Leukocyte Antigen Class 11Molecules

13 Pages·2013·2.28 MB·English

Binding of Myelin Basic Protein Peptides to Human Histocompatibility
Leukocyte Antigen Class 11 Molecules
and Their Recognition by T Cells from Multiple Sclerosis Patients
AntoniettaValli,* AlessandroSette,t Ludwig Kappos,ICarla Oseroff,t John Sidney,t
GuidoMiescher,I Martina Hochberger,11 Ekkehard D. Albert,11 and LucianoAdorini*
*PreclinicalResearch, SandozPharma Ltd., CH-4002Basel, Switzerland; tCytelCorp., SanDiego, California 92121;IDepartmentof
NeurologyandDepartment ofResearch, UniversityofBasel, Kantonsspital, CH-4031 Basel, Switzerland;andlLaboratoryof
Immunogenetics, UniversityofMunich, D-8000Munich, FederalRepublicofGermany
Abstract ity leukocyte antigen class II molecules multiple sclerosis-
-
myelinbasicprotein Tcellepitopes
-
Multiple sclerosis (MS) is an autoimmune disease in which
myelin proteins have been implicated as autoantigens recog- Introduction
nizedbypathogenicautoreactiveTcells.Tostudytherelation-
ship between human myelin basic protein (hMBP) and HLA Multiple sclerosis(MS)' isachronic,inflammatorydiseaseof
alleles associated to MS susceptibility, such as DRB1*1501, thehumancentralnervoussystem(CNS)characterizedbyde-
the binding of synthetic peptides spanning the entire hMBP myelination and by focal infiltrates ofmacrophages, plasma
sequence to 10 purified HLA-DR molecules was determined. cells, andTcellsintheCNS(1). AlthoughthecauseofMSis
All the hMBP peptides tested showed binding affinity for at unknown,aTcell-dependentautoimmuneprocess,involving
leastoneoftheDRmoleculesanalyzed, butthreehMBPpep- autoreactiveTcellsspecificforamyelinprotein,hasbeenpos-
tides,includedinsequences 13-32,84-103,and144-163were tulated (reviewed in reference 2)andthedemonstration ofin
found capable ofbindingto three ormore DR molecules. The vivo-activatedTcellsspecificforhumanmyelinbasicprotein
hMBPpeptide84-103 wasthemostdegenerateinbinding, in (hMBP)inthebloodofMSpatientshasimplicatedhMBPasa
thatitboundto9outof10DRmoleculestested.Interestingly, candidateautoantigeninthepathogenesisofMS(3).
it bound with highest affinity to DRB1*1501 molecules. To The possible role ofhMBP in MS has been further sup-
correlatethebinding patternofhMBPpeptides toHLAclass portedbytheobservationthatexperimentalallergicencephalo-
II molecules with their recognition byTcells, 61 hMBP-spe- myelitis (EAE), an animal model forMS, can be inducedby
cific T cell lines (TCL) were established from the peripheral immunizationwithMBPorMBPpeptidesinadjuvant(4, 5),
bloodof20MSpatients,whowerehomozygous,heterozygous, as well as by passive transfer ofMBP-specific CD4+ T cells
ornegativeforDRB1*1501.AnalysisofhMBPepitopesrecog- (6-8).EncephalitogenicTcellsinmiceandratsarespecificfor
nizedbytheseTCLandtheirHLArestrictiondemonstrateda immunodominant MBP epitopes presented by specific MHC
verygoodcorrelationbetweenbindingdataandTcellprolifera- class II molecules; forexample, in BIO.PL mice encephalito-
tiontohMBPpeptides.AlthoughvirtuallyallhMBPpeptides genic T cells recognize the amino-terminal MBP peptide 1-9
tested could be recognized by at least one TCL from MS pa- presentedbyI-AU molecules(9).ThevariableregionsoftheT
tients, three immunodominant T cell epitopes were apparent cellreceptor(TCR)inMBP-specificTcellshavebeenfoundof
amongtheTCLexamined,correspondingexactlytothehMBP limitedheterogeneity, withapreferential expressionofVB8.2
peptidescapableofbindingtoseveralDRmolecules.Nomajor and Va 2 or Va 4, both in miceandrats(10). Thus, inthese
differencecouldbedetectedintherecognitionofimmunodomi- species, MHC class II and TCR gene products associated to
nant hMBP peptides by TCL from DRB1*1501 positive or recognition ofencephalitogenic MBPepitopesbyTcellshave
negativeMS patients. Theseresults have implications forthe been identified. Recently, however, it hasbeen demonstrated
role ofhMBP as relevant autoantigen, and ofDRB1*1501 as thatEAEcanbeinducedbyotherautoantigens,suchasproteo-
susceptibility allele in MS. (J. Clin. Invest. 1993. 91:616- lipidprotein (11), raisingthepossibilitythatamultiplicityof
628.)Key words: immunodominance- humanhistocompatibil- myelinantigensmaybecapableoftriggeringautoimmunereac-
tions resulting in the clinical manifestations characteristic
ofEAE.
Dr. Valli is currently at Cattedra di Malattie Infettive, Universita di Asignificantassociationbetweenmajorhistocompatibility
Parma,ViaGramsci 14,1-43100Parma,Italy;Dr.Adoriniiscurrently complex (MHC) class II gene products and disease has been
atRocheMilanoRicerche, ViaOlgettina58, 1-20132 Milan,Italy. observedin MSpatients. ThehigherconcordancerateforMS
AddressreprintrequeststoDr.LucianoAdoriniathispermanent inmonozygoticcomparedtodizygotictwinsindicatesageneti-
address:PreclinicalResearch386-106,SandozPharma,Ltd.,CH-4002 callydeterminedsusceptibilitytothedisease(12).Becausethe
Basel,Switzerland. frequency ofthe HLA class II allele DRB1*1501, and ofthe
Receivedforpublication 6July 1992 andin revisedform 17Sep-
associated DQB1*0602, issignificantly increased inNorthern
tember1992.
J.Clin. Invest. 1. Abbreviations used in this paper: APC, antigen-presenting cells;
©TheAmerican SocietyforClinical Investigation, Inc. EAE,experimentalallergicencephalomyelitis;EDSS,expandeddisabil-
0021-9738/93/02/0616/13 $2.00 itystatusscale;hMBP,humanmyelinbasicprotein;MS,multiplescle-
Volume91,February 1993,616-628 rosis;TCL,Tcellline;TCR,Tcellreceptor.
616 Vallietal.
European MS patientscomparedtocontrols, DRB1*1501, or gen fluoride in the presence ofappropriate scavengers. The peptides
gene(s) in linkage disequilibrium with it, could represent a werethenpurifiedbyreversedphasehigh-performanceliquidchroma-
susceptibilitygeneforMS(13, 14).AlthoughtheDRBl*1501 tography (HPLC). Peptide purity was substantiated by amino acid
haplotype is associated with the highest relative risk ofMS, sequence and/or composition analysis. They were routinely > 95%
other HLA class II alleles are overrepresented among certain pureafterHPLC.
