pulsed with 2 mCi = 74 kBq [3H]thymidine/well for 8 h contributetotheformationofpsoriaticskinlesions. (Amersham-Buchler,Braunschweig,Germany).Incorporatedradioactivity wasmeasuredascpm(28).Negativecontrolvalues(cultivationofPBMCs Materials and Methods intheabsenceofAgorwithpreparationsfrommock-transfectedE.coli) Streptococcalimmunizationofrabbits werealwayssubtractedfromtheresultsobtainedbyspecificAgstimula- tion.Forstatisticalanalysis,meancpmornumbersofSFCswereanalyzed RabbitsofthestrainDeutscherRiesewereimmunizedwithS.pyogenes inf-andttests.Probabilityoferror(pvalue)wassettop,0.05. serotypeMT12,NCTC10085(providedbyDr.Efstratiou,PublicHealth TCR b-chain variable (TCRBV)-repertoire analysis, cloning, and se- LaboratoryService,London,U.K.)bysixs.c.injectionsat2-wkintervals quencingofTCRBVgenerearrangementsweredoneexactlyasdescribed with100mlpartiallysonicatedGASpelletsinIFA.Serumsampleswere (15).TCRrearrangementsaregivenas deducedaminoacidsequenceof collectedbeforethefirstimmunizationand2wkafterthelastimmunization. theCDR3. CD4/CD8 ratios of the T cell lines were determined as reported pre- Preparationofhumankeratinocytesandcultureofhumancell viously(15).TcelllineswereseparatedintoCD4+andCD8+Tcellsusing lines magneticbeads(Microbeads,Skedsmokorset,Norway). Preparationandcultureconditionsofhumanprimarykeratinocytesfrom Results surgicalskinsamples,theepidermoidcarcinomacelllineA431(American TypeCultureCollection,Rockville,MD),oranEBV-transformedhuman Streptococci-specificrabbitandpatientserashowcommon Bcelllinehavebeendescribed(22).Toinduceproteinsdependentonstress reactivities withvariouskeratinocyteproteins ordifferentiationtheywereheatshockedbyincubationat43˚Cfor8hor grownwithhighCa2+concentrationsfor24h(1.2mM). To identify common epitopes on proteins from GAS and kerati- nocytes, rabbits (n = 3) were repeatedly immunized with S. pyo- SDS-PAGE,Westernimmunoblotting,andisoelectricfocusing genes, serotype M12, which is frequently observed in psoriasis D o Western immunoblotting followed standard protocols (23, 24). For two- patients(10).Analysisofthereactivityofthestreptococci-specific w dimensionalgelelectrophoresis,humankeratinocyteswerelysedin150mM hyperimmune sera by Western immunoblotting and comparison nlo NaCl,1%NonidetP-40,50mMTris(pH8.0),centrifugedat10,0003gfor a 20 min, and the proteins were lyophilized. Each 50–100 mg lyophilized withthepreimmunesera(Fig.1A)demonstratedthatstreptococcal de d kgeraradtiiennot,cy1t8e0lmysmatelesnwgethre,AapmpelireshdatmoIPmhmarombailciinaeBDioryteSchtr,iUpsp(ppsHala3,–S1w0eldineena)r. ipmromteuinnsizabtuiotnnhoatdpirnodteuicnesdfsreormumoAthbesracgeallintsytpveasr,iosuuschkearastianolcyymte- from Isoelectricfocusingrunningconditionswere500Vhat500V,then3,000Vh phoblastoidBcelllineorA431cells(Fig.1B).Thisreactivitywas h at2,000V,and37,500Vhat2,500V.BlotswereblockedinPBS,0.1%Tween largely independent from conditions affecting cell differentiation ttp 2cu0b(aptHed7w.4it)h,4h%umBaSnAse,r1a0(%dilFuCteSd(1S:5ig0m0ain-APlBdrSic,h0,.1S%t.LTowueiesn,M20O[)paHnd7.i4n]-, (highcalcium concentrations) orheat stress. ://w w 10%FCS,4%BSA)orrabbitsera(1:1000)for2h.Abreactivitywasdetected Whenanalyzedwithkeratinocyteproteinlysatesfractionatedby w b(SyigHmRaP--Acoldnrjiucgha)toerdEpCroLterienaAge(n1t:1(A00m0e)rasnhdamvisPuhaalrizmeadcbiayBdiioamtecinho).benzidine two-dimensional SDS gel electrophoresis, the preimmune rabbit .jim m u Peptidesequencing n o Peptide sequencing using Edman degradation was done at Toplab (Mar- l.o tminoslroiegdy,seGaerrcmheasnwy)eraecpcoerrfdoinrmgetdoastttahnedEarxdPAprSoYceMduorleesc.uDlaartBabioalsoegyanSderhvoer- byrg/ oftheSwissInstituteofBioinformatics(www.expasy.org)ortheNational g u CenterforBiotechnologyInformation(www.ncbi.nlm.nih.gov). e s t o Proteinsandsyntheticpeptides n A Recombinant heat shock protein (hsp)27 protein and an epidermal cyto- p keratin preparation were obtained from Sigma-Aldrich or BIOTREND ril 6 (Cologne, Germany). pGEX-2T-ezrin was a gift of Dr. Monique Arpin , 2 (LaboratoiredeMorphogene`seetSignalisationCellulaires,InstitutCurie, 0 1 Paris,France)andwasexpressedasGST-fusionproteininEscherichiacoli 9 strain BL21 (DE3) (Novagen, Madison, WI). Maspin, peroxiredoxin 2 (PRDX2), and keratin 6 were amplified by RT-PCR from human kerati- nocyte mRNA, cloned into the His-tag pET16b-vector (Novagen), and expressedasHis-tagfusionproteinsinE.colistrainXL2-Blue(Stratagene, LaJolla,CA).SyntheticpeptideswereobtainedfromBiomers.net(Ulm, Germany). Patientsandcontrols Thelocalethicscommitteeapprovedthestudy.Patientsandhealthycontrols participated voluntarily and gave written informed consent. Psoriasis patientsweretypedforHLAclassIallelesandclassifiedfortypeIorII psoriasis: type I psoriasis = early onset (,40 y of age) and/or positive FIGURE 