DOI: 10.1126/scitranslmed.3001012, 41ra51 (2010);2 Sci Transl Med, et al.Jason A. Tye-Din

Celiac DiseaseComprehensive, Quantitative Mapping of T Cell Epitopes in Gluten in

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CEL IAC D I SEASE

Comprehensive, Quantitative Mapping of T CellEpitopes in Gluten in Celiac DiseaseJason A. Tye-Din,1,2,3* Jessica A. Stewart,1* James A. Dromey,1* Tim Beissbarth,1*†

David A. van Heel,4 Arthur Tatham,5 Kate Henderson,6 Stuart I. Mannering,1‡ Carmen Gianfrani,7

Derek P. Jewell,8 Adrian V. S. Hill,9 James McCluskey,10 Jamie Rossjohn,6 Robert P. Anderson1,3§

(Published 21 July 2010; Volume 2 Issue 41 41ra51)

Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens ingrain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Ourunderstanding of the epitopes of pathogenic CD4+ T cells is based primarily on responses shown by intestinal Tcells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33–aminoacid peptide from wheat a-gliadin is the immunodominant antigen, but little is known about the spectrum of Tcell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T cellepitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients byfeeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic pro-lamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patientswho ate the same cereal, but were different after wheat, barley, and rye ingestion. Unexpectedly, a sequencefrom w-gliadin (wheat) and C-hordein (barley) but not a-gliadin was immunodominant regardless of thegrain consumed. Furthermore, T cells specific for just three peptides accounted for most gluten-specific Tcells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic Tcells in celiac disease show limited diversity and therefore suggest that peptide-based therapeutics for thisdisease and potentially other strongly human leukocyte antigen–restricted immune diseases should bepossible.

INTRODUCTIONCD4+ T cell recognition of peptides derived from dietary wheat glutenand related prolamins from barley and rye is fundamental to thepathogenesis of celiac disease (CD) (1). Intestinal gluten-specific Tcells, which confer susceptibility to CD, are restricted by the majorhistocompatibility complex molecules human leukocyte antigen(HLA)–DQ2 or HLA-DQ8 (2, 3). Most known gluten-derived epi-topes are relatively resistant to digestive proteases and have been se-lectively deamidated by tissue transglutaminase (tTG), an enzymewhose activity is increased in the inflamed intestinal mucosa (4–6).Major clinical problems in the management of CD are that the diag-

nostics are suboptimal and invasive and that patients must rely on acomplex, costly, and lifelong therapy—strict dietary gluten exclusion(7). Even trace dietary contamination by gluten is injurious, and fullrecovery of intestinal histology is achieved in fewer than half of adults(8, 9). There is a demand for effective therapeutic adjuncts or alterna-tives to the gluten-free diet; several are under development and theirdesign is predicated on clear understanding of the immunotoxic glu-ten peptides. One such therapeutic candidate that is effective in variousmurine models of T cell–mediated disease requires repeated dosingwith peptides corresponding to immunodominant T cell epitopes (10).

Designing a peptide immunotherapy would be straightforward ifgluten-specific T cells from all CD patients were specific for a limitednumber of gluten peptides. Indeed, the importance of HLA-DQ2 andHLA-DQ8 in determining susceptibility suggests that the pathogenic Tcell response to gluten is highly focused. But despite important ad-vances, two persistent problems have prevented a comprehensiveunderstanding of the pathogenic T cell response in CD. First, T cellsspecific for gluten are so rare in intestinal tissue that they cannot be di-rectly evaluated. Second, there are hundreds of wheat gluten proteinsandmany others in rye and barley that are potentially toxic in CD; thus,there are toomany candidate epitopes in gluten to simultaneously assessthem all in a single unbiased T cell screening assay.

Epitope mapping studies have overcome the scarcity of gluten-reactive T cells in patient samples with T cells derived from intestinaltissue that have been expanded to measurable numbers over days orweeks by periodically restimulating with wheat gluten and mitogens(11–18). When screened against a protease-digested recombinant a2-gliadin polypeptide preincubated with tTG, intestinal T cell lines (TCLs)selectively recognize three distinct, overlapping epitopes in tandem

1Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria3052, Australia. 2Department of Medical Biology, University of Melbourne, Parkville,Victoria 3010, Australia. 3Department of Gastroenterology, Royal Melbourne Hospital,Grattan Street, Parkville, Victoria 3050, Australia. 4Barts and The London School ofMedicine and Dentistry, Queen Mary University of London, London E1 2AT, UK.5Cardiff School of Health Sciences, University of Wales Institute, Cardiff CF5 2YB, UK.6Protein Crystallography Unit, Department of Biochemistry and Molecular Biology,School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.7Institute of Food Science, National Research Council, Via Roma 52, 83100 Avellino,Italy. 8Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DU, UK.9Jenner Institute, University of Oxford, Oxford OX3 9DU, UK. 10Department ofMicrobiology and Immunology, University of Melbourne, Parkville, Victoria 3010,Australia.*These authors contributed equally to this work.†Present address: Department of Medical Statistics, University Medicine Goettingen,37099 Goettingen, Germany.‡Present address: St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy,Victoria 3065, Australia.§To whom correspondence should be addressed. E-mail: banderson@wehi.edu.au

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repeat (DQ2-a-I, PFPQPELPY; DQ2-a-II, PQPELPYPQ; and DQ2-a-III, PYPQPELPY), which are present within a single protease-resistant33–amino acid peptide (4). This 33–amino acid oligopeptide is usuallythe most bioactive gluten peptide recognized by gluten-specific TCLsfrom HLA-DQ2+ CD donors. Occasional discrepant findings amongstudies of gluten epitopes have been attributed to subtly differentexperimental protocols, the use of material from children rather thanadults, and the use of gluten from different sources prepared in var-ious ways (15, 17).

Distinct immunoreactive peptides have been successfully isolatedfrom crude hydrolysates of gluten and several pure recombinant a-and g-gliadins, but gluten is highly insoluble and, despite the fact thatall wheat gluten fractions are immunotoxic, there have been no system-atic searches for epitopes derived from the aqueous insoluble glutenins orfrom w-gliadins, nor have there been systematic epitope mapping stu-dies of prolamins in barley (hordeins) or rye (secalins).With recent im-provements in and reduced cost of peptide synthesis, epitope mappingstudies have used synthetic gluten peptides spanning ana-gliadin and ag-gliadin polypeptide (19) and also peptides corresponding to certainsequences predicted to be susceptible to deamidation by tTG and likelyto bind efficiently to HLA-DQ2 (20). Systematic searches for T cell epi-topes in the many hundreds of complementary DNA–derived wheat,barley, and rye prolamins have been limited to predictive studies; manycandidates have been proposed but there is no sense of their relativecontribution to the gluten-specific T cell response norwhether they rep-resent distinct epitopes (11).

The only known source of fresh polyclonal gluten-specific T cells atsufficiently high frequency to enumerate without expansion in vitro isperipheral blood from CD donors directly after short-term oral glutenchallenge (21). T cells specific for gluten are maximal in blood 6 daysafter volunteers commence oral gluten challenge and can be enumer-ated by overnight enzyme-linked immunospot (ELISpot) assay. Such Tcells are CD4+, secrete interferon-g (IFN-g), preferentially recognize de-amidated gluten, and express the a4b7 integrin associated with homingto the intestinal lamina propria (21, 22). A deamidated 17–amino acidoligopeptide encompassing the overlapping DQ2-a-I and DQ2-a-IIepitopes is the only peptide in A-gliadin recognized by peripheral bloodT cells that have been induced by oral gluten challenge in HLA-DQ2+

CD volunteers.To test whether the immunotoxicity of prolamins from wheat,

barley, and rye is truly limited to just a few critical peptides, we haveexploited oral challenge with wheat, barley, or rye to enable antigenpresentation to occur in vivo and the sampling of peripheral bloodT cells induced in >200 HLA-DQ2+ CD patients. By developing ahigh-throughput IFN-g ELISpot assay with peripheral blood mono-nuclear cells (PBMCs) collected 6 days after commencing oral grainchallenge (GC), we have screened customized peptide libraries encom-passing >16,000 unique candidate 12–amino acid sequences from alltoxic prolamin fractions of wheat, barley, and rye. T cell clones (TCCs)were used to confirm minimal epitopes and to determine the prom-iscuity of peptide recognition. Unbiased screening of these freshlycollected T cells revealed that the immunotoxicity of gluten is bothconsistent and reducible to three highly immunogenic peptides. Un-biased screening also reveals that promiscuous recognition of gliadin-,hordein-, and secalin-derived peptides by T cells whose optimal epi-tope is an w-gliadin/C-hordein–derived sequence, rather than epitopespresent in a-gliadin, accounts for the immunotoxicity common towheat, barley, and rye.