Affinity purification ofDR molecules. The following Epstein-
ethnic groups ofMS patients: DR4 in Southern Italians (15)
Barr virus (EBV)-transformed homozygous cell lines-LG2
and Arabs (16) and DR13 in Japanese (17) and Mexicans
(DRB1*0101), 3107 (DRB5*0101), MAT (DRBI*0301), PRIESS
(18). In contrast to the well-established association between
(DRBI*0404), BIN40 (DRBI*0404), SWEIG (DRBI*1101),
HLAgenesandMS,theassociationofTCRgenes,eitherinthe PITOUT (DRBI*0701): or transfected fibroblasts-L466.1
germline (19, 20) or in the hMBP-specific peripheral T cell (DRBI*1501),L416.3(DRB5*0101),L242.5(DRBI*1601),L255.1
repertoire (21-25), with MS isstill unclear. (DRB5*0201)-wereusedasasourceofHLAclassIImolecules.DR-
Toassessthepossible role ofhMBPasautoantigeninMS, transfectedfibroblastwereakindgiftofDr.R.W.Karr(Universityof
several groups have studied the epitope specificity ofhMBP- Iowa,IowaCity,IA).TheHLAnomenclatureisaccordingtoreference
reactiveTcellsinMSpatientsandcontrols(26-32).Autoreac- 34.CelllineswereculturedinRPMI 1640mediumsupplementedwith
tive T cells, mostly CD4' and DR-restricted, specific forsev- 10%heat-inactivatedfetalcalfserum(FCS)orhorseserum(Hazelton
Biologics,Inc.,Lenexa,KS).Cellswerelysedataconcentrationof108
eralhMBPpeptideshavebeenobtainedfrombothMSpatients
cells/mlin 50mMTris-HCI, pH 8.5,containing2%Renex, 150mM
andhealthycontrols. Although,collectively, majordifferences
NaCI, 5 mM EDTA, and 2 mM phenylmethyl sulfonyl fluoride
werenotapparentintheTcellreactivitytohMBPbetweenMS (PMSF). Thelysateswerecleared ofnuclei anddebrisbycentrifuga-
patientsandcontrols, ithasbeensuggestedthatrecognition of tionat 10,000gfor20min.
immunodominanthMBPepitopesinindividualsexpressingan DRmoleculeswerepurifiedessentiallyasdescribed(35,36)using
MS-associated HLA class II allele could be important in the themabLB3.1(37)covalentlycoupledtoproteinA-SepharoseCL-4B.
pathogenesisofthedisease. ThiswouldbeexpectedifhMBPis Aliquots ofcell lysates equivalent to - 10 g ofcellswere passed se-
the autoantigen in MS, and ifthe role ofthe MS-associated quentiallythroughthefollowingcolumns:SepharoseCL-4B(10ml),
class II alleles is to present hMBP peptides to autoreactive T protein A-Sepharose (5 ml), W6/32-protein A-Sepharose (10 ml),
andLB3.1-proteinA-Sepharose(15 ml)usingaflowrateof15 ml/h.
cells.
The columnswere washedwith 10 column volumesof10 mMTris-
To test this hypothesis, the binding ofa panel ofhMBP
HC1,pH8.0,0.1%Renex(5 ml/h);2columnvolumesofPBS,and2
peptidesto several purified DR molecules, includingthose as-
columnvolumesofPBS-1%octylglucoside. DRmoleculeswereeluted
sociatedtoincreasedfrequencyofMS,wasdetermined. Inad- from the LB3.1 column with 0.05 M diethylamine, in 0.15 M NaCI
dition, T cell lines (TCL) specific forhMBP were established containing 1%octylglucoside(pH 11.5),immediatelyneutralizedwith
from MS patients positive or negative for the MS-associated 2 M glycine, pH 2.0, and concentrated by ultrafiltration through a
allele HLA-DRB1*1501. Amicon YM-30 membrane (Amicon Corp., Danvers, MA). Protein
Asdemonstratedinthepresentstudy,somehMBPpeptides contentwasevaluatedbya BCA protein assay(PierceChemicalCo.,
bindwithhighestaffinitytoHLAclassIImoleculesencodedby Rockford, IL)andconfirmedbySDS-PAGE.
allelesassociatedtoincreasedfrequencyofMS,thusproviding BindingofhMBPpeptidestopurifiedHLAclassIImolecules.Puri-
a possible molecular explanation for the association between fiedDRmolecules(5-500nM)wereincubatedwith5nM125I-radiola-
beled peptides for 48 h in PBS containing 5% dimethylsulfoxide
class II genes and MS, and at the same time implicating the
(DMSO)inthepresenceofaproteaseinhibitormixture.Purifiedpep-
hMBPsequence84-103asapossiblecandidatefortheautoan-
tideswereiodinatedusingthechloramine-Tmethod.Thefinalconcen-
tigenic determinant. The findingthatthispeptide binds, with trationsofpeptideinhibitorswere: 1 mMPMSF, 1.3mM 1.10phenan-
loweraffinity,toseveralotherDRmolecules,mayalsoprovide troline, 73 ,M pepstatin A, 8 mM EDTA, 6 mMN-ethylmaleimide,
anexplanationfortheincompleteassociationbetweenMSand and 200 MMN-Ap-tosyl-L-lysine chloromethyl ketone(TLCK). Final
class IIgenes. detergentconcentration intheincubation mixturewas0.05% NP-40.
However, theobservation thathMBP-specific Tcellsfrom Assayswere performed atpH 7.0 with theexception ofDRB1*0301,
eitherDRB1*1501-positiveor-negativeMSpatientsrecognize which was performed at pH 4.5, and DRBI*1601, which was per-
equally well this and otherhMBP peptides cautions against a formedatpH5.ThepHwasadjustedaspreviouslydescribed(38).The
simplisticinterpretationofthebindingdata,andillustratesthe DR-peptidecomplexeswereseparatedfromfreepeptidesbygelfiltra-
tion on Sephadex G50 columns as previously described (39, 40) or
complexity and heterogeneity of T cell responses against
TSK2000 (7.8 mm X 15 cm) eluted at 1.2 ml/min in PBS, pH 6.5,
hMBP.
