Proc. Natl. Acad. Sci. USAVol. 92, pp. 10398-10402, October 1995Immunology

Similar antigenic surfaces, rather than sequence homology,dictate T-cell epitope molecular mimicry

(molecular modeling/autoimmunity/HLA-DQ)

SONIA QUARATINO*t, CHRISTOPHER J. THORPEt, PAUL J. TRAVERSt, AND MARCO LONDEI**The Mathilda and Terence Kennedy Institute of Rheumatology, Sunley Division, 1 Lurgan Avenue, London, W6 8LW, United Kingdom; and tDepartment ofCrystallography, Birkbeck College, Malet Street London, WC1E 7HX, United Kingdom

Communicated by J. F. A. P. Miller, The Walter and Eliza Hall Institute of Medical Research, Victoria, Australia, July 25, 1995 (received for reviewMay 24, 1995)

ABSTRACT Molecular mimicry, normally defined by thelevel ofprimary-sequence similarities between selfand foreignantigens, has been considered a key element in the pathogen-esis of autoimmunity. Here we describe an example of molec-ular mimicry between two overlapping peptides within a singleself-antigen, both of which are recognized by the same humanself-reactive T-cell clone. Two intervening peptides did notstimulate the T-cell clone, even though they share nine aminoacids with the stimulatory peptides. Molecular modeling ofmajor histocompatibility complex class II-peptide complexessuggests that both of the recognized peptides generate similarantigenic surfaces, although these are composed of differentsets of amino acids. The molecular modeling of a peptideshifted one residue from the stimulatory peptide, which wasrecognized in the context of the same HLA molecule byanother T-cell clone, generated a completely different anti-genic surface. Functional studies using truncated peptidesconfirmed that the anchor residues of the two "mimicking"epitopes in the HLA groove differ. Our results show, for twonatural epitopes, how molecular mimicry can occur andsuggest that studies of potential antigenic surfaces, ratherthan sequence similarity, are necessary for analyzing sus-pected peptide mimicry.

Autoimmunity may be caused by a failure in the establishmentor maintenance of self-tolerance, with subsequent peripheralactivation of pathogenic self-reactive T cells (1). Severalmechanisms have been proposed to account for this break-down in tolerance. One compelling hypothesis is based on theconcept that common epitopes are shared between self andforeign antigens and that the induction of an immune responseagainst the foreign antigen can lead to secondary recognitionof self (2). This "molecular mimicry" hypothesis is based oncomparing primary-sequence homologies between differentantigens (2, 3), and to date it is still believed that sequencesimilarities dictate molecular mimicry (4). Searches for puta-tive epitope mimics are thus usually based only on aminoacid-sequence similarities or, more recently, on similarities inbinding motifs to the major histocompatibility complex(MHC) molecules (5).Many examples of homologies based on primary sequences

have been reported and considered relevant in the pathogen-esis of different autoimmune diseases, but actual demonstra-tion of cross-reactive T cells has not been easy (2, 6). Somestudies, however, demonstrated so-called "degenerate" T-cellrecognition (7, 8), and a good example of molecular mimicryhas been recently reported, where T-cell clones specific formyelin basic protein (MBP)-(85-99) were also activated byforeign peptides (5). In this study the authors did not simplyalign sequences between the self-epitope and foreign mole-

cules but rather sought evidence for molecular mimicry on thebasis of the MHC class II binding and T-cell receptor (TCR)recognition motifs and a detailed mutational analysis (5).Among 129 sequences of viral and bacterial origin selected andchallenged with the T-cell clones, one bacterial and seven viralpeptides could stimulate the myelin basic protein-specificT-cell clones. Despite this elegant study, at present the mo-lecular mechanisms controlling the mimicry phenomena re-main poorly defined.T lymphocytes recognize antigen as short peptides in the

context of MHC molecules (9), and only recently has it becomeapparent how such epitopes interact with the MHC molecules(10-13). We hypothesized that identification of the antigenicsurfaces by molecular modeling might help gain a greaterunderstanding of how molecular mimicry occurs. In the cur-rent study we provide an example of molecular mimicry in theabsence of primary-sequence homology and identify the mech-anism controlling this mimicry in that only the two recognizedpeptides produced similar antigenic surfaces.