RESULTS

Quantitative ex vivo assessment of patients’ T cellsspecific for published epitopesGluten peptides recognized by TCCs and TCLs were assessed by IFN-gELISpot with PBMCs isolated from nine HLA-DQ2+ CD donors 6days after they began a 3-day wheat GC. IFN-g ELISpot responsesto deamidated gliadin were detected in eight donors [median, 23;range, 13 to 153 spot-forming units (SFUs) permillion PBMCs]; amongseven of these eight donors, the a-gliadin 17–amino acid oligopeptideQLQPFPQPELPYPQPQP (5 mM) and the related 33–amino acid oli-gopeptide QLQPFPQPELPYPQPELPYPQPELPYPQPQPF also eli-cited detectable responses of a similar magnitude (Fig. 1A). Yet, incontrast, DQ2-g-IV (SQPEQEFPQ) was the only one of 10 other re-ported gluten T cell epitopes to produce a detectable response, and thiswas relatively weak and seen only in one donor. We concluded that al-though T cells specific for DQ2-a-I and DQ2-a-II make a substantialcontribution to the T cell population induced by wheat GC, the specific-ity of many other gluten-specific T cells induced by GC could not beexplained by previously reported epitopes.

Comprehensive screening of wheat gluten, rye secalin, andbarley hordein peptidesConventionally, CD4+ T cell epitopes are mapped with overlapping15- to 20–amino acid oligopeptides encompassing all antigen-derived10- to 12–amino acid oligopeptide linear sequences. This traditionalapproach is impractical to map all T cell epitopes in gluten; even in2001 when this study commenced, there were several hundred candi-date prolamin genes isolated from wheat, rye, and barley. Instead, wetook advantage of the close homology between protein sequencesderived from each subgroup of gluten proteins and developed aniterative algorithm to define all unique 12–amino acid oligopeptidesencoded by gliadin and glutenin genes in Triticum aestivum (wheat),hordein genes in Hordein vulgare (barley), and secalin genes in Secalecerale (rye) and then to design a minimal number of 20–amino acidoligopeptides encompassing all of these 12–amino acid oligopeptides(23).

Because intestinal and fresh peripheral blood T cells induced by oralGC in CD patients preferentially recognize deamidated prolamins,individual 20–amino acid oligopeptides (25 mg/ml) in each of five“comprehensive” gliadin, low–molecular weight (LMW) glutenin,high–molecular weight (HMW) glutenin, hordein, and secalin libraries(table S1) were pretreated with tTG and then screened in overnight IFN-gELISpot assays with fresh PBMCs from a total of 86HLA-DQ2+CDvol-unteers after GCwith wheat (to assess the gliadin and half the LMWglu-tenin library or the HMW glutenin and the second half of the LMWglutenin library), barley (to assess the hordein library), or rye (to assessthe secalin library).

A hierarchy of stimulatory peptides was clearly demonstrated foreach first-round library (Fig. 1B). For each 20–amino acid oligopeptide,a score (out of 100) was calculated that was equal to the average relativefrequency of 20–amino acid oligopeptide specific T cells present inblood (see Materials and Methods). Overall, 61% (1666 of 2723) of20–amino acid oligopeptides had a score of 0, 13% (343) had a scorebetween 0 and 1, 18% (495) had a score between 1 and 5, 5% (125)had a score between 5 and 10, and 2% (66) had a score between 10and 30, whereas only 1% (28) had a score >30. Of the first-round

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tTG-treated 20–amino acid oligopeptides, 219 had scores >5, represent-ing 5% (125 of 2152) of wheat gliadin and glutenin 20–amino acid oli-gopeptides, 14% (59 of 416) of hordein 20–amino acid oligopeptides,and 23% (35 of 155) of secalin 20–amino acid oligopeptides.

Peptides eliciting at least 5% of the IFN-g ELISpot response of themost active 20–amino acid oligopeptide in each individual donor werefine-mapped with 16–amino acid oligopeptides (as described in Ma-terials and Methods). Design of second-round peptides predicted thatepitopes fell within 12–amino acid sequences having glutamine residuesdeamidated by tTG to create anchors for 9–amino acid epitopes at

positions 4, 6, or 7, facilitating binding to HLA-DQ2; such 12–aminoacid sequences had glutamine at position 7with proline at position 9 butnot 8 or 10, and/or a bulky hydrophobic residue at position 10. The 12–amino acid sequence candidate was then flanked at positions!1 and 13with native residues, and then at positions !2 and 14 with glycine resi-dues. The final 16–amino acid oligopeptide was then synthesized withglutamate or glutamine at positions predicted to be susceptible to de-amidation by tTG. For first-round 20–amino acid oligopeptides thatlacked glutamine residues predicted to be deamidated by tTG, two16–amino acid oligopeptides overlapping by 12 residues were synthe-

Fig. 1. IFN-g ELISpot response after GC defines a hierarchy of peptide re-sponses. Six days after commencing 3-day wheat, barley, or rye challenge,PBMCs fromHLA-DQ2+ CD donors were assessed for IFN-g ELISpot responseto previously characterized epitopes and comprehensive peptide libraries.(A) PBMCs from nine donors were assessed after wheat challenge. Dashedlines reflect the cutoff threshold for considering responses “positive,” fourtimes the response to medium alone, and >10 SFUs per well adjusted toSFUs per million PBMCs. (B) Three-day wheat, barley, or rye challenge wasundertaken in a total of 86 CD volunteers. PBMCs from donors were usedto screen one of five first-round gluten peptide libraries that included 272320–amino acid oligopeptides encompassing 16,838 unique 12–amino acidoligopeptides in 313 GenBank entries for gliadins, LMW glutenins, andHMW

glutenins (T. aestivum) (after wheat challenge), hordeins (H. vulgare) (after bar-ley challenge), and secalins (S. cerale) (after rye challenge). (C) PBMCs fromCD donors were used to screen three second-round gluten peptide librariesthat included 441 16–amino acid oligopeptides encompassing reactive 20–amino acid oligopeptides from first-roundwheat gliadin and LMWandHMWglutenin libraries (28 donors after wheat challenge), 80 16–amino acid oligo-peptides encompassing reactive 20–amino acid oligopeptides from thefirst-round barley hordein library (10 donors after barley challenge), and5316–aminoacid oligopeptides encompassing reactive 20–aminoacidoligo-peptides from the first-round rye secalin library (10 donors after rye chal-lenge). The line indicates minimum score of 5 required to be considered aconfirmed stimulatory peptide.

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sized. Second-round libraries consisted of 554wheat gliadin- and glutenin-derived 16–amino acid oligopeptides, 89 hordein-derived 16–aminoacid oligopeptides, and 64 secalin-derived 16–amino acid oligopeptides;they were screened by IFN-g ELISpot with PBMCs from a further co-hort of HLA-DQ2+ CD volunteers after wheat (n = 28), barley (n = 10),or rye (n=10)GC.Ahierarchy of stimulatory peptideswas again clearlydemonstrated for each second-round library (Fig. 1C).

We elected to focus on the most active candidate deamidated 12–amino acid oligopeptide or wild-type 16–amino acid sequences rep-resented among peptides with a score of "5 in both the first-roundand the second-round screening libraries (Fig. 1C). Overall, 37 wheatgluten–, 30 barley hordein–, and 29 rye secalin–derived 12- or 16–amino acid sequences were confirmed as distinct T cell–stimulatorysequences (Table 1). These T cell–stimulatory gluten peptides rarely eli-

Table 1. Hierarchy of peptides afterwheat, barley, or rye challenge in vivo andrecognition by TCCs in vitro. First-round library 20–amino acid oligopeptideswith a score of >5 confirmed by second-round 16–amino acid oligopep-tides (core 12–amino acid sequence in bold) with score of >5 using day6 PBMCs after gluten challenge. Recognition determined by TCCs raisedto cognate ligand and incubated with second-round peptides and veri-

fication library (25 mg/ml). Epitopes: a-I PFPQPELPY, a-II PQPELPYPQ, w-I PFPQPEQPF, w-II PQPEQPFPW, and Hor-I PIPEQPQPY. W, wheat gliadin(unless LMW or HMW indicated); B, barley; R, rye. Abbreviations for theamino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V,Val; W, Trp; and Y, Tyr.

noitingocer TCCSecond - roundFirst-roundFirst-round 20eroC Defined or predicted

identifier (second-round core) score Score Res§ DomII !-I !-I !-II !-I !-II "-I "-I "-II "-II Hor- I Sec- I Gliadin gluten epitopes