1. Rabbit immunization with GAS induces serologic re- family history and/or inheritance of HLA-Cw6, -B57, or -B13. Type II psoriasis=lateonset(.40y),negativefamilyhistoryforpsoriasis,and activities against keratinocyte proteins. Western immunoblot of protein lysatesfromprimaryhumankeratinocytes(Kc),theepidermoidcellline lackofHLA-Cw6(2). A431,andanEBV-transformedlymphoblastoidBcellline.Reactivityof Ag-specificTcellstimulationandgenerationofAg-specific apreimmune(A)orastreptococci-specifichyperimmune(B)rabbitserum. Tcelllines Toinduceproteinsdependentonstressordifferentiation,cellswereheat shockedbyincubationat43˚Cfor8hand/orgrownwithhighcalcium(Ca2+) PreparationandcultivationofPBMCs,Tcellstimulation,andgenerationof concentrations (1.2 mM) for 24 h before harvesting. Standard molecular Ag-specific T cell lines were done as described (25, 26). For T cell stimulation,13106/mlPBLsweregrowninthepresenceofproteinAgs massmarkerproteinsweremyosin(200kDa),b-galactosidase(116kDa), (5mg/ml),peptides(10mg/ml),orPHA(1:100)for40h(IFN-g–release phosphorylaseB(97.4kDa),BSA(66kDa),eggalbumin(45kDa),carbonic ELISPOT) or 5 d ([3H]thymidine incorporation). The IFN-g–release anhydrase(31kDa),andsoybeantrypsininhibitor(21.1kDa).Arrowheads ELISPOTassay(Mabtech,NackaStrand,Sweden)wasperformedasde- indicateseveralofthenewlyinducedserologicreactivities. 5394 POSTSTREPTOCOCCALPSORIATICAUTOANTIGENS D o w FIGURE2. Commonreactivitiesofstreptococci-specificrabbitseraandpsoriasispatientserawithkeratinocyteproteins.Todefinepotentialpsoriatic n lo autoantigensresultingfromastreptococcal-inducedimmuneresponse,lysatesofhumankeratinocytesfractionatedbytwo-dimensionalgelelectrophoresis a d wereprobedbyimmunoblottingwithpreimmunerabbit(A),streptococci-specifichyperimmunerabbit(B),psoriasispatient(C),orhealthycontrol(D)sera. e d E,Coomassiestain.Identicalproteinsrecognizedbythestreptococci-specificrabbitandpatientseraarecircled. fro m h sera(Fig.2A)stainedonlyafewkeratinocyteproteinswithamajor organicpyrophosphatasewereconsideredlesslikelytobetargets ttp reactivity against a protein that, according to its position in the of a tissue-specific autoimmune response, and we omitted them ://w Coomassie-stained gel (Fig. 2E), represented actin. Instead, the from furtheranalysis. w w sptrroetpetioncso(cFciig-s.p2eBc)i,fiacsrdabidbitthseesrearareoacfttehdewpsitohrisaesviserpaaltikeenrtasti(nno=cy7te) psoPrBiaMsiCssanodf 7262HheLaAlthcylaisnsdIiv-tiydpueadlspwatiitehnotustwaitfhamchilryonhiicstpolrayquoef .jim m (Fig. 2C). Seven keratinocyte proteins were stained by the strep- psoriasiswerestimulatedinvitrowiththeseproteins.Theincidence u n tococci-specificrabbitseraandallpsoriasispatients’serabutnotby ofstreptococcalinfectioninthispatientpopulationwasanalyzed o thepreimmunerabbitorthehealthycontrolsera(n=8)(Fig.2D). byWeisenseeletal.(12);anincreasedprevalenceofstreptococcal l.org Theseproteinswereconsideredpotentialtargetsofastreptococcal- infectionwasnotedinHLA-Cw6+patients.However,becauseof b/ inducedautoimmuneresponseinpsoriasisandidentifiedbyamino a mean disease duration of 15 y, a direct correlation between y g acid sequencing and alignments with the Swissprot protein se- psoriasis onset and streptococcal angina was not possible. T cell ue s quence library. They represented ezrin/cytovillin, maspin/serpin stimulationwasmeasuredbyanELISPOTassayidentifyingIFN- t o B5,PRDX2,hsp27/b-1(hsp27),keratin6,GAPDH,andinorganic g–producingcellsandexpressedasthenumberofSFCsper1.53 n A pyrophosphatase(TableI). 105PBMCs.Theresultsweredifferentiatedaccordingtothemain p psoriasis risk allele, HLA-Cw6, which was present in 38 of 74 ril 6 Ezrin, maspin,PRDX2,andhsp27areparticularly (51.4%)ofthepsoriasispatients andtwoofthehealthycontrols. , 2 immunogenic forTcellsfrompsoriasispatients 0 PHAstimulation servedaspositivecontrol. 1 9 To determine the potential role of the different proteins as auto- Numbers ofSFCs for PHAstimulation andbaseline activation antigensofthepsoriaticTcellresponse,PRDX2,ezrin,andmaspin (datanotshown)weresimilarinpatientsandcontrols.Twoofthe wereexpressedasrecombinantfull-lengthproteins,andkeratin6 proteins,PRDX2(p=0.0003)andmaspin(p=0.0091),induced wasexpressedastwooverlappingproteinscorrespondingtoaa10– asignificantlygreaternumberofSFCsinpsoriasispatientsthanin 210 and 190–559 of the keratin 6f isoform. Recombinant hsp27 healthy subjects (Fig. 3, Table II). This difference was more andacytokeratinpreparationfromkeratinocyteswerepurchased. pronouncedforHLA-Cw6+patients(PRDX2:p=0.0006;maspin: Because of their ubiquitous tissue distribution, GAPDH and in- p = 0.0026) than for HLA-Cw62 patients (PRDX2: p = 0.0301; TableI. Aminoacidsequencesofpeptidesandcorrespondingproteinsasidentifiedbyhomologysearches AminoAcidSequenceofthePeptidesandPositioninthe