Ezrin, Maspin, Peroxiredoxin 2, and Heat Shock Protein 27

12 Pages·2010·0.79 MB·English

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
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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
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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