W01 PQPFPPQLPYPQPQLPYPQP 60 75 96 69 78 58 81 100 68 59 !-II, !-III

W02 MQLQPFPQPQLPYPQPQLPY 55 74 88 58 100 74 100 100 96 62 !-I, !-II

W03 QPFPQPQQPFPWQPQQPFPQ 53 65 81 35 97 66 55 95 "-I "-II

W04 PQQPQQPFPQPQQPIPVQPQ 31 53 77 31 94 PFPQPQQPI, PQPQQPIPV

W05 PQQQQPFPQPQQPFSQQPQQ 19 39 69 15 52 "-I

W06 QQPQQPFPQPQLPFPQQSEQ 25 34 69 15 79 100 78 100 PFPQPQLPF, PQPQLPFPQ

W07 WPQQQPFPQPQQPFCQQPQQ 15 27 62 8 "-I

W08 LQLQPFPQPQLPYSQPQPFR 15 24 65 8 75 59 81 78 46 !-I

W09 LQPFPQPQPFLPQLPYPQPQ 34 23 54 0 82 100

W10 SQQPQQQFSQPQQQFPQPQQ 6 20 42 0 14 #-IV

W11 QAFPQPQQTFPHQPQQQFPQ 6 18 50 0 85

W12 CKVFLQQQCSPVAMPQRLAR 12 16 54 4

W13 LQLQPFPQPQLPYLQPQPFR 20 15 54 4 66 81 92 46 !-I

W14 QQQFIQPQQPFPQQPQQTYP 10 15 46 4 92

W15 LMW SHIPGLERPWQQQPLPPQQT 9 13 17 11

W16 SQQPQQPFPQPQQQFPQPQQ 15 13 50 0 20 #-VIIa PQQQFPQPQ

W17 QQPFPQPQQPQLPFPQQPQQ 12 10 35 0 36 PFPQPQQPQ

W18 PQLPYPQPQLPYPQPQPFRP 54 10 39 0 60 81 !-II

W19 QPFPQPQQPFPWQPQQPFPQ 53 10 35 0 93

W20 FPELQQPIPQQPQQPFPLQP 6 10 31 4 51 QQPQQPFPL

W21 HMW QGQQGYYPISPQQSGQGQQP 7 10 22 6

W22 HMW LQPGQGQPGYYPTSPQQIGQ 11 9 17 6

W23 QQFLQPQQPFPQQPQQPYPQ 15 9 33 0 #-III

W24 HMW PGQGQSGYYPTSPQQSGQKQ 5 9 17 6

W25 TPIQPQQPFPQQPQQPQQPF 15 8 35 0 100

W26 PQQPQQPFPLQPQQPFPQQP 10 8 35 0 77

W27 PFTQPQQPTPIQPQQPFPQQ 12 7 23 0

W28 PQQPFPQPQQTFPQQPQLPF 7 7 22 6 QQTFPQQPQ

W29 HMW GQGQSGYYPTSPQQSGQEAT 6 7 17 6

W30 PLQPQQPFPQQPQQPFPQPQ 19 6 19 0 #-VIa

W31 QPFPQLQQPQQPLPQPQQPQ 9 6 4 0

W32 QQPFPQQPQQPFPQPQQPIP 37 6 23 0 20 #-VIIb, PFPQQPQQPF#

W33 PQQPFPQPQQTFPQQPQLPF 7 5 17 0 71 PFPQPQQTF

W34 VAHAIIMHQQQQQQQEQKQQ 6 5 22 0

W35 PQQPFPQQPQQQFPQPQQPQ 11 5 19 0 PFPQQPQQQ, QQPQQQFPQ

W36 QQPAQYEVIRSLVLRTLPNM 9 2 11 4

W37 ATANMQVDPSGQVQWPQQQP 0 6 6 6

B01 QPFPQPQQPFPWQPQQPFPQ 50 66 88 50 86 66 80 74 "-I "-II

B02 WQPQQPFPQPQQPFPLQPQQ 53 64 75 50 80 74 "-I

B03 QPQQPFPQPQQPIPYQPQQP 32 55 75 38 QFPQPQQPF

B04 QPQQPQPFPQQPVPQQPQPY 38 45 63 25 53

B05 PQPFPQQPIPQQPQPYPQQP 38 43 50 25 70 46 Hor-I PQPFPQQPI, PFPQQPIPQ, QQPIPQQPQ

B06 QQPQPFSQQPIPQQPQPYPQ 63 40 50 25 60 Hor-I QQPIPQQPQ

B07 QSQQQFPQPQQPFPQQPQQP 8 37 63 13 59 H!2/S!2 QFPQPQQPF

B08 PQPFPQQPIPQQPQPYPQQP 38 33 38 25 59 Hor-I QQPIPQQPQ

B09 QQPFPQQPFPQQPQPYPQQP 26 32 63 13 46 #-VIa QQPFPQQPF, PFPQQPFPQ, PFPQQPQPY

B10 PQQPQQPFPQPQQPFSWQPQ 39 27 50 13 34 29 72 H!9/S!9 ("-I)

B11 QPQPYPQQPQPYPQQPFQPQ 39 26 50 0 PQPYPQQPQ, PYPQQPQPY

B12 QQPFPQQPFPQQPQPYPQQP 26 23 50 13 42 #-VIa PFPQQPQPY

B13 PQPYPQQPQPFPQQPPFCQQ 19 21 50 0 PQPYPQQPQ

B14 FQQPQQSYPVQPQQPFPQPQ 22 19 38 0 82

B15 YPQQPQPFPQQPIPQQPQPY 41 19 38 0 819 PQPFPQQPI, PFPQQPIPQ

B16 QQQPFPQQPIPQQPQPYPQQ 41 16 38 0 52 QQPFPQQPI, PFPQQPIPQ

B17 QPQQPQPFPQQPVPQQPQPY 38 15 38 0 76

B18 QPQPFPQQPIPLQPHQPYTQ 7 14 38 0 85 PQPFPQQPI

B19 LPRPQQPFPWQPQQPFPQPQ 14 13 38 0 91

B20 QQPFPLQPQQPFPQPQPFPQ 13 12 25 0 60

B21 PFPQQPQQPFPQPQQPFRQQ 19 10 25 0 44 H!9/S!9 ("-I)

B22 PQQPFQPQQPFPQQTIPQQP 8 10 25 0 64

B23 NPLQPQQPFPLQPQPPQQPF 9 10 25 0 50

B24 NPLQPQQPFPLQPQPPQQPF 9 9 25 0 63

B25 PFPQQPQQPFPQPQQPFRQQ 19 8 25 0 33 #-VIIb

B26 QPQQPFPLQPQQPFPWQPQQ 7 7 13 0 72

B27 TFPPSQQPNPLQPQQPFPLQ 18 7 13 0 81

B28 PQQTIPQQPQQPFPLQPQQP 10 6 25 0 11

B29 QPQQPFSFSQQPQQPFPLQP 9 6 25 0 29

B30 QQPFPQQPFPQQPQPYPQQP 26 5 25 0 #-VIa QQPFPQQPF

*

amino acid oligopeptide

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cited IFN-g ELISpot responses when incubated with PBMCs collectedfrom CD donors before GC (table S2) and did not elicit responses inPBMCs collected from healthy HLA-DQ2+ volunteers who had eatena gluten-free diet for 4 weeks and then had wheat GC (table S3).

Peptides were called dominant for aparticular CDdonor if they elic-ited at least 70% of the response of the most active peptide in eachlibrary for that donor (Table 1). In wheat, 20 tTG-treated second-round16–amino acid oligopeptides were dominant in at least one donor, butonly 7 of these were dominant in >10% of donors. Eleven hordein 16–amino acid oligopeptides and seven secalin 16–amino acid oligopep-tides elicited dominant responses in >10% of donors after barley and ryechallenge, respectively.Thehighest-scoring tTG-treated second-roundwheatgluten-, hordein-, and secalin-derived sequences (W01, LPYPQPQLPYPQ;B01, QPFPQPQQPFPW; and R01, QPFPQPQQPIPQ) were dominantin >50% of donors, and each was recognized by >80% of donors.

Fifty-two of the 96 immunogenic second-round peptides includedknown or predicted T cell epitopes (11, 15, 16). Included within the 44newly described T cell–stimulatory gluten peptides identified were fourclosely related HMW gluten 16–amino acid oligopeptides that re-sembled the HLA-DQ8–restricted epitope present in HMW glutenin(DQ8-GLT-I: QGYYPTSPQ) (24). Themost active of these HMWglu-tenin peptides (W21: QGQQGYYPISPQQSGQ) was recognized byPBMCs from fewer than one-quarter of donors after wheat GC butwas occasionally dominant.

Seven epitopes previously definedwith TCCs or TCLs in vitro wererepresented among the 96 confirmed immunogenic peptides: DQ2-a-I, DQ2-a-II, DQ2-a-III, DQ2-g-III, DQ2-g-IV, DQ2-g-VI, and DQ2-g-VII. Direct comparison of the relative size of the T cell populationsspecific for these individual epitopes is complex because many of theimmunogenic second-roundpeptides door are likely to encompassmorethan one distinct epitope. The second-round peptides that included theDQ2-a-II and DQ2-a-I or DQ2-a-III epitopes achieved the highestscores after wheat GC (74 and 75, respectively), but the maximal scoresof 16–amino acid oligopeptides that included other epitopes were sub-

stantially less (DQ2-g-III: score, 9; DQ2-g-IV: score, 20; DQ2-g-VI: score,6; andDQ2-g-VII: score, 13).After barley or ryeGC, 16–amino acid oligo-peptides including DQ2-g-VI were almost half as active as the mostactive peptides, and 16–amino acid oligopeptides including DQ2-g-VII were less than one-sixth as active as the most active peptides.

All but the four HMW glutenin peptides and one gliadin peptideincluded glutamine at a position predicted to be deamidated by tTGand to enhance binding to HLA-DQ2.