containing0.5% NP-40and0.1% NaN3. Becausethelargesizeofthe
radiolabeled peptide used for the DRBI*1501 binding assay makes
Methods separation ofbound from unbound peaks more difficult underthese
conditions, all DRBI*1501 assays were performed usingaTSK2000
Antigens.hMBPwaspreparedasdescribed(33).PurityofhMBPprep- (7.8mmX30cm)columnelutedat0.6ml/min.Columneluateswere
arations, as assessed by silver staining after gel electrophoresis, was passed through a model 170 radioisotope detector(Beckman Instru-
routinelyfoundtobe>90%.PeptidesweresynthetizedonanApplied ments, Inc., Palo Alto, CA), and radioactivity was plotted and inte-
BiosystemsInc. (FosterCity,CA)430Apeptidesynthetizer. Afterre- grated with a model 3396A integrator (Hewlett-Packard Co., Palo
moval of the a-amino-tert-butyloxycarbonyl protecting group, the Alto,CA).Thefractionofpeptideboundwasdeterminedaspreviously
phenylacetamidomethylresinpeptidewascoupledwithafourfoldex- described (40). The radiolabeled peptides used were: HA Y307-319
cess ofpreformed symmetrical anhydride (hydroxybenzyltriazole es- for DRB1*0101, DRB5*0201, and DRB1*1601; hMBP Y78-101
ters forarginine, histidine, asparagine, andglutamine) for I h in di- for DRBl*1501; MT 65 kD Y3-13 with Y7 substituted with F
methylformamide. Forarginine, asparagine, glutamine, andhistidine forDRB1*0301; a non-natural peptide with thesequence YARFQS-
residues,thecouplingstepwasrepeatedtoobtainahighcouplingeffi- QTTLKQKT for DRB1*0401 and DRB1*0404; TT 830-843 for
ciency. After synthesis wascompleted, the peptide wascleaved from DRB5*0101, DRB1* 101, and DRB1*0701. In thattheLB3.1 mAb
theresinandtheprotectinggroupsremovedbytreatmentwithhydro- usedforDRpurificationischain-specific,B1 moleculeswerenotsepa-
MyelinBasicProteinPeptidesBindingtoHLA-DRMolecules 617
ratedfromB3molecules.ThespecificityofthebindingassayforDRBl Tcellproliferationassays.TCLwerewashedtwiceinRPMIand5
moleculesisobviousinthecaseofDRBl*0101 wherenoB3chainis X 104cellswereincubatedwith105irradiatedautologousPBLperwell.
expressed, and has been demonstrated for DRBl*0301 (40), Alternatively, mitomycin c-treated homologous EBV-transformed B
DRBl*0401, and DRBl*0404 (A. Sette, unpublished observations), cells (2 X 104 cells per well) were used as antigen-presenting cells
DRB1** 01 (40),andDRB1*0701 (41).TheproblemofDRBchain (APC). EBV-transformedBcells(EBV-B)wereincubated(107/ml)
specificityinassaysforpeptidebindingtoDRBl*1501, DRB5*0l01, with50,ug/mlmitomycinc(SigmaChemicalCo.)for45minat370C
DRBl*1601, and DRB5*0201 is circumvented by the use oftrans- and then washed five times. Triplicate cultures were set up without
fectedfibroblasts.Inpreliminaryexperiments,eachoftheDRprepara- antigen, with 30 Mug/ml hMBP or with 1 Mg/ml ofhMBP synthetic
tionswastiteredinthepresenceoffixedamountsofradiolabeledpep- peptides.EachTCLwasalwaystestedwiththeentirepanelof16syn-
tides to determine the concentration ofDR molecules necessary to thetic hMBP peptides. After 3 d ofculture, I MCi per well tritiated
bind 10-20%ofthetotalradioactivity. Allinhibitionassayswereper- thymidine(Amersham,Bucks,UK;40Ci/mmol)wasadded,thecells
formed using these DR concentrations and inhibitory peptides were harvested6hlaterandthymidineincorporationmeasuredbyscintilla-
typically testedatconcentrationsranging from 120Mg/mlto 1.2ng/ tionspectrometry.
ml. Thedatawerethen plotted andthedoseyielding50%inhibition DeterminationofHLArestriction.Monoclonalantibodiesrecogniz-
determined.Eachpeptidewastestedintwotofourindependentexperi- ingmonomorphicdeterminantsofDR(Dl-12),DQI (BT3/4),DQ2
mentsandresultsarepresentedasarithmeticmeanofbindingcapacity (XIII358/4),DQ3(XIV466),andDP(B7/21)moleculeswereagift
expressedinnanomolarconcentration. fromDr.R.S.Accolla,UniversityofVerona,Italy(50-52).APCwere
Patients. 20patientswith clinically definite MS accordingto the incubated with monoclonal antibodies (final dilution ofascitic fluid
Schumachercriteria(42)wereincludedinthisstudy.Theywereallfree 1:1,000)for2 hbeforeadditionofTcellsandantigen.Cultureswere
ofimmunosuppressive treatment atthe time ofblood samplingand thencarriedoutasdescribedabove. ATCLwasconsideredrestricted
hadnotreceivedcorticosteroidsoradrenocorticotropin(ACTH)dur- byagivenHLAclassIIisotypewheninhibitionbythecorresponding
ingtheprevioussixmonths.Theirdisabilitystatuswasassessedbythe monoclonal antibody was > 50% of the control response. In most
sameneurologist(Dr.Kappos)andquantifiedbyKurtzke'sexpanded cases, inhibitionwasbetween80%and 100%.TheHLA-DRallelere-
disability statusscale (EDSS, 43). Diseaseactivityatthetimeoffirst strictingtheresponsewasdeterminedbystimulatingTCLwithapanel
blood samplingwasalso assessed bythesame rateraccordingtothe ofhomozygousEBV-BcellsobtainedfromtheEuropeanCollectionof
followingcriteria: Activedisease(1)wasdefinedasongoingdeteriora- AnimalCellCultures(Porton,UK).Mitomycinc-treatedEBV-Bcells
tion or relapse ofthe disease during the preceding 2 wk. Clinically were pulsedwith antigen for 4 h, then washed twicebeforeaddition
inactive(0)wereconsideredallpatientswithoutanydeterioration or ofTCL.
relapseduringthepast 12mo.Patientswhodidnotmeetthesecriteria SurfacephenotypeofTcelllines.TheCD4andCD8phenotypeof
wereconsideredprobablyactive(2).Inadditiontotheclinicalassess- TCL was determined by cytofluorografic analysis. TCLwere stained
ment, magnetic resonance imaging (44, 45)wasperformed within 2 withanti-Leu-3 fluorescein isothiocyanate (FITC)(CD4)plusanti-
mobeforethefirstbloodsamplingbya2.OTSiemensMagnetom(Er- Leu-8 PE (CD8), (Simultest, Becton, Dickinson & Co., Mountain
langen, FRG) according to the recommendations ofthe European View,CA)andanalyzedbyaFACScanflowcytometer.