PrimaryStructureoftheCorrespondingProtein NameofIdentifiedProteins;Synonyms MolecularMass(kDa) aa23 GFPTWLK aa29 Ezrin;p81,cytovillin,villin-2 90 aa162 LTRDQWEDRIQV aa173 Ezrin;p81,cytovillin,villin-2 aa426 KIALLEEARRKEDEVEEW aa443 Ezrin;p81,cytovillin,villin-2 aa84 WGDAGAEYVVESTGVFTTM aa102 GAPDH 42 aa172 LATQSNEITIPFTFESRAQ aa190 Heatshockprotein27;HSP27,heatshockproteinb-1,hspB1, 28 aa26 GQYISPFHDIPIYADK aa41 Inorganicphosphatase 35 aa360 YEELQITAGR aa369 Keratin6 60 aa159 KILVVNAAYFVGK aa171 SerpinB5;maspinproteaseinhibitor5 45 aa120 DEGIAYRGLFIIDGK aa134 Peroxiredoxin-2;thioredoxinperoxidase1,NKenhancingfactorB 21 TheJournal ofImmunology 5395 FIGURE4. RepresentativespectratypesofTCRBVgenerearrangements ofthepsoriaticskinlesion,PBL,Ag-specificandnegativecontrolTcelllines. TCRb-chaincDNAfromAg-specificTcelllineswasamplifiedbyPCRusing 26TCRBV gene-specific primer pairs.Thefragment lengthsofthePCR productsofeachTCRBVgenefamilywereanalyzedonageneticsequencer, yielding spectratypes with peaks spaced by three nucleotides. Each peak representsTCRb-chainrearrangementsofthesamelength.AlthoughCDR3 D o spectratypesfromPBLsshowlargelyGaussian-likedistributions,theAg- w specificTcelllinesandthepsoriaticskinlesionrevealabiasforTCRBV nlo rearrangements with particular lengths of the CDR3, which suggests the a d presenceofoligoclonalAg-drivenTcellexpansionsinthesesamples. ed fro m Thus,PRDX2andezrinseemparticularlyimmunogenicforthe h Tulactieolln-:mmedasiaptiendinimHmLuAn-eCwre6s+poannsdehisnp2t7heinoHveLrAal-lCpws6or2iapssisorpiaospis- ttp://w FIGURE3. StimulationofPBMCsfrompsoriasispatientsandhealthy w controls with the different proteins, a crude preparation of keratinocyte patients. As partial-length proteins, keratin 6f seems to be im- w cytokeratins,orPHA.Resultsaregivenforhealthycontrols(H;n=22),all munogenic forselect patients. .jim psoriasispatients(PV;n=74),anddifferentiallyforHLA-Cw62(Cw62, m Ezrin,maspin,PRDX2,andhsp27promoteanoligoclonal u 36/74)orHLA-Cw6+(Cw6+,38/74)psoriasispatients.Theyareexpressed n asmeanSFCsper1.53105PBMCsaftersubtractionofthecorresponding Tcellexpansioninvitro ol.o negative controls. Vertical bars indicate SD. ♦, patient with particularly InvivoandinvitroAgstimulationofTcellsmaypromotetheoli- rg strongresponse. goclonalexpansionofAg-specificTcellpopulations,whichcanbe b/ y identifiedbyarestrictedTCRrepertoire(29).Moreover,Tcellre- g u maspin: p = 0.3768) (Table II) and was reflected by particularly sponsestoimmunodominantantigenicpeptidesmaybecharacter- es strongresponses ofindividual patients (Fig.3). ized by identical or homologous TCR CDR3 motifs in different t on Onaverage,hsp27andezrin(Fig.3)alsoinducedanincreased individuals(30–32).WeusedtheseattributesofTcellAgspecificity A p ssotpniomlynudfleoadrtiohtonspa2in7griepnastotehrreiaesHxistLeAnpt-aCatiswenw6t2se.llp,Battehiceeandutsisfef(eprhe=enac0let.h0wy30ac7so;sniTgtarnobillfisecIarIen)-t. ttgoheefnueprrstahotereirdactihicnarivmacitmtreourinzfereortmheseptrhoeenlesvPea.BnFMcoerCotsfhtiohsfeppauorppteoantsiteeia,nlTtawucteioltlhanlietnixgetesennwsseifvoreer ril 6, 20 1 Stimulation with the cytokeratin preparation reached statistical streptococcal-driventype-1psoriasisbyperiodicrestimulationwith 9 significanceonlyforHLA-Cw6+patients(p=0.047).Theaverage thedifferentrecombinantproteinsorPHA.AsdeterminedbyFACS Tcellstimulationinducedbythepartial-lengthkeratin6proteins analysis, the Ag-specific T cell lines were dominated by CD8+ wasnotincreasedinthegroupofpsoriasispatientsversushealthy Tcells,withaCD4/CD8ratioof0.13,0.32,0.28,0.23,0.43,and subjects, although individual patients responded quite strongly 0.92fortheezrin-,maspin-,PRDX2-,keratin6-,hsp27-specific,and (Fig. 3). None of the proteins induced an increased T cell stim- PHA-stimulatedTcelllines,respectively,comparedwith0.89for ulationin thetwo HLA-Cw6+healthy subjects. thecorrespondingbloodTcells. TableII. Ag-specificstimulationofbloodlymphocytesfromhealthycontrolsandpsoriasispatients. ProteinUsedfor AllPsoriasisPatientsversus HLA-Cw62PsoriasisPatients HLA-Cw6+PsoriasisPatients HLA-Cw6–PositiveversusHLA-Cw6– Stimulation HealthyControls versusHealthyControls versusHealthyControls NegativePsoriasisPatients hsp27 0.0708 0.0307* 0.3274 0.0729 Ezrin 0.0600 0.0681 0.1569 0.3886 Maspin 0.0091* 0.3768 0.0026* 0.0119* PRDX2 0.0003* 0.0301* 0.0006* 0.1177 K6-N 0.2242 0.3572 0.1587 0.3440 K6-C 0.4581 0.3397 0.3171 0.2572 CK 0.0641 0.1335 0.0468* 0.2802 SignificanceofdifferencesbetweenthemeansofSFCsmeasuredbyELISPOTandanalyzedbyttest(pvalues). pp#0.05. CK,cytokeratin;K6-C,C-terminalproteinpartofkeratin6;K6-N,N-terminalproteinpartofkeratin6. 