Despite its logistical complexity and the requirement for a large num-ber of CD volunteers, this large-scale, high-throughput peptide libraryscreen exploiting fresh peripheral blood T cells was highly informative;each prolamin fraction of wheat, barley, and rye contained a definedhierarchy of immunogenic peptides.

Dependence of the frequency of peptide-specific T cellsin blood on the cereal ingestedThe most active T cell–stimulatory second-round library peptides weresynthesized to high purity and characterized in greater detail withPBMCs from further HLA-DQ2+ CD donors after wheat (n = 14), barley(n = 11), or rye (n = 8) GC. The dominance of stimulatory peptides wasremarkably different after wheat, barley, or rye GC (Table 2). Peptidesderived fromwheata-gliadin, including the DQ2-a-I, DQ2-a-II, or DQ2-a-III epitopes, were dominant only after wheat GC.Other frequently dom-inant peptides were almost exclusively dominant after the ingestion of justone grain. For example, the C-hordein 16–amino acid oligopeptidePQQPIPEQPQPYPQQP (B08-E7) was active only after barley challenge,and the w-secalin sequence PFPQQPEQIIPQ (R11-E7) only after rye chal-lenge (Fig. 2, B to D). For other peptides such as those including the motifQPFP(W,L,Y,V,I)QPEQPFPQ and also the protease-resistant g-gliadin26–amino acid oligopeptide FLQPEQPFPEQPEQPYPEQPEQPFPQ(11), T cell responses were relatively stronger after barley or rye chal-lenge but still could be detected after wheat challenge (Table 2).

In contrast, peptides sharing the sequence QPFPQPEQP(F,I)P(W,L,Y,Q)(Q,S) were equally active and frequently dominant after wheat,

R01 QQLPLQPQQPFPQPQQPIPQ 48 67 82 64 PQPQQPIPQ

R02 SIPQPQQPFPQPQQPFPQSQ 30 64 82 55 18 51 H!2/S!2, H!9/S!9 ("-I)

R03 QPFPQPQQPTPIQPQQPFPQ 51 62 91 55 PFPQPQQPT, PQPQQPTPI

R04 QPFPQPQQPTPIQPQQPFPQ 51 29 73 0 78 IQPQQPFPQ

R05 PAPIQPQQPFPQQPQQPFPQ 18 23 55 9 93 IQPQQPFPQ

R06 PQQPFPQQPEQIIPQQPQQP 42 34 82 27 98

R07 QYSPYQPQQPFPQPQQPTPI 19 27 64 0 100

R08 SQQPQRPQQPFPQQPQQIIP 14 32 70 10 123 #-VIa, PFPQQPQQI

R09 QQLPLQPQQPFPQPQQPIPQ 48 23 73 0 85

R10 FPLQPQQPFPQQPEQIISQQ 29 26 45 18 99

R11 PQQPFPQQPEQIIPQQPQQP 42 24 55 9 90

R12 FPQQPQQPFPQPQQQLPLQP 29 47 80 40 PQPQQQLPL

R13 SPQPQQPYPQQPFPQQPQQP 11 18 64 0 38 PYPQQPFPQ, QQPYPQQPF

R14 QQPQQPFPLQPQQPVPQQPQ 33 17 64 0 62 107

R15 QPQQIIPQQPQQPFPLQPQQ 25 15 64 0 11 88

R16 PQQPFPQQPEQIIPQQPQQP 42 14 55 0 39

R17 QTQQSIPQPQQPFPQPQQPF 24 14 55 0 84 H!2/S!2

R18 QQPFLLQPQQPFSQPQQPFL 9 13 55 0 76

R19 QQPQQPFPLQPQQPVPQQPQ 33 13 55 0

R20 EQIISQQPFPLQPQQPFSQP 10 12 45 0 61 19

R21 NMQVGPSGQVEWPQQQPLPQ 10 11 36 0

R22 PQQLFPLPQQPFPQPQQPFP 64 13 36 9

R23 PQTQQPQQPFPQPQQPQQLF 49 11 55 0 33 #-VIIb

R24 SPQQPQLPFPQPQQPFVVVV 21 11 64 0 H!9/S!9 ("-I)

R25 FPQQPEQIISQQPQQPFPLQ 6 9 45 0 16 40

R26 PAPIQPQQPFPQQPQQPFPQ 18 9 55 0 33 29 #-VIIb

R27 PQEPQQLFPQSQQPQQPFPQ 7 6 27 0 36 #-VIIb

R28 SPQPQQPYPQQPFPQQPQQP 11 6 36 0 3361 #-VIa

R29 PTPIQPQQPFPQRPQQPFPQ 8 5 40 0 11

Duplicate core sequences: W03 and B01; W19 and B19; W25 and R04; W26 and B20; W30 and R28; and W32, B25, and R26.

II "Dom" is percent of second-round donors with response to tTG-treated 16

Cell color denotes clone specificity: !$% or !$%% "$% or "$%% Hor-I Sec-I Gliadin peptide.

Number in cell is the IFN-# response of the clone to second-round peptide, expressed as a percentage of the response to cognate ligand.

"Res" is percent of second-round donors responding to tTG-treated 16

!-I PFPQPQLPY, !-II PQPQLPYPQ, !-III PYPQPQLPY, #-IV SQPQQQFPQ, # -VIa QQPFPQQPQ, # -VIIb QQPQQPFPQ, H!2/S!2 PQPQQPFPQ, and H!9/S!9 PFPQPQQPF predicted and confirmed

by Vader et al. (#) or epitopes predicted by Shan et al (italics).

Epitopes

amino acid oligopeptide more than four times in response to medium alone and at least 10 SFUs per well.

amino acid oligopeptide >70% of maximal response to any second-round tTG-treated 16 amino acid oligopeptide

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barley, or rye challenge. The w-gliadin/C-hordein–derived sequenceQPFPQPEQPFPW(W03-E7)was consistently themost active of this pep-tide family (Fig. 2A). PBMCs isolated from CD donors after GC alloweddetailed characterization of the extendedw-gliadin sequence, includingW03 [AAG17702(81–102)], with lysine-substituted variants andoverlapping, glutamate-substituted 15–amino acid oligopeptides; the se-quence PFPQPEQPFPWwith a single deamidated glutaminewas criticalfor recognition by T cells induced by wheat, barley, or rye GC (fig. S1).These findings in Australian CD volunteers corroborated earlier obser-vations when a pilot 652-member 20–amino acid oligopeptide gliadinpeptide library (table S1) was screened with PBMCs from EnglishHLA-DQ2+ CD donors after either wheat (n = 13) or purified ryechallenge (n = 6) (see Materials and Methods and table S4).

Collectively, these unanticipated findings show that the specificitiesand relative importance of T cell responses generated in vivo dependon the cereal ingested. This unbiased analysis showed that it is anw-gliadin/C-hordein–derived sequence that is the most consistentlystimulatory gluten peptide after wheat, barley, and rye ingestion in vivo.

Distinct families of gluten peptides recognized by TCCsspecific for dominant peptidesTCCs were isolated from intestinal lamina propria mononuclear cells(LPMCs) or PBMCs cultured with deamidated gliadin or peptidesshowing distinct patterns of immunodominance after cerealGCs: wheata-gliadin (W02-E7), wheat w-gliadin/barley hordein (W03-E7), barleyC-hordein (B08-E2E7), and rye w-secalin (R11-E4E7). All 12 TCCswere HLA-DQ2–restricted and had T helper 1 (TH1) or TH0 cytokineprofiles (table S5). Epitopes recognized by TCCs were determined withlysine-substituted variants of the parent peptide. As expected, the DQ2-

a-I or DQ2-a-II epitopes were the core 9–amino acid sequences recog-nized by the five TCCs raised against the a-gliadin–derived peptideW02-E7. Two of the four TCCs raised against thew-gliadin/C-hordeinpeptideW03-E7 recognized the core 9–amino acid sequencePFPQPEQPF(herein named DQ2-w-I) and the other two recognized the overlap-ping 9–amino acid sequence PQPEQPFPW (hereinnamedDQ2-w-II).The singleTCCraised against the C-hordein peptide B08-E2E7 recog-nized the 9–amino acid sequence PIPEQPQPY (herein named DQ2-Hor-I), whereas the 9–amino acid core epitope for the solitary TCCraised to thew-secalin peptide R11-E4E7was not determined but namedhere as DQ2-Sec-I. When screened against the second-round and verifi-cation libraries, the TCC raised against deamidated gliadin was found tobe specific for the g-gliadin–derived peptideW11-E7 (9–amino acid coresequence not determined).