MATERIALS AND METHODST-Cell Clones. T-cell clone 37 was established from a Graves

disease thyroid infiltrate in the absence of antigen, accordingto our published procedure (14). Briefly, activated thyroid-infiltrating T lymphocytes were cultured in the presence ofrecombinant interleukin 2 (20 ng/ml, from Hoffmann-LaRoche) for 1 week in RPMI 1640 medium/10% human serumand then expanded for a further week with irradiated (4500rads; 1 rad = 0.01 Gy) autologous peripheral blood leucocytes,OKT3 anti-CD3 mAb (30 ng/ml) and recombinant interleukin2. Cells were cloned by limiting dilution in the absence of anyantigen, as described (15). Further expansion of the T-cellclones was achieved by restimulation every 2 weeks withHLA-unmatched irradiated peripheral blood mononuclearcells and phytohemagglutinin at a 1:1000 dilution. Every 4 daysrecombinant interleukin 2 at 10 ng/ml was added.

Antigens. Peptide thyroid peroxidase (TPO)-(535-551) cor-responding to amino acids 535-551 of human TPO (LDPLIR-GLLARPAKLQ), eight overlapping synthetic peptides of 12amino acids spanning from residue 534 to 552 of human TPO,as well as the truncated peptides within the region TPO-(536-547) and TPO-(539-550), were synthesized by Anergen (Red-wood City, CA) and Chiron Mimotopes (Clayton, Australia).

T-Cell Proliferation Assay. The TPO peptides were used todefine the minimum epitope specificity of T-cell clone 37. TheT-cell clone was tested 15 days after the last stimulation withirradiated feeders and phytohemagglutinin. The reactivity tothe TPO-(534-552) synthetic overlapping peptides was as-sessed by coculturing 104 T cells with 3 x 104 glutaraldehyde-

Abbreviations: TPO, thyroid peroxidase; MHC, major histocompat-ibility complex; TCR, T-cell receptor; EBV, Epstein-Barr virus.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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EBV + T + 535-551 Clone 37EBVonl5

TF+ T+L2EBV+T

EBV + T + 534-545EBV + T + 535-546EBV +T + 536-547EBV + T + 537-548EBV - T + 538-549EBV + T + 539-550_EBV + T + 540-551EBV + T + 541-5523

*- P3

-0 P6

0 10 20 30 40

[ H]Thymidine incorporation, cpm x 10->

FIG. 1. Fine epitope mapping of T-cell clone 37 using eight nestedoverlapping peptides of 12 amino acids. Data are the means oftriplicate cultures, and error bars show the SEs of the mean. The graphis representative of seven experiments, in which similar results wereobtained. Peptides were used at 5 ,uM because previous experimentsshowed that this was the optimal dose response for p535-551 (data notshown). IL2, interleukin 2; T, T-cell clone 37.

fixed autologous Epstein-Barr virus (EBV)-transformed pe-ripheral blood mononuclear cells as antigen-presenting cells.Cells were cocultured in triplicate for 72 hr in round-bottom96-well microtiter plates and then pulsed with [3H]thymidine[1 ,tCi (37 kBq) Amersham] during the last 8 hr of culture. Thefixed autologous EBV-transformed cells were pulsed with orwithout the indicated dose of peptides for 1 hr at 37°C beforeincubation with T cells.

RESULTSRecognition of Two Distinct Peptides Within the 535-551

Region ofTPO by T-Cell Clone 37. Characterization of humanself-reactive T-cell clones established from the thyroid infil-trate of a Graves disease patient identified a 17-residue peptideof TPO molecule (residues 535-551) as immunodominant inthe context of the HLA DQB1*0602/DQA1*0102 molecule(S.Q., M. Feldmann, C. M. Dayan, 0. Acuto & M.L., unpub-lished work). The fine epitope mapping of clone 37 (whichexpresses only one TCR af3 heterodimer) provided a consis-tent but unusual profile (Fig. 1). Two nonsequential peptidesp536-547 and p539-550 (P3 and P6, respectively) were rec-ognized by clone 37 to a greater extent than the interveningpeptides. Because different highly purified peptide prepara-tions gave identical results (data not shown), we could excludethe possibility of artifacts.The Sequence IRGLLARPA Shared by P3 and P6 Is Not

Relevant for TCR Recognition. The primary sequence ofpeptide 535-551 reveals that P3 and P6 share a commoncentral region IRGLLARPA (Fig. 2), which, based on prima-ry-sequence homology, should be the epitope recognized bythe T-cell clone. However, peptides P4 and P5 (Fig. 2), whichalso contain this sequence, stimulated clone 37 poorly (Fig. 1).

