An allo-specific peptide derived from HLA-A2 is presented by HLA-DQ7

18

Abstracts

076 AN ALLO·SPECIFIC PEPTIDE DERIVED FROM HLA·A2 IS PRESENTED BY HLA.DQ7

077 P eptide binding specificit)' of major histocompatibility complex (MH C) clas s I resolved into an array of apparently ind ependent sub Qu ant itatian by peptide libraries and im pr oved prediction

Feugeas IP I.2, Lemaire S2, Khalil II , Haentjens G2, Toubert AI, DerappeC2, Neel D2. AuberyM2, Charron DI .

sp ecificities.

IINSERM U396, 2lNSERM U180 Paris France.

Bisgaard Holm', ArneHolm' and Seren Buus", IMMI' and KVL', Copenhagen, Denmark

or binding. Anette Stryhn*, Lars 0:stergaard Pedersen" , Tina Romme - , Charlott e

Considerable interesthas focused on understandinghow MHC specificity is generated

The peptide moti fs of two HLA cla ss II molecules, DRII and DQ7, were determined from natural peptides. The EBY transformed B cells IVM (HLA-A2-DRBI *1102-DQAI ·OSOI-DQBI*0301 ) were cultured to a final yield of2 1010cells. DRll and DQ7 molecules were irnmunopurifiedand peptides were extracted after acid elution and separated by reverse-phase HPLC. Five peptides from DR 11 and five from DQ7 were sequenced using Edman degradationand other peptides were analysed by pool sequencing. Peptides were from II to 15 amino acids in length and P often occurred at the second residue for both DRS and DQ7_The peptide motif for DRII was I at position i and K or R at p0sition i + 4. For DQ7 the most signifi cant signal was A in the middle of the peptide as also described by Falk et al. The source of one peptid e eluted from DQ7 was a polymorphic part of the HLA -A2 heavy chain (from 56th to 69th amino-acid). It was al so exactly the same peptide than the synthetic peptide used by Kren sky et al in the 10th international workshop to modulate lysis by HLA -A2- specifIc cytotoxic T lymphocytes. The biochemical characterisation of this peptide from HLA-DQ7 strongly supports functional tests showing an indirect presentation of alloantigens by MHC molecules .

078 PEPTIDE BINDING TO THE DIJi'FERENTIALLY DISEASE-ASSOCIATED HLA-B*2704 AND B*2706 AND TO MUTANTS MIMICKING THEIR

.and characterizing the specificity of MHC molecules with the ultimate goal being to predict peptide binding. We have used a strategy where all possible peptides of a particular sue are d istributed into "positional scanning combinatorial peptide libraries" (PSC PL) to develop a highl y efficient, universal and unbiased approach to address MHC $pe9ficity. The PSCPL

approach was qualitatively and quantitatively superior to other currently used strategies. lbe "average" effect of any eachacid in each position was readily quantitated allowing a detailed

description of extended peptide binding motif, jncluding primary andsecondary anchor residues. It even identified disfavoured residues, which were found to be surprisingly important in shaping MHC class I specificity. Assuming thai MHC class ] specificity is the result of largely independently acting sub-sites. the binding of unknown peptides could be predicted. Conversely, this argues that MHC class I specificities consist of an array of

sub-speci ficities largely acting in a combinatorial mode, We suggest that PSCPL might be the method of choice for a complete mapping of the

specificities of all human MHC class I molecules.

079 A NALYSIS OF ENDOGENOUS PEPTIDES ELUTED FROM HLA-B27 VA R IANTS : IMPLICATIONS IN THE SUSCEPTIBILITY TO

POLIMORPHISM

S PO~DY LA RTHROPATHffiS

Lamas J.R., GaJoc:ha B.,Vmadaagoo J .A. ud LOpez de Cut", J.A Centro de BiologlaMolocular "Severo Ochoa", Consejo Superior de Investigaciones Cientificas and Universidad Aul6noma de Madrid,Spain.

Tou bert Anto ine, Boi sger ault Florence, Tien g Va nna ry, Dulphy Nicol as, Sto lzenberg Marie-Cl a ude , Khalil Iman and Ch arr on Dominique INS ERM U.396, Paris, Fran ce

Peptide binding to the disease-associated antigen HLA·B27 and its modulation by subtype polimorphism was addressed in this study. The effect of subtype changes was analyzed using a quantitative stabilization assay in which the swfaoeexpression of HLA-B27 on RMA-Scells was measured as a function of the coocentratlcc of peplides naturally presented by B'27 05, 0'2702 and their analogues. Binding to B"2704, 0"2706 and to mutants mimicking the changes between these subtypes and B'2705 was analyzed. Bulky aliphatic (Leu) , aromatic (phe or T}T) or basic (Arg,. Lys)C-terminalresiduescontributesimilarlyto binding to B"2705. For B"2704 aliphatic C-terminal residues are the most suitable, but aromatic and even basic residues can also be accomodated. B'2706 has strong preference for bulky aliphatic C-terminal residues and moderate suitability for aromaticones. The effectsof individualchanges in the subtypes accountonly partially for the binding propenies of 0'2704 and 0'2706, suggestinginteractiveeffectsof the changes in determining the peptide specificil}' of these subtypes that are not fully accoontedfor by simpleadditiveeffectsof the individualchanges.