Next, we determined whether TCCs were highly specific or degen-erate in their recognition of the other immunostimulatory gluten pep-tides that we had previously identified with fresh polyclonal PBMCsfrom CD donors. TCCs were screened against 697 tTG-treated 16–amino acid oligopeptides in the second-round library and alsoagainst 3028 18–amino acid oligopeptides in a freshly synthesizedverification library encompassing all unique deamidated and wild-type10–amino acid sequences from gliadin, hordein, and secalin (table S1).TCCs were screened by IFN-g ELISpot with a concentration of peptideequivalent to that previously used to screen the first- and second-roundlibraries with PBMCs fromCDdonors. Therewas little cross-recognitionof the four dominant peptides (W02-E7, W03-E7, B08-E2E7, and R11-E4E7) by TCCs but substantial redundancy of peptide recognition formany of the subdominant gluten peptides (Tables 1 and 2 and tableS5). Remarkably, 11 clones specific for six epitopes (DQ2-a-I, DQ2-a-

Table 2. Grain specificity of select stimulatory sequences after in vivo wheat, barley, or rye challenge, and recognition by T cells in vitro.

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II, DQ2-w-I, DQ2-w-II, DQ2-Hor-I, andDQ2-Sec-I) recognized 22 of 32 gliadin,26 of 30 hordein, and 22 of 29 secalin se-quences that we had previously definedas T cell–stimulatory peptides for poly-clonal T cells from CD donors (Fig. 3, Aand B). TCCs specific for w-gliadin/C-hordein–derived W03-E7 were the mostcross-reactive and recognized 54 of the 91gliadin/hordein/secalin T cell–stimulatorypeptides. Among the 54 peptides recog-nized by TCCs specific for W03-E7 were18 peptides that are also recognized byTCCs specific for other dominant pep-tides (W02-E7, B08-E2,E7, or R11-E4E7).In contrast, there was almost no overlapbetween peptides recognized by TCCsspecific for W02-E7, B08-E2,E7, or R11-E4E7 (Fig. 3B). Peptides recognized byTCCs specific for W02-E7, B08-E2,E7, orR11-E4E7 were typically derived from thesame cereal as the cognate ligand; forexample, TCCs specific for DQ2-a-I orDQ2-a-II selectively recognized wheatgliadin-derived peptides (Tables 1 and 2).

Hence, 70 of 96 T cell–stimulatory glu-ten peptides identified from PBMCs fromHLA-DQ2 CD donors after oral GC wererecognized by TCCs raised against fourpeptides.

Defining the majorimmunodominant peptides in CDIf a few dominant immunostimulatorypeptides are the preferred ligands formostgluten-specific T cells in vivo, it would beexpected that a mixture of these peptideswould elicit a substantial proportion ofthe response to gluten and also that re-sponses to individual peptides would be ad-ditive if assessed with fresh polyclonal Tcells fromCDdonors after oral GC.We as-sessed IFN-g ELISpot responses tomixturesof up to 12 peptides by using PBMCs fromCD donors; the peptides included the fourdominant peptides from wheat, barley, andrye (W02-E7,W03-E7,B08-E2E7, andR11-E4E7). Other peptides were selected be-cause they were occasionally dominantand from diverse gluten fractions includ-ing wheat glutenins and gliadins, or oatavenins. Because T cells specific for severalpeptides were elicited only after wheat,barley, or rye GC, the oral GC was mod-ified to include equal amounts of wheat,barley, and rye. IFN-g ELISpot responseselicited by the equimolar mixture of thethree dominant peptides encompassing

Fig. 2. The hierarchy of immunodominance is dependent on the cereal grain ingested. (A to D) Fourimmunodominant stimulatory sequences were tested after wheat (n = 10), barley (n = 8), or rye (n = 6)challenge to examine grain specificity. SFUs were expressed as a percentage of the most active de-amidated prolamin peptide tested for each donor. The median is shown.

Fig. 3. Immunostimulatory peptides for PBMCs stimulated most TCCs, but TCC cross-reactivity with otherdominant peptides was restricted to clones specific for w-gliadin (W03). Recognition of confirmed stimula-tory sequencesby11TCCs raised toW02-E7 (n=5),W03-E7 (n=4), B08-E2E7 (n=1), andR11-E4E7 (n=1). (A) Invivo GC followed by IFN-g ELISpot characterized 96 immunogenic gluten sequences. TCCs were tested against91 sequences and found toproliferate or secrete IFN-g in response to70. (B) The relationbetween theTCCs andthe 70peptides they recognize is depicted. Five TCCs recognizingW02 reacted to a total of eight sequences. Ofthese, five of eight sequences were also recognized by TCCs raised to W03. The four TCCs specific for W03recognized a total of 54 sequences, of which 13 were also recognized by TCCs raised to B08 and/or R11. TheTCCs specific for B08andR11 responded to9and18 sequences, respectively,withonly 1 sequence common toboth.

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DQ2-a-I, DQ2-a-II, DQ2-w-I, DQ2-w-II, and DQ2-Hor-I epitopes[W02-E7,W03-E7, and B08-E2E7; cocktail 2 (C2)]were comparable to ex-panded cocktails of 6 or 12 peptides, butmore than twice themixture of thedominant a- and w-gliadin wheat peptides (W02-E7 and W03-E7) andeight times greater than the single peptide encompassing DQ2-a-I andDQ2-a-II (W02-E7) (Fig. 4A).When assessed with PBMCs collected fromCDdonors after singleGC, IFN-g ELISpot responses to C2 (50 mM)wereequivalent to as much as 90% of that elicited by optimal concentrationsof tTG-treated wheat gliadin, barley hordein, or themost immunogenicsecalin fraction (w-secalin) (320 mg/ml), respectively (Fig. 4, B to D).

Immunostimulatory sequences: Focused in a-gliadin butdistributed among other prolaminsThere has long been interest from the food industry in naturally non-toxic cereals and gluten proteins or in designing modified gluten pro-

teins devoid of toxicity inCD. Furthermore,it would be ideal for food tests to be able todetect each class of toxic protein in CD.Physical maps were constructed for pro-lamins in wheat, rye, and barley; thosethat contained the most immunogenicsequences identified in the second-roundscreening libraries are shown in Fig. 5.The a-gliadins, historically the focus of ef-forts to map the gluten peptides that aretoxic in CD, were unusual because theyhad a single, polymorphic region with po-tent T cell–stimulatory activity. Our find-ings are consistent with those of others(25): The a-gliadin 33–amino acid oligo-peptide, currently ascribed a central role inHLA-DQ2–associatedCD, is the longest ofa variety of polymorphicT cell–stimulatorysequences;many sequences but not all haveDQ2-a-I, some include DQ2-a-II, and afew such as the 33–amino acid oligopeptidealso includeDQ2-a-III in tandem repeats.TCCs specific for DQ2-a-I and DQ2-a-IIrarely recognized sequences in g- and w-gliadins anddidnot recognizepeptides fromother prolamin families in wheat, barley,or rye.Most other strongly immunogenicg-gliadin, w-gliadin, secalin, and hordeinpolypeptides contained groups of overlap-ping T cell–stimulatory sequences withinseveral discrete regions. T cell–stimulatoryregions in polypeptides from a-gliadin,g-gliadin,w-gliadin, B-hordein, C-hordein,75-kD secalin, andw-secalin generally in-cluded sequences recognized by TCCs spe-cific for DQ2-w-I and DQ2-w-II (Fig. 5).T cell–stimulatory sequences shown in Fig.5 localize to regions rich in glutamine andproline and are predicted by the ExPASypeptide cleavage algorithm to be resistantto peptidases (table S6).

DISCUSSION

Since Dicke first described gluten from wheat and gluten-related frac-tions from barley and rye as the toxic factor in CD (26, 27), the ultimategoal of many researchers’ efforts has been the definition of the toxiccomponents of gluten. The complexity of gluten and the difficulty indeveloping disease-relevant, high-throughput bioassays have preventedcomprehensive definition of gluten toxicity, now understood to belargely but not exclusively mediated by CD4+ T cells (28). Morethan 100 gluten peptides have been predicted in silico to be epitopeson the basis of their expected resistance to proteolysis, susceptibility todeamidation by tTG, and conforming to the HLA-DQ2–binding motif(11).

We have now comprehensively characterized the T cell response toall toxic prolamins in adults with CD by using peripheral blood collected

Fig. 4. A minimal three-peptide mixture can stimulate an optimal IFN-g ELISpot response. IFN-g ELISpotwas used to compare the frequencies of T cells from CD donors specific for individual dominant glutenpeptides (50 mg/ml) or mixtures of peptides (constituent peptides each 50 mg/ml) after combined chal-lenge with equal amounts of wheat, barley, and rye (n = 13) (A). Each donor’s responses to peptidesand mixtures of up to 12 peptides were normalized against their response to the 12-peptide mixture.Maximal T cell responses were observed to the mixture of three peptides (C2). (B to D) Next, IFN-gELISpot was used to compare frequencies of T cells from CD donors after wheat (n = 10) (B), barley (n = 5)(C), or rye GC (n = 6) (D) to the mixture of three peptides (C2) (50 mg/ml) to the respective deamidatedwheat gliadin, barley hordein, or rye w-secalin fraction (320 mg/ml). Responses to C2 were equivalent to asmuch as 90% of the response to prolamin. Peptides assessed were capped by anN-pyroglutamyl residue (pE)and C-amidated. The peptides in C1 were capped variants of a-gliadin W02-E7 (pELQPFPQPELPYPQPQ-NH2) and w-gliadin/C-hordein W03-E7 (pEQPFPQPEQPFPWQP-NH2); C2 was expanded to also includehordein B08-E2E7 (pEPEQPIPEQPQPYPQQ-NH2); C3 was further expanded to include secalin R11-E4E7(pEQPFPEQPEQIIPQQP-NH2), homolog of oat avenin–derived T cell–stimulatory peptide (pEYQPYPEQEQPILQQ-NH2), and g-gliadin W36 (pEYEVIRSLVLRTLPN-NH2); and C4 was expanded to 12 peptides with the ad-dition of HMW glutenin W21 (pEGQQGYYPISPQQSGQ-NH2), LMW T cell epitope reported previously(pEQPPFSEQEQPVLPQ-NH2), g-gliadin W11-E7 (pEQAFPQPEQTFPHQP-NH2), LMW glutenin W15-E8(pEGLERPWQEQPLPPQ-NH2), g-gliadin W17-E6E9 (pEPFPQPEQPELPFPQ-NH2), and a-gliadin W09-E7(pEPQPFLPELPYPQP-NH2).