We used peptides truncated at the amino and carboxyl terminiof P3 and P6 (Fig. 3) to define the core recognized in thesepeptides. This experiment also suggested that the sharedoverlapping region within these two peptides was unlikely to beresponsible for the stimulation observed. Recognition of pep-tide P3 required a minimum length of 10 amino acids, residues536-545 (DPLIRGLLAR) (Fig. 3A), whereas 9 amino acids(residues 540-548, RGLLARPAK) were sufficient for clone37 to respond to P6 (Fig. 3B). In particular, Lys-548 wasessential for recognition of P6 and yet was absent from P3, aresult inconsistent with the presence of a shared epitope. Thisresult is further substantiated in Fig. 3B, in which the shared9-amino acid-long peptide IRGLLARPA induced no activa-tion.

Peptides TPO-(536-547) (P3) and TPO-(539-550) (P6)Bind with Different Registers to HLA DQB1*0602. The rec-ognition of both peptides P3 and P6 by the same T-cell clonecannot be rationalized in terms of a shared epitope. Wetherefore hypothesized that our enigmatic results might beexplained by the way the TCR of clone 37 might "see" theMHC-peptide complexes. The first question to answer washow P3 and P6 peptides could be accommodated withinHLA-DQ6 (DQB1*0602-DQA1*0102). Because the molecu-lar structure of DQ6 has not yet been crystallized, a molecularmodel of the HLA-DQ6 (DQB1*0602-DQA1*0102) was con-structed from the coordinates of the HLA-DR1 molecule (16),using the program COMPOSER, implemented as part of theSYBYL suite of programs (17, 18). The P3 and P6 peptides werebuilt into this model following the backbone of the influenzahemagglutinin peptide crystallized with HLA-DR1 (16), and

DPLIRGLLARPA

EBV + T

EBV + 535-551 +T

536-547 DPLIRGLLARPA

+ 536-544 DPLIRGLLA

+ 536-545 DPLIRGLLAR

+ 537-545 PLIRGGLLAR

0

IRGLLARPAKl.Q

EBV+ T

EBV + 535-551 +T

PEPTIDE P3

A

-A_

10 20 30 40 50 60 7

PEPTIDE P6

B

539-551 IRGLLARPAKCLQ N

+ 541-549 GLLARPAKL

+ 540-548 RGLLARPAK

+ 539-547 IRGGLLARPA

-I

10

P1P2P3P4P5P6P7P8

534-545535-546536-547537-548538-549539-550540-551541-552

GLDPLIRGLLARLDPLIRGLLARPDPLIRGLLARPAPLIRGLLARPAKL IRGLLARPA KLIRGLLARP LQRGLLARPAKLQVGLLARPAKLQVQ

FIG. 2. Schematic representation of the overlapping peptides used

in Fig. 1. Amino acids are indicated by the single-letter code. In the boxare the nine residues that are common to peptides P3 and P6, as wellas to the central peptides P4 and P5.

0 1. 0 3 4 5O l 0 20 30 40 :50

660 70

[3H]Thymidine incorporation, cpm x 10-

FIG. 3. Core peptides within P3 and P6 were assessed by using a setof truncated peptides at 5 ,uM in proliferation assays. (A) Theminimum epitope within P3 spans between Asp-536 and Arg-545. (B)The minimum epitope within P6 spans between Arg-540 and Lys-548.The IRGLLARPA peptide, common to both peptides P3 and P6, is notrecognized by clone 37, confirming its irrelevant role in the definitionof mimicry. These results belong to the same assay and are represen-tative of three different experiments. Different peptide concentrationsprovided identical patterns of responsiveness (data not shown). T,T-cell clone 37.