ConcertedActiononMS(46).Patientsweredividedaccordingtothe
presence(1)orabsence(0)ofgadoliniumenhancinglesions(45,47). Results
Informed consent was obtained from MS patients according to the
requirementsoftheDeclarationofHelsinki. Binding ofhMBP peptides to HLA class II molecules. We
HLA typing. Typing forthe polymorphism ofHLA class II loci wishedtoexaminethecorrelationbetweenDRmoleculesasso-
DRB1, DQA1,andDQBI wasperformedusingastandardtechnique ciatedwith MS and their capacityto bind hMBPpeptides. In
for oligonucleotide typing of polymerase chain reaction-amplified
addition,wewishedtocomparethecapacityofhMBPpeptides
DNA.Primersandoligonucleotidesaswellashybridizationandwash-
to bind to DR molecules with their antigenicity in vitro for
ingconditionswerethoserecommendedbythe 1thInternationalHis-
tocompatibilityWorkshop(48).Nonradioactivelabelingofoligonucle- hMBP-specific TCL derived from MS patients. Therefore, a
otidesandchemiluminescencedetectionwasperformed accordingto panel of overlapping peptides spanning through the entire
Nevinny-Stickeletal.(49). hMBPmoleculewassynthetizedandtestedforbindingtosev-
Establishment ofMBP-specific TCL. Peripheral blood mononu- eral different HLA class II molecules (Tables I and II). The
clearcells(PBL)fromMSpatients,separatedbyFicoll-Hypaqueden- binding affinity ofhMBP peptides to six different DR mole-
sity gradient centrifugation, were resuspended in culture medium at culesispresentedinTableI.Asexpected,differenthMBPpep-
106cells/mland200Mlwasculturedinround-bottomedwellsofmi- tides bind, with different affinities, to distinct DR molecules.
crotiter plates (Nunc, Copenhagen, DK) together with 30 ,g/ml
For example, three nonoverlapping peptides (84-103, 134-
hMBP. CulturemediumwasRPMI 1640(Gibco, Basel,CH)supple-
153, 153-170) bound with high affinity to DRB1*0101,
mentedwith25mMHepes,2mML-glutamine,50,gM2-mercaptoeth-
whereasnohigh-affinitybindersweredetectedforDRB1*0301
anol,50,g/mlgentamicin(SigmaChemicalCo.,St. Louis,MO)and
10% heat-inactivated humanABpositive serum. After6dofincuba- molecules. Overall, as documented in Tables I and II, 8/16
tionat 37°Cinahumidifiedatmosphere of6% CO2in air, 100Ml of (50%) hMBP peptides bound with high or medium affinity
culture medium was replacedby fresh medium containing 20U/ml (KD 5-500 nM) to at least one DR molecule. Similar results
human recombinant IL-2 (Hoffmann-La Roche, Basel). After 3-4d wereobtainedwhenthebindingcapacityofhMBPpeptidesto
the cultures were inspected microscopically for growth and positive differentDR2allelicproductsandisotypeswasmeasured(Ta-
culturesweresplitincompletemediumwithorwithoutinterleukin2 ble II). Although all hMBP peptides examined showed some
(IL-2).Afteranadditional3-4d,thelattercultureswerewashedtwice degree ofbindingaffinity foratleastoneoftheDRmolecules
andsplitintotwoadjacentmicrotiterwells.Irradiated(3,000rad)au-
tested, the three peptides corresponding to sequences 13-32,
tologousPBL(10'cellsperwell)wereaddedtobothwellsandhMBP
(30Mg/ml)tooneofthem.hMBP-specificproliferationwasmeasured 84-103, and 144-163 were capable ofbindingwith relatively
highaffinity(KD< 500nM)toseveralHLAclassIImolecules.
bytritiatedthymidine incorporation forthe last 6 h ofa 3-d culture
period.Atday 15,hMBP-specificcelllinesgrowninmediumcontain- This degenerate binding capacity was particularly evident for
ingIL-2 wererestimulated withautologousirradiated PBL(106/ml) peptide 84-103,abletobindto8 outof10HLAclassIImole-
andhMBP(30 Mg/ml)inmediumcontainingIL-2. Thereafter,these cules tested with a KD of 100 nM or less. Very high affinities
cultureswererestimulated inthe samewayevery7-10d. were measuredinthebindingofhMBPpeptide 84-103toDR
618 Vallietal.
TableI. BindingofhMBPPeptidestoDifferentDRBJ Molecules
Sequence Position DRBI*0101 DRB1*0301 DRBI*0401 DRBI*0404 DRBI*1101 DRBI*0701
nM
Ac-ASQKRPSQRHGSKYLATAST Ac-1-20 5,000 2,273
KYLATASTMDHARHGFLPRH 13-32 13,000 100 87 5,000 157
HARHGFLPRHRDTGILDSIG 23-42 - 4,000
RDTGILDSIGRFFGGDRGAP 33-52 2,153 169
RFFGGDRGAPKRGSGKDSHH 43-62 5,000
GSGKDSHHPARTAHYGSLPQ 55-74 _
RTAHYGSLPQKSHGRTQDEN 65-84 3,333
QKSHGRTQDENPVVHFFKNI 74-93 1,667 900 1,515 12,500
NPVVHFFKNIVTPRTPPPSQ 84-103 13 7 36 17 66
VTPRTPPPSQGKGRGLSLSR 94-113 10,000 - 5,625 16,667 10,000
GKGRGLSLSRFSWGAEGQRP 104-123 833 726 2,632 1,667 4,167
FSWGAEGQRPGFGYGGRASD 114-133 2,045
GFGYGGRASDYKSAHKGFKG 124-143 13,000 - 15,000 139 -
YKSAHKGFKGVDAQGTLSKI 134-153 70 5,000 2,000 107
VDAQGTLSKIFKLGGRDSRS 144-163 25 4,545 9,000 314 18 1,136
IFKLGGRDSRSGSPMARR 153-170 25 10,000
Dashindicatesbinding> 20,000nM.
molecules associated with increased MS susceptibility ThealleleDRB1*1501 (formerlydenominatedDR2Dw2),in
(DRB1*1501 and DRB1*0401). Interestingly, this peptide linkage disequilibrium with DQB1*0602, occurs with signifi-
could also bind, although with loweraffinities, to DR2 mole- cantly increased frequency in MS patients ofNorthern Euro-
culesencodedbyalleles,suchasDRBI*1601 andDRB5*0201, pean ancestry(14). Amongthe20MSpatientswehavestud-
not associated with increased frequency ofMS. Conversely, ied, 3 were DRB1*1501 homozygous, 6 heterozygous, and 11
peptide 13-32didnotbindtoDRB1*1501 moleculesand 144- negative. Therefore, theDRB1*1501 allelewaspresentin45%
163 boundwith verylowaffinity. oftheMSpatientsweanalyzed, inagreementwithotherstud-
HLADRandDQallelesofMSpatients. Thesalientclinical iesofHLA-MSassociation (13, 14).
featuresoftheMSpatientsanalyzedaresummarizedinTable Establishment ofhMBP-specific TCLfrom MSpatients.