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from donors (who were normally on strict long-term, gluten-free diet)after oral challenge with grain to screen all potential T cell–stimulatory12–amino acid sequences from wheat gliadins and glutenins, barleyhordeins, and rye secalins. Our algorithmic approach to designing com-prehensive peptide libraries of a size that is manageable in an optimizedhigh-throughput IFN-g ELISpot assay was a critical technical advance(23). However, progress in sequencing prolamin genes has meant thatthe number of proteins initially screened was less than the numberincluded in GenBank by the end of the study.Ultimately, wewere ableto use TCCs and distinct epitope hierarchies after wheat, barley, andrye challenge to define a discrete set of immunodominant, nonredun-dant peptides consistently capable of recapitulatingmuchof theT cell–stimulatory capacity of gluten.

Three key observations from our study reshape our understandingof gluten toxicity in CD. First, the contributionmade by T cells specificfor many nonredundant, immunodominant gluten peptides, includinga-gliadin peptides encompassingDQ2-a-I andDQ2-a-II, the C-hordeinpeptide encompassing DQ2-Hor-I, and the w-secalin peptide includ-ingDQ2-Sec-I, is critically determined by the grains consumed by a CD

donor. Therefore, mapping epitopes for T cells raised against prolaminsfrom a single cereal will provide an incomplete assessment of relevantT cell–stimulatory peptides.

Second, many gluten peptides stimulate T cells. We confirmed 96T cell–stimulatory peptides and found that 38 distinct peptides weredominant for at least one donor, but the T cell population activated bya single dominant peptide is capable of recognizing and responding toa large number of related gluten sequences. In vitro, we showed thatTCCs specific for four dominant peptides recognized 74% of all Tcell–stimulatory peptides and 68% of dominant peptides. The diversityof sequences cross-reactive with the most important T cell–stimulatorypeptides results partly from having epitopes overlapping within a single11–amino acid sequence and also because some T cells, especially thosespecific for thew-gliadin/C-hordein–derived sequence PFPQPEQPFPW,are highly redundant in their recognition of gluten peptides. Althoughdegenerate recognition by a single T cell receptor (TCR) could explainthe diversity of responses from T cells specific for a single dominant pep-tide, the close sequencehomologyof reactivepeptides indicates that epitopecross-reactivity is a more likely explanation.

Fig. 5. The distribution of immunodominant sequences is restricted ina-gliadins but not in g-gliadin, w-gliadin, hordein, or secalin. Shadedboxes indicate the location and T cell stimulation score (vertical height)of peptide sequences derived from selected immunogenic gluten pro-teins in wheat, barley, and rye (dotted line indicates signal sequence).The box color indicates peptide recognition by TCCs specific for dom-inant wheat a-gliadin epitopes (DQ2-a-I or DQ2-a-II) (red), wheat w-gliadin/barley hordein epitopes (DQ2-w-I or DQ2-w-II) (blue), B/C-hordein epi-

tope (DQ2-Hor-I) (yellow), rye w-secalin epitope (DQ2-Sec-I) (brown),and g-gliadin epitope contained in W11-E7 (green). Colored hatched boxesindicate sequences recognized by TCCs specific for more than one of thesepeptides. Black/white hatched boxes indicate sequences not recognizedby any of these clones. The number in brackets after GenBank accessionnumbers indicates the number of other polypeptides found in GenBankthat share similar sequences and possess the same T cell–stimulatory pep-tides in the same relative positions.

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Third, although a-gliadin–derived peptides encompassing DQ2-a-I/III and DQ2-a-II are immunodominant in wheat gluten after wheatchallenge, our comprehensive screen of prolamins from wheat, barley,and rye indicates that the w-gliadin/C-hordein–derived peptide encom-passing DQ2-w-I and DQ2-w-II is recognized by most T cells inducedby oral challenge with a mixture of wheat, barley, and rye. Hence, it is anw-gliadin/C-hordein–derived peptide that might be considered the ca-nonical dominant T cell–stimulatory peptide in HLA-DQ2–associatedCD. The immunogenicity of DQ2-w-I, in particular, may relate to its over-lapping and repeated presence throughout w-gliadin and through cor-responding homologous sequences in barley hordein and rye secalin(29), plus its unusual conformationally mobile secondary structure (30, 31).

Although thew-gliadin fraction ofwheat gluten is toxic inCD (32), itis not surprising that the importance of DQ2-w-I and DQ2-w-II in CDhas been overlooked. There has been a historical focus on wheat glutenand a-gliadins in CD immunotoxicity studies. The A-gliadin com-ponent of a-gliadin was the first fully sequenced wheat prolamin (33),and when we commenced our study, there was just one complete wheatw-gliadin sequence in GenBank compared to 60 for a-gliadins. Previousstudies have also used TCLs raised against wheat gluten and gliadinrather than against barley hordein or rye secalin. It might be speculatedthat TCLs against hordein or secalin would have led to the DQ2-a-I orDQ2-a-II epitopes being overlooked, whereas the importance of DQ2-w-I andDQ2-w-II, and also DQ2-Hor-I andDQ2-Sec-I epitopes wouldhave been appreciated earlier. Indeed, the historical emphasis on wheatgluten has arisen because wheat is themore frequently consumed cerealinmostWestern diets today, yet CD is as common in Scandinaviawhererelatively larger amounts of rye and barley are consumed.

Our findings with fresh polyclonal T cells from blood after individ-ual GCs and isolated clones support both DQ2-w-I and DQ2-a-I, andDQ2-w-II and DQ2-a-II, as four distinct epitopes despite their close struc-tural similarities. This further supports our recent findings that some in-testinal TCLs recognize the w-gliadin sequence encompassing DQ2-w-Iand DQ2-w-II but not DQ2-a-I or DQ2-a-II (25). These observationscontrast with those of Vader et al. (14), who considered DQ2-w-I a func-tional homolog of DQ2-a-I (namedHora9/Seca9) and, similarly, that thesequence PQPEPFPQ (named Hora2/Seca2), closely related to DQ2-w-II, was a functional homolog of DQ2-a-II. Vader et al. attributedthe toxicity of barley hordeins and rye secalins to the presence ofHora9/Seca9 and Hora2/Seca2 that were recognized by T cells spe-cific for DQ2-a-I and DQ2-a-II; our findings provide an alternate ex-planation for the toxicity of rye and barley.

Our findings generally support established principles of epitope selec-tion in HLA-DQ2–associated CD. For instance, the importance of pro-tease resistance in shaping the repertoire of immunogenic gluten peptideswas supported by our data, which showed that cleavage sites for trypsin,chymotrypsin, and pepsin are largely restricted to immunologically silentregions of the prolamin polypeptides. However, the explanation for thedominance of just a few peptides rather than the many others that sharesimilar chemical properties that predict epitope selection is unclear.Moredetailed studies will be needed to measure HLA-DQ2 binding, suscepti-bility to deamidation by tTG, and protease resistance.

The epitopes DQ2-a-I/III, DQ2-a-II, DQ2-g-IV, DQ2-g-VI, andDQ2-g-VII reported for TCCs were confirmed with PBMCs in theIFN-g ELISpot, but many others were not. This should not be inter-preted as meaning that previously defined epitopes such as DQ2-g-Iare not recognized by relevant T cells, but simply that such T cells arenot sufficiently frequent in blood or consistently recognized by CD do-

nors to reach the threshold applied in this study (~7% of the responseelicited after wheat challenge by peptides encompassing DQ2-a-I/IIIand DQ2-a-II). Furthermore, we assigned a hierarchy to peptides accord-ing to their recognition by polyclonal T cells; it was not practical to dis-tinguish between peptides containing two or more epitopes and thosewith a single epitope; hence, dominance was assigned to peptides ratherthan epitopes.