P:

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the side-chain positions were initially fitted manually; theresulting ensemble was subjected to energy minimization. Aside view of the modeled P3 and P6 peptides is shown in Fig.4 A and B, respectively. In these models the HLA-DQ6(DQB1 *0602-DQA1*0102) chains have been removed to showmore clearly the peptide conformation. The first finding wasthat both peptides could be accommodated within the HLA-DQ6 (DQB1*0602-DQA1*0102) groove, although they used adifferent register of binding. This phenomenon, of a shortpeptide containing two registers of binding for the same MHCallele, has been observed (19). The second, and more surpris-ing observation, was the remarkable similarity between themolecular surfaces generated by the two peptides bound to theHLA-DQ6 molecule, as shown in Fig. 4C, where the twopeptides are overlaid. It is likely that this structural homologyis responsible for the recognition of both peptides by the sameT-cell clone.

Peptides TPO-(536-547) (P3) and TPO-(539-550) (P6)Display Similar Antigenic Surfaces in the Context of HLA-DQB1*0602/DQA1*0102. T cells do not recognize primarysequences directly but rather recognize the composite molec-ular surface of the peptide in the context of a given MHCmolecule. It is clear, when the surface of the two peptide-MHC complexes is examined, as in Fig. 5 A and B, that thepredicted electrostatic surfaces of the P3 and P6 complexes arealmost identical. Indeed, the two complexes display similarbasic, hydrophobic, and acidic patches within the upper (point-ing toward the TCR) surface of the HLA-peptide complex. Itis also clear from the side, as well as the top, view of the models(Figs. 4 and 5A and B) that Arg-545 is an essential residue forrecognition of P3, whereas Lys-548 is essential for recognition

a

of P6, consistent with data in Fig. 3. The high level of similaritybetween these two peptides becomes even more stringentwhen compared with the molecular model of another peptiderecognized in the context of DQ6 (DQB1*0602-DQA1*0102).Peptide P4 was the best candidate because it is well recognizedby a different T-cell clone. We had, therefore, the uniqueopportunity to test our hypothesis with a peptide that isrecognized in the same HLA context and is even more similarin its primary sequence to peptide P3. As shown in Fig. SC, theantigenic surface of P4 in the HLA groove is completelydifferent from those displayed by P3 and P6 (Fig. 5A and B),both in terms of electrostatic patches as well as in respect tothe amino acids pointing toward the TCR.

DISCUSSIONThe two natural peptides reported here provide a specificexample of molecular mimicry dependent upon the antigenicsurface available for recognition by the T cell, rather thansimple sequence homology. Although the two immunogenicpeptides share nine residues (Fig. 2), this region is irrelevantto the TCR, as the register of binding of P3 and P6 to HLADQ6 differs. Indeed, this common region provides a cleardemonstration that sequence homology as normally defined(2, 4) may be misleading in identifying potential molecularmimicry. A simple sequence homology analysis would havesuggested that the common core was the element dictatingmolecular mimicry. But the low stimulatory activity of peptidesP4 and P5, both containing the core of nine residues (Fig. 1),and the lack of stimulation by the core peptide IRGLLARPAitself (Fig. 3B) indicate that this region is not relevant for

Leu543

Ile539

b

Gly54l Leu543Po4

%rPk4-4s 5L

Ile539 Ala544 Ala547

Leu542

c

ArglLys

Asp/IleIle/Leu

Gly/Ala

FiG. 4. Conformation of TPO peptides P3 and P6, as bound to DQB1*0602, is shown, looking toward the al domain; the amino terminus ofthe peptide is at left. (A) Peptide P3: residues Leu-538, Arg-540, Leu-542, Leu-543, and Arg-545 are capable of being seen by the TCR. (B) PeptideP6: residues Gly-541, Leu-543, Arg-545, Pro-546, and Lys-548 are capable of being seen by the TCR. (C) Overlay of peptides P3 and P6; note thatthe overall distribution of positively charged groups and hydrophobic side chains is similar in the two peptides.