Population studies suggest that some HLA ·B27 subtypes (HLA-B*2705, B*2702) could be more strongly associated with the deve lopm ent of spondylarthropa thies than others (B*2703, B*2706, B*2709). Differences in the pept ide bindin g groo ve could impose difference s in the nature of peprides bound by these di fferent alleles. We have elu ted endogenous peptides from CIR-B*2705 and B*270 3 tran sfectant s. The B*2705 HPL C profile was more comp lex than the B- 2703 one . Severa l B · 2705 and B· 270 3 individu al peak s were seq uenced by Edma n degradatio n and mass spectrometry . Some peptide s were shared by both su btypes . One B· 2705 elute d peptide pr esent in a major HPLC frac tio n was not foun d in the B*2 70 3 peptides. Th e corresponding synthetic peptide bound in vitro specifica lly to T2 -B*2 705 an d not to T 2-B *2703. Th is result emphasiz es that even one amin o-acid diferenc e outside the major anch or bindi ng pockets at po sition 59 betw een B· 270 5 and B*2703 could notab ly influence the endogenous peptides natur ally presented, This could have consequences in terms of T cell repertoire selection and development of autoimmunity.

080

081

IDENTIflCAnON OF HLA-A2 BINDING PEPTWES FROM CYTOMEGALOVIRUS AND IT S RECOGNITION IIY CYTOTOXIC T LYMPHOCYTES..

~. ,

A. K. Rupmi'.. J. E. Grundy"., J. A. Madrigat·

• BMTI Unit. Anthon y Nolan Research C CIIlrc and "Immunoogy Dcparu nent, Royal Free Hospital School Mcdtcicnc. London.Ulc .

or

The human pathogen CMV, is a major cause of morrallt y in the case of immunocompromiscd recipients or allogeneic bone marrow transplants. The CDS+ class [ restricted response to CMV plays a cru cial role in the control ofC MV infection in asyrnpmmatlc immuno competent hosts; however, the viral antigen recognised by CDR+ CTLs arc nol ","ellclmractcriscd. The lower matrix M !ill phuspbopnncin is a prime candKt\1c fOf production of CMV antigenlc peptides and it beeo basshown that it can act :L\ largel for Cf'L's. We h.1VC used an in vitro a..say (0 investigate pote ntial viral anti gen s rccognrscd by HLA-A2 re strict ed Crt,s. Syn thetic pepndes were

designed using (he published rr65 protein sequence 10 romuiu the consensus binding mouf Iur HLA·A2, These pepridcs were used in a standard 1'2 bind ing assay. T2 cells (HL A -A2, 8 5) were incu bated overnig ht in the presence o f the syn thetic peptides .

The positive control HLAoA2-bindi ng influenza matrix peptide, AE41, resulted ill II 3 fold increase in cell surrncc HLA-A2 expression. Incu txuion with the designed CMV pp65 peptides resulted in va rying degrees of }fLA-A ~ expression. In particular, peptid e AE45 showed a two-fold increase in expression. T he aim of our project is to de fine CMV specific cpitopes recognised by cytotoxic T ce lts (CTL) . Using the T2 bindin g as say we have identi fied certain CMV pp6 5 syn thetic pepude s that bind specifi cally to the HLA-A2 molecul e . We are now in the process of analysing the recognitionof sud! pp65 peptides by C'Fl.ts especific for thopp65 protein. Further

definition of CMV specific peptide cpuopcs presented by particular Class I molecules will allow studie s of the CTL response to CMV in infected patients with defined HLA haplotypes,

UNUSUAL EXPRESSION OF A LINE-I REfROTRANSPOSABLE ELEMENT IN Mill Ipr MICE. N. Kiger, P. Bobe. K. Benihoud and C. Chischportic h, INSERM U267, 94807 ViUejuifCedex. France During aging, the MRUlpr mice develop a syndrome similar to human SLE and rheumatoid arthrit is, both patholo gies in which the natur e of the autoantigen and the event triggering the autoimmune response are k unknown . We have observed in some aged MRUlpr (H-2 ) mice an unexpected reactivity between lymphoid ceUs and antibodies directed against the AJ3d MHC class II chain. Using an anti- AJ3d mAb, 45 and 12 kD molecules were immunoprecipitated or affinity purified from lymphoid cells. These molecules are recognized by an anti-132m antiserum, sugge sting that a peptide which mimics an epitope of AJ3d MHC class II antigen is presented at the cell surface by a MH C class I molecule. To define the molecular nature of this peptide we have cloned the cDNA coding for the "AJ3' " specificity in MRLIlpr mice. All cDN As isolated share high sequence homology (>80%) with the ORF2 (rever se transcriptase) of the LINE -I retrot ransposon . Northern blot analysis using polyA" RNA and antisens RNA probes showed a strong transcription of LINE - l coding sequences in MRLIlpr lymphoid organs and especially in new born thymuses. There fore, an autoimmune response to LINE-I produc ts would result from either a defect in thymic negative selectio n and/or an absence of peripher al deletion of reactive T lymphocytes as a consequ ence of the defective Fas lytic pathway in this strain.