This study supports a central role in the immunopathogenesis of CDfor T cells specific for two overlapping epitopes found in w-gliadin.TCCs raised to these epitopes recognize sequences derived from virtu-ally all prolamin fractions in wheat, barley, and rye (we were unable toassess glutenins for cross-reactivity). In our hands, gluten peptide recog-nition by clones specific for dominant T cell–stimulatory sequencesderived from a-gliadin, C-hordein, or w-secalin was far less cross-reactivethan clones specific for w-gliadin. It will also be important to deter-mine whether deamidation of the w-gliadin peptide encompassingDQ2-w-I and DQ2-w-II epitopes enhances both the affinity of bindingto HLA-DQ2 and the diversity of cognate TCRs, suggested as a pivotalevent in the genesis of gluten immunity in CD (34).

Repeating this study in childrenwouldbe impractical, because the vol-ume of blood required to screen all potential gluten epitopes is too large,but it will be possible to test whether peptides immunodominant inadults also make a substantial contribution to the gluten-specific T cellresponse in childhoodCD. Such studies in childrenwould require themto follow a strict gluten-free diet and then undergo short-term GC.Whether the specificity of T cell responses to gluten begins focusedand then diversifies, or vice versa, is unlikely to be revealedwith oral GCin children with established CD because immune responses evolve overweeks rather than years. However, we have noted that the specificity ofgluten peptide-specific serum immunoglobulin G (IgG) is identical inadults with active CD and in children immediately after tTG IgA sero-conversion before they have diagnosed CD. Equally, our data arederived from volunteers with diagnosed CD, rather than from indi-viduals who might be seropositive for tTG IgA and have unrecognizedor asymptomatic CD; future studies will need to address each of theseclinically distinct disease phenotypes to confirm the consistency ofgluten-specific T cell responses.

Detailed understanding of the T cell–stimulatory sequences in CDwill facilitate food tests, design of functional foods to reduce the tox-icity of gluten, diagnostics to detect CD-specific T cells, and therapeu-tics to reduce exposure of T cells to toxic peptides. Because wholegluten is a complex mixture of aqueous insoluble proteins that stimulateboth innate and acquired immunity (35), it is unattractive for use in theprotein-based desensitization therapy that is effective in allergic diseases(36). Thus, the practical application that motivated us to undertakethis study was the possibility of a peptide-based immunotherapy.

Peptide-based immunotherapy with altered peptide ligands (APLs)has shown encouraging therapeutic results in preclinicalmodels with clo-nal T cell populations (37–39), but in clinical trials formultiple sclerosis, ithas proven ineffective and occasionally associated with a shift in T cellphenotype from TH1 to TH2 (40, 41). We previously explored the possi-bility ofAPLs to antagonize TH1 responses of polyclonal peripheral bloodT cells to the 17–amino acid a-gliadin oligopeptide encompassing DQ2-a-I and DQ2-a-II. However, this approach was only modestly effectiveex vivo (42). More recently, antigen-specific immunotherapy with esca-latingmicrogram doses of peptides predicted to be T cell epitopes (forexample, from allergens such as cat dander protein Fel-d-1 impli-cated in asthma), administered by repeated intradermal injection,

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has shown efficacy in phase II clinical trials (43). It appears that peptide-based immunotherapy of this type depends onpeptideuptake andpresen-tation by immature dendritic cells to promote antigen-specific regulatoryT cells and spreading of tolerance (44, 45).

Previously, the diversity and lack of consistency of T cell–stimulatorypeptides in gluten compromised the design of a peptide-based immu-notherapy for CD. Our study now allows the design of a potential im-munotherapywithpeptides confirmedas immunodominant in a commonhuman immune disease. The lead compound consists of three immu-nogenic gluten peptides, which are now in phase I clinical development.A critical step toward a peptide-based immunotherapy for CD will beto show that such a compound is bioactive and targets relevant T cellswhen administered to volunteers with HLA-DQ2–associated CD. Pro-vided these fundamental immunological properties and safety areestablished, such a compound promises to provide unique insights intothe therapeutic potential of peptides confirmed to be disease-specificT cell agonists.

MATERIALS AND METHODS

Subjects and controlsThe studywas approved by theOxfordshire Regional Ethics Committeeand by the Melbourne Health Human Research Ethics Committee.Together, 226 oral GCs (wheat, 113; barley, 41; rye, 43; and combined,29) were undertaken in CD volunteers [median age, 50 years; range, 19to 70 years; female, 165 (73%);HLA-DQB1*02 homozygous, 68 (30%)]and 10 wheat challenges in healthy volunteers [median age, 47 years;range, 22 to 64 years; female, 5 (50%); HLA-DQB1*02 homozygous, 3(30%)]. Nineteen CD volunteers were recruited in Oxford (UK) fromthe Coeliac Clinic at John Radcliffe Hospital, and the remainder wererecruited in Melbourne (Australia) by advertisement in the VictorianStateCoeliac Societynewsletter. Leukocyte-derivedDNAfromvolunteerswas genotyped with a panel of sequence-specific primers to determineHLA-DQA andHLA-DQB alleles (Victorian Transplantation Immunol-ogy Service, Parkville,Victoria,Australia) (46–48).CDandhealthy volun-teers were included if they had HLA-DQA1*05 and HLA-DQB1*02(encoding HLA-DQ2) but did not have either DQA1*03 or DQB1*0302(alleles encoding HLA-DQ8). CD volunteers had biopsy-proven CDconforming to the European Society of Paediatric Gastroenterologyand Nutrition diagnostic criteria (49). Healthy volunteers following anormal gluten-containing diet, and alsoCDvolunteers following a strictgluten-free diet, were confirmed to have normal levels of serum trans-glutaminase IgA (INOVA Diagnostics). At the time GC was undertaken,CD volunteers had been strictly gluten-free for 3 months, and healthyvolunteers for 4 weeks.

Grain challengeOral challenges extended over 3 days unless otherwise stated. Wheatchallenge consisted of two 50-g slices of gluten-containing wheatbread (Oxford: Sainsbury’s standard white bread; Melbourne: Baker’sDelight white bread block loaf cut to toasting size thickness) for break-fast, followed by two slices for lunch. Barley challenge consisted ofpearl barley (Ward McKenzie) cooked into a risotto (150 g dry weightdaily). Rye challenge involved rye (Oxford: manually sorted rye cultivarMotto and milled on an experimental mill at Long Ashton ResearchStation; Melbourne: Biodynamic Rye flour, Eden Valley BiodynamicFarm) baked into muffins (100 g dry weight rye flour daily). Partici-

pants undertaking the combined cereal challenge consumed two muf-fins consisting of 25 g of wheat flour (White Wings, Goodman FielderAustralia), 22 g of barley flour (Four Leaf Milling), and 22 g of rye flour(Four Leaf Milling) each day. Blood (50 to 300 ml) was drawn in themorning before (day 0) and/or 6 days after commencing GC (day 6),with the total volume collected on both days not exceeding 300 ml. Par-ticipants were allocated randomly to each challenge.

AntigensSynthetic peptides (screening grade and >70% purity) were purchasedfrom Research Genetics, Mimotopes, or Pepscan. Unless otherwise stated,peptide libraries were assessed at a concentration of 25 mg/ml. Hordeinand secalin fractions were prepared from rye and barley grown in iso-lation from other grains and hand-milled flour and fractionated ac-cording to published methods (50). Deamidation with guinea pig livertTG (Sigma) was as described previously (21, 22). Gliadin (Sigma) andother prolamins were incubated for 4 hours at 37°C in 10-fold excesswith chymotrypsin (Sigma) in ammonium bicarbonate (pH 8) and final-ly boiled for 15 min. Prolamin protein concentrations were determinedby bicinchoninic acid method (Pierce).

Peptide library designWheat gliadin sequences present in GenBank in 2001 were alignedby Lasergene MegAlign software (DNAStar) to allow visual selectionof 20–amino acid oligopeptides encompassing unique 12–amino acidoligopeptides (Pilot library). Subsequently, wheat, barley, and rye glutenpeptide libraries were designed with a customized algorithm, as pre-viously described (23), to entries for gliadins, glutenins, hordeins, andsecalins in National Center for Biotechnology Information GenBank intheir genome-encoded (wild-type) sequence (comprehensive library)or both wild-type and in silico transglutaminase-deamidated sequence(verification library) according to defined deamidation motifs (20, 51)(table S1).

Second-round wheat, rye, and barley libraries were designed byreducing selected 20–amino acid oligopeptides to nine overlapping12–amino acid oligopeptides. If any 12–amino acid sequence incor-porated glutamine at position 7 and it conformed to the deamidationmotif defined for tTG (QX1PX3, or QX1X2[F,Y,W,I,L,V], where X1 andX3 are not proline) (20, 51), then a 16–amino acid oligopeptide wasdesigned whereby the 12–amino acid sequence with glutamine at po-sition 7 was flanked by the native residues at positions !1 and 13 and byglycine at positions !2 and 14. This strategy allowed the central, poten-tially deamidated glutamine residue to be accommodated at anchorpositions 4, 6, or 7 in any potential 9–amino acid sequence HLA-DQ2peptide-binding sequence, consistent with the HLA-DQ2–binding motif(52, 53). If selected 20–amino acid oligopeptides did not include any12–amino acid sequences with glutamine at position 7, then two 16–amino acid oligopeptides overlapping 12 residues were synthesized. Somesecond-round 16–amino acid oligopeptides with a central glutamine res-idue susceptible to tTG-mediated deamidation were also synthesizedwith glutamine replaced by glutamate (in silico deamidation).