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a b

Arg 545

ArgS45

c

A 545

FIG. 5. The molecular surfaces of the MHC-peptide complexes of P3 (A), P6 (B), and P4 (C) in the context of the DQB1*0602-DQA1*0102heterodimer. Surfaces were calculated and displayed by the program GRASP (20), and the surface of the peptide has been colored by surfacepotential to highlight the similarity between the two peptides.

mimicry. T-cell cross-reactivity in animal models of autoim-munity, such as experimental acute encephalomyelitis, hasbeen shown (21, 22). Most recent reports demonstrated thatviral and bacterial peptides can activate human autoreactiveT-cell clones (5, 23). These studies were possible because theMHC anchor residues and TCR-binding motifs were known.Therefore appropriate substitutions, considering electrostaticcharges and other stringent criteria, allowed the recognition ofone bacterial and seven viral peptides by T-cell clones specificfor myelin basic protein-(85-99) (5). A similar approach wouldnot have been possible in this study because (i) HLA-DQ6anchor motifs are not yet properly defined and (ii) peptides P3and P6, although strong stimulants of T-cell clone 37, have verylow affinities for MHC (S.Q., D. C. Wraith & M.L., unpub-lished work), contrary to myelin basic protein-(85-99), whichis a high-affinity binder. The molecular modeling of thepeptide-MHC complex was the most useful strategy for pre-dicting the TCR-binding residues and understanding the mo-lecular mechanism enabling T-cell cross-reactivity within P3and P6, due to the similarity within the antigenic surfaces. Thetertiary-structural model could also explain the experimentalobservations that Arg-545 is essential for T-cell recognition ofP3 and that Lys-548 (which is not present in P3) is essential forthe recognition of P6. Thus, our study provides an example ofmolecular mimicry with an autoantigen-reactive T-cell cloneand clarifies how it occurs. Clone 37 and other T-cell clonesrestricted by the same genetic restriction element and recog-nizing different epitopes within p535-551 will be instrumentalin defining general rules of MHC-binding motifs forDQB1*0602 molecules, by using single-substituted peptides.This set of experiments will also provide useful information

about the TCR contact residues of the different T-cell clones.Our analysis provides strong evidence that this phenomenonrelies on conformational features of the epitopes. Further-more, our analysis shows that simple sequence comparisons ofpeptides, without consideration of the relevant MHC, may notbe a worthwhile enterprise. Recent studies demonstrated thatantigen analogs might act as powerful and specific inhibitors ofT-cell activation (24-26), as well as allowing thymic-positiveselection of T cells (25). Although the rules defining how apeptide behaves as an analog are not yet completely known,synthetically prepared (24, 27) or naturally occurring analogs(28,29) work only in the context of the specific MHC and TCR.Therefore, the approach used in this study may be central inthe search for analogs, as well as to assess the role of molecularmimicry for peptides with low affinity for MHC, which mayhave an important role in the development of autoimmunediseases (30). To the best of our knowledge, this molecularmodeling is the only analysis of how peptides may fit into a DQgroove becauseDQ crystal structure has not yet been reported.The definition of mimic peptides and/or analogs is even morecompelling for HLA-DQ-restricted self-epitopes. DQ mole-cules, unlike DR, have two polymorphic chains; hence, bothsides of the peptide-binding groove are polymorphic andsubject to more heterogeneity than in the case ofDR (31, 32).Furthermore, HLA-DQ molecules have been considered cen-tral in several autoimmune diseases, such as diabetes, multiplesclerosis, and celiac disease (33), and little is known aboutDQ-binding motifs. Thus our report provides another inter-pretation of the concept of T-cell mimicry and is relevant instudying HLA-DQ-restricted mimicry, as well as analog pep-tides possibly involved in autoimmunity.

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We thank Profs. Edward A. Clark and Marc Feldmann for criticalreading of the manuscript. We also thank Drs. S. Sharma (Anergen,Redwood City, CA) and M. Geysen (Chiron Mimotopes, Clayton,Australia) for the synthesis of the peptides used in this study. This workwas supported by the Arthritis and Rheumatism Council (ARC), theItalian Association of Multiple Sclerosis (AISM), and the ImperialCancer Research Fund (P.J.T.). C.J.T. is supported by a CollaborativeAwards for Science and Engineering (CASE) award from RocheProducts. P.J.T. is a Royal Society University Research Fellow.

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