5396 POSTSTREPTOCOCCALPSORIATICAUTOANTIGENS TheTCRusageofthedifferentTcelllineswascomparedwiththat TheTCRrearrangementsfrombloodTcellsandthePHA-driven of the psoriatic skin lesion and blood lymphocytes by fragment- T cell line were heterogeneous. In contrast, many of the TCR lengthspectratypingoftheTCRb-chainrearrangements,whichhad rearrangements from the Ag-specificT cell lines were highly re- been amplified by 26 PCR reactions specific for the different petitiveandindicatedAg-drivenclonalTcellexpansions.Clonal TCRBVgenefamilies.ThisapproachmayidentifyrestrictedTcell TCR rearrangements within a given TCRBV gene family repre- populationsastheresultofabiasedusageofTCRlengthswithin sented up to 93% of the analyzed TCR sequences (Table III). A agivenTCRBVgenefamily. similardominanceofindividualTcellcloneswasseenwithinthe TCRBVgenespectratypingofnonstimulatedPBLsandthePHA- psoriatic skin lesion, with up to 89% identical TCR rearrange- drivenTcelllineshowedapredominanceofquasi-Gaussianb-chain ments in a given TCRBV gene family (Table IV), confirming lengthsinmostTCRBVgenefamilies,whichreflectedunselected formerfindingsofanoligoclonalpsoriaticTcellexpansion(7–9). Tcellpopulations.RepresentativespectratypesaregiveninFig.4. TCRoftheAg-specificTcelllinesandthelesionalpsoriatic VariousTCRBVgenefamiliesoftheAg-specificTcelllinesand TcellinfiltratesharehomologousclonotypicCDR3 b-motifs the psoriatic skin lesion displayed strongly biased patterns of fragment lengths, with discrete prominent peaks indicating Ag- HomologiesintheCDR3ofexpandedTCRb-chainsmayindicate drivenoligoclonalTcellexpansions.Severalofthesepeaksinthe T cell specificity for the same Ag. Comparison of the deduced Ag-specific T cell lines and the psoriatic skin lesion overlapped, aminoacidsequencesshowedobvioushomologiesintheCDR3of whichindicatedthat,inthedifferentsamples,TcellswithTCRsof severalclonalTCRrearrangementsoftheAg-specificTcelllines identical lengths had been selected. The rearrangements of these andthelesionalpsoriaticinfiltrate(TablesIII,IV).Aclonallyex- TCRBVgenefamilieswereanalyzedbycloningandsequencingof pandedCDR3motifoftheezrin-specificTcelllinewiththeamino theTCRcDNA.TheywereTCRBV6,BV13.1,BV13.2,andBV14 acid sequence SSSGS (clone E1; Table III) was observed in two D for theezrin-specificTcell line;TCRBV3 for thehsp27-specific variations,SSSGandLSSG(clonesPV20and21;TableIV),inthe o w T cell line; TCRBV8, BV9, BV17, and BV21 for the maspin- skin lesion. The PRDX2- (clones P2–6), hsp27- (clone H4), and n lo specificTcellline;andTCRBV3,BV8,BV13.2,andBV21forthe ezrin-specific(clonesE2andE5)Tcelllinesandthepsoriaticskin a d PRDX2-specific T cell line. None of the TCR fragment-length lesion(clonesPV1,2,9–13,and23)containedclonallyexpanded ed patternsofthekeratin6-specificTcelllinesoverlappedwiththose CDR3-amino acid motifs with variations around a core of four fro ofthepsoriaticskinlesion. amino acids: SSGTG (Tables III, IV). A clonotypic TCRBV8- m h ttp TableIII. ClonallyexpandedTCRb-chainrearrangementsintheAg-specificTcelllinesandCDR3homologieswithTCRrearrangementsofthe ://w w correspondingpsoriaticskinlesion w .jim DeducedAminoAcidSequence Identical/Total ClonesWith m TCRBV (OneLetterAminoAcidCode) TCR HomologousCDR3 u n Gene Rearrangements Identical Clone inthePsoriatic o TCL Family BV N-D-N BJ BJ (n)a TCRs(%) Designation SkinLesion(seeTableIV) l.o rg Ezrin 3 CAS SSSGS YNEQFFG 2.1 13/38 34.2 E1 PV20,21 b/ CASS SGTGSK YNEQFFG 2.1 15/38 39.4 E2 PV9–13,23 y CASSL QDNAN GYTFGS 1.2 5/38 13.1 E3 gu 6 CAS RVRGRVSYSRD EQYFG 2.7 18/32 56.2 E4 es CAS NRTPGTGDK QYFG 2.7 3/32 9.4 E5 PV9–13 t o n 13.1 CAS TRTGGLLL NTEAF 1.1 24/40 60.0 E6 A CASS FLAGGP NEQFFG 2.1 12/40 30.0 E7 PV4,7,14,16–19,24,25,30,31 p 14 CCCAAASSSSSS FFPLGLARSPAGGAGNGLV ENTQDEFQTFQFGFYGFG 222...113 126///44900 6256...506 EEE1890 PV17,25,30,31 ril 6, 20 Maspin 8 CASSL VT EQFFG 2.1 3/22 13.6 M1 PV8,B12 19 CASS FSFSPGA NTEAFFG 1.1 4/22 18.2 M2 9 CASS PLAGG SYNEQFFG 2.1 6/15 40.0 M3 PV4,7,14,16,17–19,24,25,30,31 CASSQ EMYRNPN TGELF 2.2 4/15 26.6 M4 17 CAS E NQPQHFG 1.5 7/34 20.5 M5 CASS IRG YNEQFFG 2.1 6/34 17.6 M6 CASS FSM NTEAFFG 1.1 6/34 17.6 M7 CAS KETGGQ TQYFGP 2.5 5/34 14.7 M8 21 Noclonaldominance 1–3/37 PRDX2 3 CAS RSRVQ EQYFG 2.7 4/36 11.1 P1 CAS SGTGR ETQYFGP 2.5 10/36 27.7 P2 PV1,2,9–13,23 CAS SGTG QETQYFGP 2.5 2/36 5.6 P3 PV1,2,9–13,23 8 CASSL ISGTPSD EQFFG 2.1 22/62 35.5 P4 PV1,2,9–13,23 CASSL ITGTPSD EQFFG 2.1 2/62 3.2 P5 PV1,2,9–13,23 CASSL VSGTPSD EQFFG 2.1 1/62 1.6 P6 PV1,2,9–13,23 CASS TGV NTEAFFG 1.1 17/62 27.4 P7 13.2 Noclonaldominance 1–2/38 21 CASS FLN EQYFG 2.7 43/46 93.4 P8 hsp27 3 CASSL NTG NTEAFFG 1.1 8/35 22.8 H1 PV12 CAS TQKDRG PQHFG 1.5 9/35 25.7 H2 CASS FHGVGLR GYTFGS 1.2 7/35 20.0 H3 CASS RTGTGN TGELFFG 2.2 5/35 14.3 H4 PV9–13 CAS NPPGQGAG EQYFG 2.7 3/35 8.6 H5 PV2 