III. The HLA DRand DQ alleles ofMS patients from whom hMBP-specific TCL were obtained from 20 out of28 (71%)
hMBP-specific TCL were established are shown in Table IV. MSpatientstested. Outofthe61 TCLrecognizingatleastone
TableI. BindingofhMBPPeptidestoDifferentDR2AllelesandIsotypes
Sequence Position DRBI*1501 DRB5*0101 DRBI*1601 DRB5*0201
nM
Ac-ASQKRPSQRHGSKYLATAST Ac-1-20 545 9,100
KYLATASTMDHARHGFLPRH 13-32 - 10,000 4,200
HARHGFLPRHRDTGILDSIG 23-42
RDTGILDSIGRFFGGDRGAP 33-52 11,000 -
RFFGGDRGAPKRGSGKDSHH 43-62 7,300 10,000
GSGKDSHHPARTAHYGSLPQ 55-74 6,400
RTAHYGSLPQKSHGRTQDEN 65-84 2,900 -
QKSHGRTQDENPVVHFFKNI 74-93 43 1,850 341 125
NPVVHFFKNIVTPRTPPPSQ 84-103 5 156 21 98
VTPRTPPPSQGKGRGLSLSR 94-113 -
GKGRGLSLSRFSWGAEGQRP 104-123 8,600 6,600 2,100 6,200
FSWGAEGQRPGFGYGGRASD 114-133 3,800
GFGYGGRASDYKSAHKGFKG 124-143 2,000 136 16,000
YKSAHKGFKGVDAQGTLSKI 134-153 1,300 5,500 4,100
VDAQGTLSKIFKLGGRDSRS 144-163 7,700 6,600 388 1,500
IFKLGGRDSRSGSPMARR 153-170 1,600 3,300 15,000 4,700
Dash indicatesbinding> 20,000 nM.
MyelinBasicProteinPeptidesBindingtoHLA-DRMolecules 619
TableIII. ClinicalCharacteristics ofMSPatientsIncludedin TheStudy
Diseaseactivity
Disease Disease Gd Clinical
Patient Age Sex duration course* EDSSt enhancements assessment"
yr yr
BV 23 F 2 2 4.0 0 0
BC 44 F 11 3 6.0 0 1
SE 38 F 17 4 6.0 1 1
BJ 66 M 41 4 6.0 0 2
0I 55 F 11 3 3.5 0 0
SJ 43 M 9 2 3.0 0 0
HU 44 F 1 1 1.5 1 0
TB 57 M 18 2 2.5 0 0
GA 39 F 12 2 3.5 0 0
LA 63 F 39 2 2.5 0 0
SI 54 F 7 4 2.5 0 0
KH 48 M 16 4 6.0 0 1
DV 45 F 1 2 2.0 1 0
SP 55 M 21 3 4.5 0 2
NA 35 F 7 4 4.0 1 2
SM 48 F 20 2 5.5 0 0
DM 37 M 17 1 1.5 0 0
SV 40 F 12 3 5.0 1 1
WB 58 F 8 4 6.0 1 1
MG 55 F 1 2 3.5 1 2
*Diseasecourse: (1)relapsing-remitting withcompleteremissions, (2)relapsing-remittingwithresidualneurologicaldeficit,(3)relapsingwith
progression between relapses, (4)secondarychronicprogressive (diseaseinitiatedasrelapsing-remittingandcontinuedaschronicprogressive).
tDisabilityisshowningradesofKurtzke'sEDSS. IDisease activityevaluatedbyGadolinium(Gd)- enhancedMRI:(1)enhancinglesions,
(0)noenhacinglesions. 1lDiseaseactivityevaluatedbyclinicalassessment:(1)active,(2)probablyactive,(0)inactive(seetext).
of the hMBP peptides tested, 26 (43%) were isolated from the45 TCLtestedwerefoundtoberestrictedbyasingleHLA
DRB*1501o-positive MS patients and 35 (57%) from class II isotype. The majority ofhMBP-specific TCL(38/45,
DRBI*1501-negativeMSpatients(TableIV).ATCLwascon- 85%) were DR restricted. Few TCL (3/45, 6%) recognized
sideredtobehMBP-specificwhenthestimulationindexinthe hMBPpeptidespresentedbyDQmolecules.Interestingly,DQ-
presence of30 ,gg/mlhMBPwas> 2 inatleasttwo indepen- restrictedTCLwereonlyobtainedfromMSpatientshomozy-
dent proliferation assays. The T cell proliferative response to gous for DQAl*0102/DQBI*0602. Some TCL (4/45, 9%)
MBPwasveryvariableamongdifferentTCL,withstimulation wererestrictedby DPmolecules.
indexes ranging from 2 to 1,100 (Table IV). Ingeneral, TCL hMBPepitopesrecognizedbyhMBP-specificTCLfromMS
were stable and maintained a similar level ofproliferation to patients. 61 hMBP-specific TCL established from 20 MS pa-
hMBP over a period ofseveral months. No relationship was tientsrespondedtoone ormoreofthehMBPpeptidestested.
observed between expression ofthe allele DRBI*1501 by the hMBP epitope(s) were recognized by 75% ofthe hMBP-
-
MS patientanddegreeofresponsivenesstohMBPinindivid- specificTCLtested. MostTcelllineswerespecificforasingle
ual TCL. 15 DR-restricted TCL specific for hMBP peptides sequence ofhMBP, but in some cases T cell proliferation to
weretestedforsurfaceexpressionofCD4andCD8 molecules: nonoverlapping hMBP peptides was observed. The prolifera-
5 TCL were mostly composed (> 70%) of CD4' T cells, tiveresponsetohMBPandtosynthetichMBPpeptidesoffour
whereas 10 TCL contained, in addition to single-positive representative TCL is shown in Fig. 2. The hMBP Tcell epi-
CD4'cells,asubstantialproportion(46-92%)ofdouble-posi- topes recognized by all TCL tested, as well as the degree of
tive CD4+/CD8+Tcells(data not shown). responsiveness expressed as stimulation index, ispresented in
ThemajorityofhMBP-specific TCLfromMSpatientsare TableIV. TcellproliferationtohMBPandtohMBPpeptides
restrictedbyDRalleles. TheHLAclass IImoleculepresenting wasdosedependent, andwasusuallyinducedbylowermolar
thehMBPpeptidestohMBP-specificTcellswascharacterized, concentrations ofthe appropriate synthetic peptide, as com-
in termsofisotype, for45 TCL(Table IV). The HLAclass II paredtohMBP(Fig. 3).