ELISpot assayPBMCs were isolated from heparinized whole blood with Ficoll-PaquePlus in 50-ml Leucosep tubes (Greiner Labortechnik). After beingwashed three times, PBMCs were resuspended in complete RPMI con-taining 10% heat-inactivated human AB serum. Overnight IFN-g ELISpotassays (Mabtech) using 96-well plates (MSIP-S45-10; Millipore) were

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performed by a modification to the manufacturer’s instructions (21, 22).Peptide libraries were assessed with a single well per peptide. Tetanustoxoid (CSL) (10 light-forming units per milliliter) and phytohemagglutinin(5 mg/ml) were used as positive control antigens. SFUs in individualwells were counted with an automated ELISPOT reader (AID ELISPOTReader System, AID Autoimmun Diagnostika GmbH). Our data show thatresponses in the IFN-g ELISpot assay with the 17–amino acid a-gliadinoligopeptide encompassing DQ2-a-I and DQ2-a-II is qualitatively re-producible with PBMCs in the same donor collected on days 6 and 7after commencing oral wheat GC, and also when the same donor is chal-lenged on two occasions between 6 and 12 months apart (22). Here,PBMCs of six donors challenged with wheat between 6 and 12 monthsapart to screen the first-round comprehensive library and subsequentsecond-round library showed that the 20–amino acid oligopeptides andthen 16–amino acid oligopeptides encompassing DQ2-a-I/II/III or DQ2-w-I/II were consistently immunodominant after each challenge in fiveof six subjects, whereas peptides encompassing the sequence W033remained subdominant (table S7).

Isolation and characterization of TCCsPBMCs were isolated as for the ELISpot assay. LPMCs were isolatedby treating small intestinal tissue biopsies with 1 mM dithiothreitolin phosphate-buffered saline (PBS), followed by two incubations at37°C for 30 min in Dispase II (2.4 U/ml) (Roche). Biopsies wereminced and incubated at 37°C for 1 hour in Liberase Blendzyme 3(2 U/ml) (Roche) and RPMI, washed three times in PBS, and mixedwith 1.5 ! 106 to 3 ! 106 autologous PBMCs irradiated at 20 gray(Gy). Cells were stained with 0.1 mM carboxyfluorescein diacetatesuccinimidyl ester and plated out at 2 ! 105 per well in 96-well plates.Peptide and protein antigens were used at 32 and 100 mg/ml, respec-tively. Antigen-specific CD4+ TCCs were isolated as previouslydescribed (54) and expanded with anti-CD3 as described (55). Antigenspecificity was determined by [3H]thymidine proliferation assay orIFN-g ELISpot with irradiated PBMCs (20 Gy) from HLA-DQ2+

HLA-DQ8! donors.Expanded antigen-specific clones were tested for clonality with the

IOTest Beta Mark (Beckman Coulter). Negative clones were confirmedas clonal by polymerase chain reaction of the TCR Vb chains. HLArestriction was determined with HLA-DR (10 mg/ml, clone L243) andHLA-DQ (10 mg/ml, clone SPVL3) antibodies. Secretion of IFN-g,interleukin-4 (IL-4), IL-5, IL-10, IL-13, and IL-17 by clones to cognateantigen was determined in ELISpot assays. Lysine scans of peptidesW02-E7, W03-E7, and B08-E2E7 were carried out in IFN-g ELISpotor proliferation assays with clones specific for these peptides.

Statistical analysisIFN-g ELISpot responses were considered significant and, unless other-wise stated, included for analysis when SFUs per well were greater thanfour times the response to medium alone and >10 SFUs per well. Over-all, PBMCs from 91 of 113 (81%) donors undergoing wheat challenge,30 of 41 (73%) undergoing barley challenge, 32 of 43 (74%) undergoingrye challenge, and 13 of 29 (45%) undergoing combined challengeyielded significant IFN-g ELISpot responses to at least one peptide. In-terim analysis of all donor ELISpot responses to first-round libraries(irrespective of donor’s maximal peptide responses) used a customizedexpectation maximization algorithm described earlier and also a sim-plified analysis to identify all 20–amino acid oligopeptides eliciting aresponse equivalent to at least 5% of the most active peptide in each

individual donor and in the group as a whole to enable design ofsecond-round libraries (23). A final simplified assessment used torank reactive peptides used a score between 0 and 100 equal to themean normalized response of donors who responded to at least onepeptide.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/2/41/41ra51/DC1Fig. S1. Fine mapping the T cell–stimulatory sequence in tTG-treated w-gliadin AAG17702(81–102)PQQPQQPQQPFPQPQQPFPWQP by IFN-g ELISpot with PBMCs from HLA-DQ2+ CD donors after3-day oral wheat challenge.Table S1. Gluten peptide libraries.Table S2. IFN-g ELISpot responses to peptides before and after 3-day wheat gluten challenge.Table S3. IFN-g ELISpot SFU/106 PBMC healthy HLA-DQ2 individuals on gluten-free diet 4 weeksbefore and after 3-day wheat gluten challenge.Table S4. Gliadin peptide hierarchy after 3-day wheat or rye challenge in English celiac donors.Table S5. TCC characterization.Table S6. Cleavage sites localize to nonstimulatory regions of prolamins.Table S7. IFN-g ELISpot responses in six celiac donors after consecutive wheat challenges.

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56. Acknowledgments: We thank all the volunteers who participated in this study; C. Pizzeyand the Coeliac Society of Victoria for the recruitment of volunteers; G. Tanner (Plant ScienceDivision, Commonwealth Scientific and Research Organization, Black Mountain, Canberra,Australia) for the gift of purified hordein; the Victorian Transplantation and ImmunogeneticsService for expert HLA typing of patients; and M. Stewart for the technical assistance inperforming ELISpot assays. Funding: J.A.T.-D. was supported by a National Health and Med-ical Research Council (NHMRC) Postgraduate Medical Scholarship and by a grant from theAustralian and New Zealand Coeliac Research Fund; T.B. was supported by grant PBF-S19T10of the German National Genome Research Network by the Federal Ministry of Educationand Research; D.A.v.H. was funded by a Wellcome Trust Clinician Scientist Fellowship(GR068094MA); S.I.M. is supported by the Juvenile Diabetes Research Foundation (10-2006-261); J.R. is supported by an Australian Research Council Federation Fellowship; andR.P.A. holds the Ian Mackay Fellowship from the Walter and Eliza Hall Institute and MelbourneHealth and also the Lions Cancer Council Fellowship. This work was supported by NHMRCProject grant 406656, Coeliac UK Project grant, the Graham Bird Memorial Fund (Oxford),the Oxford University College Challenge Seed Fund, BTG International plc, Nexpep Pty Ltd.,the NHMRC Independent Research Institutes Infrastructure Support Scheme grant 361646,and Victorian State Government Operational Infrastructure Support. Author contributions:J.A.T.-D., J.A.S., and J.A.D.: study design, data collection, analysis, and manuscript preparation;T.B.: study design and analysis; D.A.v.H.: data collection; A.T.: study design and data col-lection; K.H., S.I.M., and C.G.: data collection and analysis; D.P.J. and A.V.S.H.: study design;J.M. and J.R.: study design and manuscript preparation; R.P.A.: study design, data collection,

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analysis, and manuscript preparation. Competing interests: J.A.T.-D., J.A.S., J.A.D., T.B., D.A.v.H.,D.P.J., A.V.S.H., and R.P.A. are co-inventors of patents pertaining to the use of gluten peptidesin therapeutics, diagnostics, and nontoxic gluten. R.P.A. is a substantial shareholder and di-rector of Nexpep Pty Ltd. and Nexgrain Pty Ltd., companies developing peptide-basedtherapeutics and diagnostics and nontoxic gluten suitable for CD. R.P.A. is also the chiefexecutive of Nexpep Pty Ltd. J.A.T.-D. is a shareholder of Nexpep Pty Ltd. and Nexgrain PtyLtd. and a consultant to Nexpep Pty Ltd. J.A.S. and J.A.D. were former consultants to NexpepPty Ltd. D.A.v.H. and J.M. have consulted for and are shareholders in Nexpep Pty Ltd.Accession number: Clinical trial http://clinicaltrials.gov/ct2/show/NCT00879749.

Submitted 26 February 2010Accepted 2 July 2010Published 21 July 201010.1126/scitranslmed.3001012

Citation: J. A. Tye-Din, J. A. Stewart, J. A. Dromey, T. Beissbarth, D. A. van Heel, A. Tatham,K. Henderson, S. I. Mannering, C. Gianfrani, D. P. Jewell, A. V. S. Hill, J. McCluskey, J. Rossjohn,R. P. Anderson, Comprehensive, quantitative mapping of T cell epitopes in gluten in celiacdisease. Sci. Transl. Med. 2, 41ra51 (2010).

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