AminoacidsequencehomologieswiththeCDR3ofotherTCRrearrangementsareunderlined. aNumberofidenticalversustotalnumberofsequencedTCRrearrangementinagivenTCRBVgenefamily. BJ,joiningregiongene;BV,Vregiongene;N-D-N,rearrangementsite;TCL,TCLcelllineraisedagainsttheindicatedprotein. TheJournal ofImmunology 5397 TableIV. ClonallyexpandedTCRb-chainrearrangementsinthepsoriaticskinlesionandCDR3homologieswithTCRrearrangementsofthe Ag-specificTcelllines DeducedAminoAcidSequence Identical/Total CloneswithHomologous TCRBV (OneLetterAminoAcidCode) TCR Identical CDR3intheAg-Specific Gene Rearrangements TCRs Clone TCellLine(seeTable Source Family BV N-D-N BJ BJ (n)a (%) Designation III) PVskin 3 CAS RRKGQGRT YEQYFG 2.7 16/38 42.1 PV1 H5 lesion CAS GRKGQGRT YEQYFG 2.7 1/38 PV2 H5 CA IQGVL NTEAFFG 1.1 9/38 23.6 PV3 CASS LAGRG STDTQYFG 2.3 8/38 20.5 PV4 E7,E8,M3 6 CAS GRGG TDTQYFG 2.3 9/31 29.0 PV5 8 CASSL SLEG NQPQHFG 1.5 12/63 19.0 PV6 CASS FAGG YEQYFG 2.7 12/63 19.0 PV7 E7,E8,M3 CASS VT DTQYFG 2.3 18/63 28.5 PV8 M1,B1,B2 CASSL FGTGSSRGAEHK TQYFG 2.5 2/33 24.2 PV9 E2,P2–6 CASS RGTGVW EQYFG 2.7 1/33 24.2 PV10 E2,P2–6 CASS SGTGVW EQYFG 2.7 1/33 24.2 PV11 E2,P2–6 CASS AGTGNV NEQFFG 2.1 1/33 24.2 PV12 E2,P2–6,(H1) CASS SGTGD SGANVLT 2.6 3/33 24.2 PV13 E2,P2–6 CASS PLAGGPS YNEQFFG 2.1 1/33 3.0 PV14 E7,E8,M3 9 CASSQ EEVL YFG 2.7 17/19 89.4 PV15 13.1 CASS YGAGG TGELFFG 2.2 5/37 13.5 PV16 E7,E8,M3 CASS GGLAGV YNEQFFG 2.1 4/37 10.8 PV17 E7–9,M3 D CAS SLAGG SYNEQFFG 2.1 1/37 2.7 PV18 E7,E8,M3 ow CASS YHGLAGSG ETQYFG 2.5 4/37 10.8 PV19 E7,E8,M3 n CASSY SSSG NTEAFFG 1.1 7/37 18.9 PV20 E1 loa CASSY LSSG NTEAFFG 1.1 2/37 5.4 PV21 E1 de 14 ACASSSLSY PSGGGTVAYN TYGEEQLYFFFGGE 22..27 65//3279 1167..22 PPVV2223 E2,P2–4,P6 d fro m AS RLLAGE YNEQFFG 2.1 3/29 10.3 PV24 E7,E8,M3 h 17 CCAAASSSSS ALSLNPARGGDGVSPEQSTPM QNETYTQEFAYGFFFGG 212...715 12402///255999 131673...928 PPPVVV222765 E7–9,M3 ttp://w 21 CAS NKAG EQYFG 2.7 10/19 52.6 PV28 w w CAASSS NPLSAGGDV GETYQTYFGFGSG 12..25 71//1199 3160..85 PPVV2390 E7–9,M3 .jim ASS SLAGV ETQYFG 2.5 1/19 10.5 PV31 E7–9,M3 m u PBL 8 CASSL VT EQFFG 2.1 2/58 6.9 B1 PV8,M1 n o CASS VT DTQYFG 2.3 1/58 6.9 B2 PV8,M1 l.o CASSL VT EQYFG 2.7 1/58 6.9 B3 PV8,M1 rg AminoacidsequencehomologieswiththeCDR3ofotherTCRrearrangementsareunderlined. b/ y aNumberofidenticalversustotalnumberofsequencedTCRrearrangementinagivenTCRBVgenefamily. g BJ,joiningregiongene;BV,Vregiongene;N-D-N,rearrangementsite. u e s t o CDR3motifcomposedoftheaminoacidsVTofthemaspin-spe- arrangements couldbeassigned exclusivelyto theCD8+fraction n A cificTcellline(cloneM1)correspondedtothelesionalpsoriatic of the respectiveT cell line and documented that Ag-stimulation p TcellclonePV8.ClonalTCRrearrangementsofthemaspin-spe- had expandedCD8+Tcellclones. ril 6 cific(clone M3)and theezrin-specific (clones E7–9) Tcelllines , 2 Keratinocyteproteinsandstreptococcal proteinsshareamino 0 sharedavariationwithadominantCDR3motif:(F/G/P)LAG(G/V) 1 acidsequencehomology 9 ofthepsoriaticskinlesion(clonesPV4,7,14,16–19,24,25,30, and 31).Uptofive aminoacids were identical.These CDR3ho- The streptococcal-induced autoimmune response against kerati- mologies of the Ag-specific T cell lines and the corresponding nocyte proteins might result from protein regions sharing amino psoriaticskinlesionindicatethattheproteins,whichinducedthe acidsequencehomologies.Toidentifypotentialmimicryepitopes clonalTcellexpansionsinvitro,mayberelevantfortheactivation of streptococci with hsp27, ezrin, maspin, or PRDX2, a direct thelesionalpsoriaticTcellinfiltrate. sequence comparison with the whole genome of GAS was per- Only one of these CDR3 motifs, VT, was found in the corre- formed. It identified various homologous regions of up to seven sponding bloodsampleof thepatient (B1-3;Table IV).No other identical consecutive amino acids. Several of them referred to clonal selection or bias toward homologous CDR3 motifs was streptococcal Ags other than M-proteins, such as Ig-Fc– or fi- seeninblood,thePHA-drivenTcellline,orthenegativecontrol bronectin-binding proteins, RecF, RopA, or proteins from the Tcelllines. lantibioticgene cluster region. Examplesaregiven inTable V. When used for stimulation of blood lymphocytes, various The specificallyexpandedTcellclonesmayrepresentCD8+ peptidesfromthekeratinocyteproteinsdesignedaccordingtothese Tcells homologies (Ezrin-Pep2, PRDX2-Pep2, hsp-Pep2, and Maspin- To assess the phenotype of select clonally expanded T cells, the Pep1)inducedastatisticallyincreasedTcellactivationinpsoriasis ezrin- and maspin-specific Tcell lines were separated into CD4+ patients(n=32)comparedwithhealthycontrols(n=17),asdid andCD8+Tcells.TCRb-chaincDNAfromtheseTcellsubsets several peptides from the corresponding streptococcal proteins: was amplified with primer pairs corresponding to the TCRBV9 RecF, RopA, and Maspin/Strep (Table VI). Differentiation ac- rearrangementPLAGG(cloneM3)ofthemaspin-specificcellline cording