isotyperestrictingtheanti-hMBPTcellresponsewasassessed The61 hMBP-specificTCLtestedresponded,overall,to78
byinhibitionofTcellproliferationbyanti-DR,anti-DQ,and hMBPpeptides. ThehMBPpeptidesrecognizedbyTCL, irre-
anti-DPmonoclonalantibodiesspecificforHLAclassIImono- spectiveoftheirHLArestriction,arepresentedinFig.4.Ofthe
morphic determinants. Representative examples ofblocking 16 hMBP peptides tested, 14 were recognized by at least one
byanti-classII monoclonalantibodiesareshowninFig. 1.All hMBP-specificTCL.OnlytwohMBPpeptides,23-42and74-
620 Vallietal.
TableIV EpitopeSpecificityandHLA ClassIIRestriction ofhMBP-speciJfic TCLfromMSPatients
Line hMBP
Patient DRBI* DRB3/4 DQA1* DQBI* no. (SI) Peptide Si Restriction
BV 1302,07 w52,w53 0102,0201 0604,0201 11 553 84-103 685 DRB1*1302
BC 1501,07 w53 0102,0201 0602,0201 27 65 84-103 69 DR
41 133 84-103 210 DR
44 214 43-62 64 DR
SE 1051, 0102, 0602,- 3 114 55-74 176
BJ 0101,0401 0101,0301 0501,0302 3 3 144-163 3
9 5 84-103 15
10 299 144-163 294 DRB1*0101
153-170 75
01 1101, w52 0501,- 0301, 15 14 144-163 11
19 170 33-52 73
84-103 25
144-163 82
SJ 1501, 0102, 0602, 1 434 13-32 455 DQ
28 9 104-123 4 DQ
HU 1501,0401 w53 0102,0301 0602,0302 2 5 13-32 3
9 10 13-32 6
TB 1501,- 0102, 0602, 2 2 13-32 2 DQ
7 2 84-103 2 DR
9 3 84-103 3 DR
12 7 104-123 3
GA 1101,0801 w52 0401,0501 0301,0402 2 500 144-163 560 DR
7 120 33-52 20 DR
10 180 13-32 12 DR
22 3 124-143 10 DR
LA 0404,1101 w52,w53 0301,0501 0302,0301 1 135 84-103 65 DR
8 12 84-103 12 DRB1*1101
144-163 14
9 90 84-103 10 DR
13 133 84-103 150
14 6 13-32 3 DR
21 279 84-103 350 DRB1*1101
33 140 144-163 190 DR
SI 1101, 1001 w52 0101,0501 0501,0301 2 319 33-52 27 DRB1*1101
4 30 84-103 6 DRB1*1101
6 13 124-143 25 DR
9 110 84-103 17 DR
134-153 120
14 98 84-103 33 DR
134-153 79
17 15 84-103 10
KH 1301,07 w52,w53 0103,0401 0201,0402 1 12 84-103 17 DP
20 60 144-163 37 DR
21 18 144-163 22 DR
DV 0301,0803 w52 C)501,0301 0201,0601 2 80 1-20 20 DP
24 330 65-84 350
26 1100 124-143 65 DR
29 110 94-113 55
SP 1501, 1301 w52 C)103,0102 0602,0603 9 980 144-163 800 DRB1*1301
10 230 144-163 15 DRB1*1301
38 19 33-52 10 DRB1*1501 (DRB5*0101)
39 10 134-153 6 DR
153-170 9
NA 1501, 1301 w52 0)102,0103 0602,0603 15 110 84-103 70 DR
144-163 120
18 12 144-163 38 DP
SM 1501, 1101 w52 01102,0501 0602,0301 3 40 13-32 76
-1 - ----I ----
MyelinBasicProteinPeptidesBindingtoHLA-DRMolecules 621
TableIV. (Continued)
Line hMBP
Patient DRB1* DRB3/4 DQA1* DQBI* no. (SI) Peptide SI Restriction
DM 1501,0401 w53 0102,0301 0501,0302 9 90 84-103 12 DRB1*1501 (DRB5*0101)
144-163 22
11 220 84-103 80 DR
144-163 105
17 42 84-103 40 DR
144-163 51
30 102 1-20 188 DP
35 38 84-103 5 DR
134-153 33
144-163 7
42 51 104-123 9 DRB1*1501 (DRB5*0101)
58 65 84-103 17 DRB1*1501 (DRB5*0101)
144-163 25
SV 0401, w52,w53 0301,- 0302,- 14 13 65-84 3
17 120 84-103 130
144-163 150
WB 01, 1301/2 w52 0101,0501 0501,0603 32 70 84-103 40 DR
144-163 20
42 40 114-133 90 DR
124-143 70
MG 1302,08 w52 0401,0102 0402,0604 7 120 124-143 8
Dashindicateshomozigosity. SI,stimulation index.
93, failed to induce a proliferative response in any of the DRB1*1501-positive patients. However, three out ofthe five
hMBP-specificTCLexamined. Therefore, practicallyallTcell TCL from these MS patients, when tested for HLA class II
epitopes ofhMBP can be recognized by T cells from MS pa- isotype restriction, were foundtobe DQ restricted(TableIV,
tients.However,twohMBPepitopesareclearlyimmunodomi- Fig. 1). This indicates that the hMBP peptide 13-32 can be
nant. OneisincludedinthehMBPsequence84-103,inducing recognized in DRB1*1501/DQB1*0602 MS patients com-
25/78 (32%) peptide-specific Tcellresponses. ThishMBPre- plexedtoeitherDRorDQmolecules,thusaccountingforthe
gion appears to contain a nested set ofoverlapping epitopes increasedfrequency ofrespondingTCL.
(53), inthatthetruncatedsequence 87-99 wasrecognizedby HLA class II molecules presenting hMBP epitopes to
only 30% ofthe TCLspecific for84-103 (not shown). Asec- hMBP-specific TCLfromDRBJ*1501 heterozygousorhomo-
ondimmunodominantepitope,comprising20/78(25%)pep- zygous MSpatients. For some hMBP-specific TCL the HLA
tide-specific Tcell responses, islocatedinthehMBPsequence classIImoleculepresentingthehMBPpeptidewasdetermined
144-163. In addition, a third, less dominant T cell epitope, bystimulatingTcellswithapanelofhomozygousEBV-Bcells.