to HLA-Cw6 showed that the T cell response was par- or the TCRBV13S1 rearrangement FLAGGP (clone E7) of the ticularlystronginHLA-Cw6+patients(TableVI).Thedifferential ezrin-specific T cell line, cloned, and sequenced. Both TCR re- immunogenicity of select peptides was particular evident for 5398 POSTSTREPTOCOCCALPSORIATICAUTOANTIGENS PRDX2:ashiftof3aainthepeptidesequence(PRDX2-Pep1:aa 18),severalpeptideswithhomologiestostreptococcalM-proteins 24–35 of PRDX2; PRDX2-Pep2: aa 27–38) increased the stimu- (keratin-Pep2,-Pep3,and-Pep5andMaspin-Pep2)inducedapro- latory capacity significantly. nouncedTcellactivation,whichwasgreaterthanTcellactivation No differences between psoriasis patients and healthy controls byTT.Unlikethetwohalf-lengthkeratinproteins,definedkeratin6 wereobservedforstimulationwithtetanustoxoid(TT),awidely peptidespromotedTcellactivationusingthisapproach(Fig.5). used tetanusvaccine (33),or PHA. In a second approach, which also included keratin 6, potential mimicryepitopesweredefinedaccordingtopreferredaminoacidsat Discussion theanchorpositionsfortheHLA-Cw6–bindinggroove,whichareL, Severallinesofevidencesupportthatthekeratinocyteproteinsezrin, I,V,orYatposition9or6andI,L,F,orMatposition5(34).Several maspin, hsp27, PRDX2, and potentially keratin 6 may serve as nona-peptides within keratin 6, maspin, and ezrin exhibited both autoantigensofapathogenicTcellresponseinpsoriasisinducedby anchor motifs for HLA-Cw6 and amino acid homologies with streptococcal infection.Streptococci-specific rabbitsera and pso- streptococcalproteins(Keratin6-Pep1to-Pep8,Maspin-Pep2and riasispatients’serareactedagainsttheseproteins.Thereactivityof -Pep3,andEzrin-Pep4)(TableVII).Uptosevenofnineaminoacids the streptococci-specific rabbit sera was tissue selective against wereidentical.HomologieslargelyreferredtoM-proteinsbutalso proteinsfromkeratinocytesbutnotfromalymphoblastoidBcell to other streptococcal proteins, such as serum opacity factor, fi- lineortheepidermoidcarcinomacelllineA431.Theproteinsezrin, bronectin-bindingproteins,orstreptococcalhsp70.Asdetermined maspin,hsp27,andPRDX2inducedsignificantlyincreasedTcell bytheIFN-g–ELISPOTassayinHLA-Cw6+psoriasispatients(n= activation in PBMCs from psoriasis patients. Upon repetitive TableV. Potentialmimicryepitopesofthedifferentkeratinocyteandstreptococcalproteinsdefinedbysequencealignments D o w n PeptidesUsedfor HomologousAmino PositionofLast lo Stimulation AcidSequences AminoAcid GASProteinswithHomologousPeptideRegions,AccessionNo.,orGeneID a d e Ezrin-Pep1 LSSSEELLS+QQAARR 542 d fro SELTQAR 239 Ig-Fc-bindingproteingi|506943| m Ezrin-Pep2 LNIIIYYYEEE-KDDDDDDKKKLLL 192435440 puatalltifvreomregGuAlaStoMry1p,rgobte|AinE-00R4o0f9A2.r1e|lated http://w ++++ KDDKL w V+D+LIFYNEKKDDD+KL 191134 exotoxinGprecursor w.jim VDIYEKDGR 459586 putativesignalrecognitionparticle m Ezrin-Pep3 AKEELERQA 407 un EELERQ o PRDX2-Pep1 AFKEEEEVVLKKELLR+SQDDYYKKG 13357 Mproteingi|37595322 byl.org/ EVKLGDYKN 493 RopA(ropA)genegb|AF073922.1|AF073922 gu A K+ KLSDY G e s ALKQAKLSDYIG 1837273 RecFprotein,gb|AE004092.1|M1GAS(RecF) t o EVKL+DYK n A EVKLGDYKN 1572913 transcriptionregulatortriggerfactorfrom p PRDX2-Pep2 E+VKKLLSSDDYYKGGKYV 38 gb|AE004092.1|M1GAS,completegenome ril 6, 2 0 QAKLSDYIG 1837273 RecFprotein,gb|AE004092.1|M1GAS 19 EVKL DYK V EVKLGDYKNLVV 1572904 RopAgenegb|AF073922.1|AF073922(RopA) SDYKGKY+ SDYKGKYL 310252 putativerRNAmethylase,gb|AE004092.1|M1GAS +KLSD +G IKLSDVRG 1490802 hypotheticalproteingb|AE004092.1|M1GAS,completegenome hsp27-Pep1 SEIRHTADRWRVSL 99 SEI H+ADRWRV++ SEIEHIAD RVGI 8657 FF22lantibiotic(scn)geneclusterregiongb|AF026542.1|AF026542 (hsp/Strep1) hsp27-Pep2 QLSSGVSEIRH 90 QL+SG++E+ QLTSGLTEL 1808819 hypotheticalprotein,ID:15674250NC_002737 L SGV+ RH LGSGVASFRH 1539948 putativeantibioticresistanceproteinNorA +SS +-SEI H ID:15674250NC_002737 ISSQILSEIEH 8630 FF22lantibiotic(scn)geneclusterregiongb|AF026542.1|AF026542 (hsp/Strep2) MaspinPep1 MGNIDSINCK 215 +NIDS DIK YSNIDSCDIK 4462 FF22lantibiotic(scn)geneclusterregiongb|AF026542.1|AF026542 (Maspin/Strep) Alignmentsareindicatedforeverypositionbytheidenticalaminoacidswhenhomologous,orincaseofconservativesubstitutionswith“+.”Peptidesusedforstimulationare underlined. TheJournal ofImmunology 5399 TableVI. StimulationofPBLsofpsoriasispatientsandhealthycontrolswithmimicrypeptidesasdeterminedby3H-Tdtincorporationandstatistical analysis Stimulation(cpm[Mean6SD])a pValue Controls PV PVCw6+/ PVCw62/ Peptide (n=17) (n=32) PVCw6+ PVCw62 PV/Controls Controls Controls PVCw6+/2 Ezrin-Pep1 4,82363,970 5,92766,305 7,33568,079 4,44463,479 0.234 0.144 ND 0.126 Ezrin-Pep2 4,16862,054 6,44665,527 7,74266,792 5,80463,604 0.024* 0.033* 0.140 0.228 Ezrin-Pep3 5,32364,168 8,401610,147 8,708612,428 9,49368,212 0.075 0.165 0.113 ND PRDX2-Pep1 3,33062,605 4,81165,416 5,81067,001 4,40862,962 0.106 0.106 0.189 0.257 RecF 3,48462,862 5,18364,408 6,22165,362 4,18962,752 0.059 0.046* 0.287 0.125 PRDX2-Pep2 3,50363,019 6,12466,216 7,39367,719 5,19164,411 0.029* 0.042* 0.147 0.231 RopA 3,28762,467 5,97264,768 6,90765,464 5,23663,899 0.007* 0.014* 0.121 0.226 hsp27-Pep1 4,47662,946 5,52366,567 7,39168,570 3,89262,624 0.228 0.113 ND 0.082 hsp/Strep1 3,76462,187 4,96663,902 5,95864,781 4,06462,507 0.091 0.060 0.385 0.152 hsp27-Pep2 3,42762,130 6,22166,611 7,81868,515 4,37362,546 0.019* 0.035* 0.178 0.083 hsp/Strep2 4,18162,184 5,20566,889 6,79169,103 4,06663,082 0.227 0.147 ND 0.154 Maspin-Pep1 3,75762,296 5,48264,650 6,19165,445 4,90563,070 0.048* 0.062 0.162 0.270 Maspin/Strep 4,29262,432 6,16264,465 7,02965,038 5,89463,670 0.035* 0.035* 0.153 0.290 TT 20,589613,501 19,655613,505 20,004612,849 16,529610,475 ND ND ND ND PHA 94,575628,376 67,034634,750 68,388628,531 56,284634,116 ND ND ND ND aResultsaregivenasmeanoftriplicatecpmaftersubtractionofthenegativecontrol. PBLswereincubatedwithpeptides(10mg/ml).Stimulation-inducedproliferationwasmeasuredonday5by3H-Tdtincorporation.Foraminoacidsequenceofpeptidesrefer Do toTableV. w pp,0.05;calculatedbyfandttests. nlo ND,notdetermined(becauseresultsforpsoriasiswerelowerthancontrols);PV,psoriasispatient. a d e d restimulation,theypromotedanoligoclonalexpansionofTcells, PRDX2isanotherstress-inducedcytoplasmaticprotein.Itmay fro whichusedTCRb-chainrearrangementssharingahighdegreeof beupregulatedintheskinbyIFN-g,whichisabundantlyexpressed m h CDR3 homology with clonal TCR rearrangements of the corre- within psoriatic lesions(46). ttp sponding psoriatic skin lesion. The antigenicity of the proteins Theparticularantigenicity of thedifferent proteins forpsoriasis ://w could be assigned to peptides sharing amino acid sequence ho- patientswasreflectedbyasignificantlyincreasedTcellactivationand w mologieswithstreptococcalproteins. byselectoligoclonalTcellexpansionsfollowingperiodicAg-specific w Threeoftheseproteinswerekeratinocytespecific(keratin6)or restimulationofbloodTcellsfromapatientwithstreptococcal-driven .jim selectiveforepithelialsurfaces(ezrinandmaspin).Keratin6had psoriasis.CDR3sofseveraldominantTCRb-chainclonotypesfrom m u formerly beenproposed asa psoriaticautoantigen because ofse- theseTcelllinesshareduptofourorfiveaminoacidsinsequencewith no quencehomologieswithstreptococcalM-protein(16).However,in TCRclonotypesfromthecorrespondingpsoriaticlesion.Giventhe20 l.o rg ourapproach, overlappingkeratin6fproteinswere immunogenic different amino acids, the overall likelihood that two TCR re- b/ onlyforselectpatientsorwhenusedaspeptidesdesignedaccording arrangements share four or five identical amino acids is 1:204 = y toHLA-Cw6–bindingmotifs.Thiscouldreflectalackofabilityof 1:160,000or1:205=1:3,200,000.Accordingly,theCDR3homolo- gu e theproteasometocleaveappropriatepeptideswithintheAPCs. giesoftheAg-specificTcelllineswiththelesionalpsoriaticclono- st o Ezrin,acytoskeletallinkerprotein,isaparticularlyinteresting typesseemtobenonrandomandsupportthatmaspin,ezrin,hsp27, n A candidate as a psoriatic autoantigen. A high expression in kera- andPRDX2mightrepresenttargetsofthelesionalpsoriaticimmune p tinocytes, the apical microvilli of the enteral mucosa, and the response.Thisconclusionisfurthersupportedbytheobservationthat ril 6 anteriormyoepitheliallayeroftheiriscouldconstituteapotential oneofthedominantCDR3motifs,(F/G/P)LAG(G/V),oftheTcell , 2 autoantigeniclinkbetweenpsoriasis,inflammatoryboweldisease, lines and the psoriatic skin lesion were previously identified as 01 9 and autoimmuneuveitis,which are clinicallyassociated(35–38). aconservedclonotypicCDR3inHLA-Cw6+psoriasispatients(9). Maspin is a soluble cytoplasmic, membranous, and secreted Furthermore,severalclonotypicTCRswithhomologousCDR3sin extracellular serine protease inhibitor (serpin) and potent tumor theAg-specificTcelllines(E7,E8,andM3)andthepsoriaticskin suppressorinhibitingcellularinvasion,motility,andproliferation. lesion(PV14,17,18,and24)useddifferentTCRVbgenefamiliesbut Maspin is restricted to epithelial cell types and is abundantly hadrearrangedidenticalJbgenes.Becauseonlyanestimated10%of expressed in epidermis, stratified squamous epithelium of the HLA-Cw6+ individuals develop psoriasis (47), this BV gene vari- tonsils,andthemucosalepitheliumofthesmallintestine(39).Its ability, but conserved Jb gene usage, may reflect the private CD8 expression isincreased inhyperplasticpsoriatic epidermis (40). TcellrepertoiresandcontributetothedifferentTcellresponsesof Hsp27,anevolutionarilyhighlyconservedmolecularchaperon,is different individuals to the different Ags, as reported for cross-re- homogeneously distributed throughout normal epidermis and