inducing 7/78 (9%) peptide-specific responses, is present in AnexampleisshowninTableV.Inthiscase,theDR-restricted
thehMBPsequence 13-32. TCLno. 9 from patientDM(DRBI*1501/DRB1*0401)was
Immunodominant hMBP epitopes do not differ in TCL onlyrestimulatedbyhMBPpresentedbyEBV-Bcellshomozy-
from DRBI*1501 positive or negative MSpatients. Next, we gous for DRB1*1501 or for the highly homologous allele
wished to test whether a skewing in the immunodominant DRB1*1502. These results confirm the DR restriction ob-
hMBP epitopes recognized was present in TCL derived from tained by monoclonal antibodyblocking. Sinceitwasnotde-
MS patients expressing DRB1*1501, as compared to patients termined whether the HLA molecule presenting the hMBP
notexpressingit. Thus, TCLweresubdividedaccordingtothe peptide is encoded by DRB1 or DRB5 genes, this TCL is re-
presence orabsence ofthisallele in the MS patientblooddo- stricted by either DRB1*1501 or DRB5*0101 class II mole-
nors. 34 hMBP peptides were recognized by the 26 TCL de- cules, as both molecules can present hMBP peptides to TCL
rivedfromnineDRB1*1501-positiveMSpatients,whereas44 (31, 54). In this assay the whole hMBP protein was used as
hMBPpeptideswererecognizedbythe35TCLobtainedfrom antigen, indicating that both dominant hMBP epitopes (84-
the 11 MS patients not expressing DRB1*1501 (Table IV). 103 and 144-163) recognized by this TCL are restricted by
Results in Fig. 5, presented as percent hMBP peptides recog- DRB1*1501 orDRB5*0101 molecules.Thiswasconfirmedby
nizedbyTCLin thetwo subsetsofMSpatients, demonstrate presentation oftheappropriatesyntheticpeptidesbyhomozy-
that recognition of the two immunodominant epitopes in- gousEBV-B cells(notshown).
cluded in the hMBP sequence 84-103 and 144-163 does not The DRB1 allele presenting hMBP peptides was deter-
differ in TCL derived from DRB1*1501-positive or-negative minedbyhomozygousEBV-Bcell-inducedTcellproliferation
patients. As to the third dominant epitope, defined by the for 12 TCL from DR-heterozygous patients (Table VI). In
hMBPpeptide 13-32,fiveoutofsevenTCLwerederivedfrom addition, five TCL from homozygous MS patients for which
622 Vallietal.
cpm x 10^3 capacity (Tables I and II). In particular, the peptide 84-103
can be presented to TCL by DRBl*1302, DRB1* 1101, and
DRB1*1501 (or DRB5*0101) molecules. The peptide 144-
163 can be presented by at least four DR molecules:
DRB1*0101, DRBl*l 101, DRBl*1301, andDRB1*1501 (or
DRB5*0101), furtherdemonstratingthefunctional relevance
ofbindingdata.
Theseresultshighlightthreepointsrelevanttorecognition
ofhMBPepitopesbyTCLfrom DRB1*1501-positiveMSpa-
tients. First, APC from MS patients carryingtheDRB1*1501
haplotypecanpresenttoTCLatleastsixdifferenthMBPpep-
tides. Second, these peptides are not all presented by
DRBI*1501 (orDRB5*0101)molecules.Third,insomecases
noag thesamepeptide,e.g., 104-123, canbepresentedbyeitherDR
NA line 15 orDQmolecules.
Discussion
Based on experimental models ofMS, hMBP is considered a
likelycandidatefortheselfantigenrecognizedbyautoreactive,
pathogenic human Tcellscapable ofmediating MS(2, 55).
In the present study we have systematically analyzed the
binding of overlapping peptides encompassing the entire
hMBPmoleculetoseveral HLAclassII molecules, andcorre-
lated their binding capacity to the recognition ofhMBP pep-
tides by HLA class II-restricted TCL. In particular, we have
focusedonthebindingofhMBPpeptidestoHLAclassIImole-
culesassociatedornottoincreasedsusceptibilitytoMS,andon
theanalysis ofhMBP-specific TCLfrom MSpatientsexpress-
ing or not these susceptibility alleles. Although the emerging
D=3VPX &Xnti-DR antein-antM-DDQ Mtanli-M pattern iscomplex, somepointsappeartobequiteclear.
no ag MEBP anti-DR anti-DO1 enti{)Q2 anti-DO3 entl{DP
Several hMBP peptides bind with high affinity to one or
SJ line 1 two ofthe HLA class II molecules tested. In addition, three
hMBP determinants, included in sequences 13-32, 84-103,
cpmx 10^3
and 144-163exhibitdegeneratebinding, sincetheyareableto
form complexes with several ofthe HLA class II molecules
25- examined.DegeneratebindingofpeptidestoHLAclassIImol-
ecules is not unprecedented because it hasbeen described for
20- peptidesderived from malariacircumsporozoiteprotein(56),
tetanustoxoid(57),andinfluenzahemagglutinin(41,58,59).
15- Analysis ofalarge unbiased sampleofnaturallyoccurring se-
quenceshasrevealedthatdegeneratebindingisdetectableonly
10- fora minority ofpeptides capable ofbinding to HLA class II
molecules (39), and subsequent studies have illustrated that
5 degenerate DRbindingischaracteristic ofpeptidescapableof
bindingwithhighaffinitytoatleastoneDRmolecule(59).To
our knowledge, the present study shows the first example of
0 _
no ag anti-DOl antf-002 anti-D03 anti-DP degenerate binding to HLA class II molecules ofpeptides de-
NA line 18 rivedfrom acandidate autoantigen possibly involved inahu-
Figure1. CharacterizationofHLAclassIIisotypicmoleculespresent- manautoimmunedisease.
ingnaturallyprocessedhMBPpeptidestohMBP-specificTCL.The AstrikingparallelexistsbetweenbindingofhMBPpeptides
HLA class II isotyperestricting theanti-hMBPTcell responsewas to HLA class II molecules and hMBP epitopes recognized by
assessedbyinhibitionofTcellproliferationbyanti-DR,-DQ,and hMBP-specific TCLfrom MSpatients. First, theperipheral T
-DPmonoclonal antibodies specific forHLA class II monomorphic cell repertoire ofMSpatients, aspredicted frombindingdata,
determinants,asdetailedinMethods.AdescriptionoftheseTCLis includes T cells able to recognize almost any hMBP peptide.
includedinTableIV. Second, thethreehMBPpeptidescapable ofdegeneratebind-
ing(13-32, 84-103,and 144-163)correspondpreciselytothe
threeimmunodominantepitopeswehaveidentifiedinhMBP-
class IIisotype restrictionbyantibodyblockingwasavailable, specific T cell lines from MS patients. These results confirm
arealsoincluded.Theseresultsdemonstratethatthedominant previous studies indicating the immunodominance ofhMBP
epitopes 84-103 and 144-163 can be presented by different peptides84-103 and 144-163 (27, 29-32),andsuggestingthe
class II molecules, as expected from their degenerate binding existenceofathirdTcellepitopeintheamino-terminalregion
MyelinBasicProteinPeptidesBindingtoHLA-DRMolecules 623
O) -4
10
N-
10~~~
0
N
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T-
tICo) 0
CO,
x
6 o- EEl 6 .Vo <CXO-;o EU 0
c
0
-
z 0 60 0
-1 CU
01 .10 Cj
-J a.
c 0
7 I 0 Li-i -T--o-i-i-, -T- -i o- -T 1- .LTI
CU
-0o ONCNONVN100CYlWIV0mm1_-CCN00100 CocoW10rO1- 00.Cm 0 0NO1Cec1o'-YNICYeNIN01NIW1INr-6IwN~ce0TCoC'*C1,1o-'cNTt)01To'vtTV0'-o-T-C000-f-0-0a0CO0l
C c Q mm~~~~~~O~N~~~o~~~v~~~~~-~~~~~~
0aC , Nt 0 0OD O-N03 V 9 a *2-0 SNlb1 0-0CO.N01I
(QI mC2O x ._
,co
zr
CD
0
0
N
CY_
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0
to
OCU
1~ 0 0) ~ 4
6 W-OO xX
E 6 I 0.
a
c
0 0~~~
V-T--T-v4r-q-T o_ ._C._
-o >
10 0 e =0.
>0j C oo L)
C p~~qwpwJ~.qp~~qpii I
J C*_0_ - C)
m o
Zs
irCYii Iq Iq I3 Timc -o -ff- 'Ir .L
-0o CYC- -~~~0~~~G -V-3 -Y_c_m_o00~.V 0 O0ICINXYl(INNCYIN1cImIICVIRsV0)0et20(033~~cC'o~o~0-~-N0O-)-c0_c_00_Cfl r0C0DfCfL.
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4- a
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624 Vallietal.
cpmx 1O'3 peptideexpressingthemostdegeneratebindingpattern,sinceit
bindsto8outofthe 10DRmoleculestested.Inparticular,this
peptide binds with highest affinity, in the low nM range, to
30- Figure3. Dose-depen- class II molecules encoded by DRB1*1501 and DRB1*0401
dentTcell proliferation alleles. Because these alleles are associated to increased fre-
25- tohMBPandtothe quency ofMSin Northern (14)and Southern (15) European
hMBPpeptide 144-163.
populations, respectively, the results would suggest that high
20 pTahteieTntCBLJn(o5.x101f0r4ocmells affinity binding ofthis hMBPpeptide may be relevant to the
pathogenesisofMS.Conversely,theothermajorimmunodom-
perwell)wascultured
15- with theindicated con- inantpeptide, 144-163,bindsveryweaklytoDRB1*1501 and
centrationsofhMBP DRB1*0401 molecules.
10- (e) orhMBPpeptide A point ofdebate is the binding affinity ofautoantigenic
144-163 (+)in the peptidesfortheclassIImoleculesassociatedtodiseasesuscepti-
5- 1 / presence ofmitomycin bility. Accordingtoonetheory,autoreactivepathogenicTcell
c-treated autologous clonescouldrecognizelowaffinitybinders,andforthisreason
EBV-transformed B have escaped thymic deletion during negative selection (61).
0- I cells(2 xl104perwell).
Theoppositeviewconsidersautoantigenicpeptidesashighaf-
0.018 0.05 0.17 0.5 1.5 Resultsareexpressed
finity binders, and therefore highly antigenic. When events
[antigen]pM asinFig. 2.
suchasmolecularmimicry(62)orregulatoryimbalances(63)
leadtobreakdownoftolerance,theirantigenicpotentialcanbe
expressed. The hMBP peptide centered on the sequence 84-
of hMBP (30). In particular, the immunodominant hMBP 103 is a high-affinity binder to the DR molecules associated
epitopes corresponding to sequences 84-103 and 144-163 with increased frequency ofMS. In analogy with binding of
were previously identified, usingoverlapping hMBPpeptides, MBP peptides to mouse class II molecules (64), this result
byOtaetal.(29). Petteetal. (31)assignedatleastthreeinde- wouldsuggestthathighaffinitybindingmaybenecessaryfora
pendent T cell epitopes within the thrombic hMBP fragment selfpeptidetobecomeadominantdeterminantrecognizedby
131-170, anddefinedafourthoneusingthesyntheticpeptide autoreactiveTcells.
80-99. Martinetal. (30)identifiedtwoimmunodominantre- Consideringthebindingdata,andespeciallydegeneracyof
gions in the hMBP sequences 87-106 and 154-172. In addi- binding, it is therefore perhaps not surprisingto find thatthe
tion, Martinetal. (53)haveshownthatthedominantpeptide hMBPpeptides 84-103 and 144-163representdominantepi-
correspondingtothesequence 84-103containsanestedsetof topesrecognizedbyabout30%ofhMBP-specificTCL. Inthat
epitopes presented to T cells by different HLA class II mole- only 84-103 binds with high affinity to DR molecules asso-
cules, thereby implying degenerate binding ofthis hMBP se- ciatedwithincreasedfrequencyofMS,itwouldbetemptingto
quence. infer that this epitope may be relevant to disease induction
Immunodominance of T cell epitopes is certainly in- and/orprogression. However, bothimmunodominanthMBP
fluencedbymanymechanisms(60),butthepresentdataindi- epitopesarerecognizedwiththesamefrequencybyTCLfrom
cate that, atthe population level, degeneracy ofbindingplays DRB1*1501-positive or -negative MS patients. Moreover, in
an important role in determining immunodominance ofse- the three MS patients homozygous for DRB1*1501 many
lected T cell epitopes. The immunodominant T cell epitope hMBPpeptidesarerecognizedbyTCLinadditionto84-103,
included in the hMBP sequence 84-103 corresponds to the andinatleastthreeoftheseTCLtheresponseisrestrictedby
hMBPpeptide Table V. HLA ClassIIRestriction ofTCLDMNo. 9
Ac-1-20
13-32 EBV-B No
23-42 cells DRB1* DRB3/4 antigen hMBP
33-52
43 -62 cpm
55-74
65-84 Autologous 1501,0401 w53 3,557 31,593
74-93 MGAR 1501, 1,410 27,165
84- 103 E4181324 1502, 582 16,281
94- 113
KASO1 1601, 351 575
104-123
114- 133 RML 1602, 3,155 2,340
124- 143 PRIESS 0401, w53 138 83
134-153
PE117 0404, w53 255 159
144- 163
153-170-:: BSM 0401, w53 351 443
MOU 0701, w53 1,140 577
0 5 10 15 20 25 30 35
percentofTCLrecognizinghMBPpeptides
TheindicatedEBV-transformedBcelllines,aftertreatmentwithmi-
Figure4.hMBPepitopesrecognizedbyhMBP-specificTCLfromMS tomycinc,werepulsedwith30,g/mlhMBP,washedandincubated
patients. Resultsareexpressed aspercentage ofTCLrespondingto (2 X 104cellsperwell)withTCLno. 9frompatientDM. Resultsare
agivenhMBPpeptide(n = 78). presentedasinFig.2.
MyelinBasicProteinPeptidesBindingtoHLA-DRMolecules 625