activeimmuneresponsesinheterologousimmunity(48). stronglyincreasedinpsoriaticepidermis(41).Animmuneresponse Sequencealignmentsofthedifferentproteinswiththewholege- against microbial hsp may cross-react against the corresponding nomeofGASidentifiedvarioushomologouspeptidesofuptoseven autologoushspbecauseofmolecularmimicryandtriggerautoim- identicalaminoacids.Thesehomologieswerenotconfinedtostrep- munepathology(42).Enhancedexpressionofhsp27understresscan tococcalM-proteinsbutinvolvedotherstreptococcalproteins,suchas unveil previously hidden antigenic determinants, perpetuate auto- RopA,RecF,orFcRproteins.Homologouscandidateepitopesfromthe immune reactivity (43), and, thus, might promote stress-related putativeautoantigensorthestreptococcalproteinswereequallyef- worsening of psoriasis. Additionally, hsp27-specific T cells could fectiveininducingTcellresponses,supportinganimmunologiclink contributetotheincreasedriskforcardiovascularmorbidityseenin betweenforeignandself-Ags.Thiswiderspectrumofstreptococcal psoriasis(44).Increasedendothelialhsp27expressionandintimal correlates may explain why psoriasis, unlike other nonsuppurative infiltrationofhsp27-reactiveTcellsareconsideredimportantearly streptococcal sequelae, such as rheumatic fever or poststreptoco- eventsintheimmune-mediatedpathogenesisofarteriosclerosis(45). ccalglomerulonephritis,whichrequireparticularrheumatogenicor 5400 POSTSTREPTOCOCCALPSORIATICAUTOANTIGENS TableVII. PotentialmimicryepitopesofthedifferentkeratinocyteandstreptococcalproteinsdefinedbyHLA-Cw6anchorpositionsandsequence alignments Positionof AASequences, LastAmino KeratinocyteProteins Homologies Acid GASProteinswithHomologousPeptideRegions,AccessionNo.,orGeneID Scorea Keratin6-Pep1 NMQDLVEDL 249 6000 +M D+ E+L SMHDIYEEL 35 M-proteinprecursorpir|S60805 Keratin6-Pep2 DAKNKLEGL 430 8800 DAK KLE L DAKVKLEVL 43 M42proteingi|951444 Keratin6-Pep3 KLEGLEDAL 434 3000 +LEGL+DA ELEGLKDA 98 Mproteintype41emb|CAA41167 Keratin6-Pep4 KAKQDLARL 444 4400 K+KQD+ L KSKQDIGAL 280 Mproteingi|1263021 K+KQDL L KSKQDLGAL 181 geneproductgi|552001 Keratin6-Pep5 WYQTKYEEL 363 4000 QTKY+EL QTKYDEL 52 Mproteingi|3335229 TKYEEL D TKYEEL 42 Mproteinprecursorgi|S60805,M27 ow Keratin6-Pep6 ALQKAKQDL 441 4400 n ALQK +Q+L lo a ALQKKEQEL 92 Mproteingi|2981490 de AAAE+KKAAKKK+DDLL 243 heatshockprotein70gi|1750267 d fro AL+K K+DL m ALEQQKKKE+SKKKEYQDDDLLL 111798 Mprotgeeinneprpercoudruscotrggii||59582006021.ST2.2 http://w Keratin6-Pep7 GASGVGSGL 507 2200 w w AA+AGGVVGGSSGGLL 410 serumopacityfactorgb|AAD31503.2 .jim SGV G m u SGVGPG 32 Mproteinprecursorpir|S60833,M57 n o Keratin6-Pep8 RLLLLKKEEYQ+EELL 43581 2000 l.org LLKENEEL 142 Mproteingi|4235269 b/ KEYQ+L 244 y KEYQDL M3proteindbj|BAA03311 gu RL+ Y+EL es RLMTFYREV serumopacityfactorgb|AAD31485.2 t o Maspin-Pep2 YSLKLIKRL 92 14520 n A KL+K+L p SSLLKKKLSLLKKLRKRLLL 161058 Fn-biMndipnrgotperinotedibnj|BIAgbA|A83A9D953.31086.1 ril 6, 20 Maspin-Pep3 GLEKIEKQL 250 7260 19 ++K+EKQL VDKLEKQL 75 M25proteinemb|CAA63114 LEK+EKQ Mproteingb|AAC84046.1 LEKLERQ 120 Ezrin-Pep4 EYTAKIALL 431 8000 EY AKIA L EYNAKIAEL 672 isolate216Mproteingb|AY263387.1 Alignmentsareindicatedforeverypositionbytheidenticalaminoacidswhenhomologousorinthecaseofconservativesubstitutionswith“+.”Anchorpositionsforthe HLA-Cw6bindinggrooveareunderlined.PreferredaminoacidsareL,I,V,orYataminoacidposition9or6andI,L,F,orMatposition5ofpeptides. aPredictedbindingscoreforHLA-Cw6atwww-bimas.cit.nih.gov/molbio/hla_bind/. nephritogenicMproteinserotypes,maybeinducedbydifferentstrains HLA-Cw6 might reflect a select capacity of this HLA allele to ofLancefieldgroupA,aswellasgroupCandG,streptococci(10,49). present common epitopes of streptococcal Ags and their skin- Unfortunately,theTcelllinescouldnotbetestedwiththedifferent associated mimicry correlates. Ag presentation via MHC class I peptides,becausetheydiedoffafterfourroundsofAg-specificre- moleculesissupportedbythepredominanceofCD8+Tcellsinthe stimulation,probablyasaresultofthepredominanceofCD8+Tcells Ag-specificTcelllinesandtheCD8phenotypeoftwodominant andthelimitedlifespanofeffectorcells(50). TCRclonotypes(M3andE7)fromtheezrin-andmaspin-specific Theantigenicityofthedifferentproteinsandtheirpeptideswas Tcelllines.ThestimulationofCD8+TcellsbysolubleAgssuggests particularlyevidentforHLA-Cw6+patients.Severalpeptideregions that, invivo, the proteins may be subjected to cross-presentation within the proteins showed bindingcapacities for HLA-Cw6 and following a release from apoptotic keratinocytes, which may be homologiestostreptococcalproteins,andtheyactivatedTcellsin furtheraugmentedbyincreasedcelldamageinpsoriasis(51).Cross- HLA-Cw6+patients.Accordingly,theassociationofpsoriasiswith presentationiscrucialforthedevelopmentofcytotoxicCD8+Tcell
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