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Shapes TCR Usage in Celiac Disease Posttranslational Modification of Gluten Posttranslational Modification of Gluten Shapes TCR Usage in Celiac Disease Shuo-Wang Qiao, Melinda Ráki, Kristin S. Gunnarsen, Geir-Åge Løset, Knut E. A. Lundin, Inger Sandlie and This information is current as Ludvig M. Sollid of September 29, 2021. J Immunol 2011; 187:3064-3071; Prepublished online 17 August 2011; doi: 10.4049/jimmunol.1101526 http://www.jimmunol.org/content/187/6/3064 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/08/18/jimmunol.110152 Material 6.DC1 http://www.jimmunol.org/ References This article cites 31 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/187/6/3064.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Posttranslational Modification of Gluten Shapes TCR Usage in Celiac Disease Shuo-Wang Qiao,* Melinda Ra´ki,†,1 Kristin S. Gunnarsen,‡,1 Geir-A˚ ge Løset,‡ Knut E.A. Lundin,†,x Inger Sandlie,‡ and Ludvig M. Sollid*,† Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced en- teropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational con- version of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide–MHC complex. In this study, we report that there is a biased usage of TCR Vb6.7 chain among TCRs reactive to the immunodominant DQ2-a-II gliadin epitope. We isolated Vb6.7 and DQ2-aII tetramer-positive CD4+ T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semi- Downloaded from public response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3b loop. Functional testing of a prototype DQ2-a-II–reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-a-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags. The Journal of Immu- nology, 2011, 187: 3064–3071. http://www.jimmunol.org/ osttranslational modification of Ag is a key element of the T cells derived from celiac lesions that recognize the immu- pathogenesis of celiac disease, and posttranslational mod- nodominant DQ2-restricted gliadin DQ2-a-II epitope respond to P ifications of Ags have been similarly implicated in several deamidated gliadin peptides at concentrations that are at least 100- other autoimmune diseases (1–5). Celiac disease is a human in- fold less than concentrations required for similar T cell responses flammatory condition of the small intestine precipitated by the elicited by corresponding native peptides (9). This improved T ingestion of cereal gluten proteins in wheat that consist of glia- cell recognition has been attributed to increased binding affinity din and glutenin subcomponents. The disease has a very strong of deamidated peptides to HLA-DQ2. Indeed, deamidation of the by guest on September 29, 2021 HLA association, and T cells of celiac lesions recognize gluten DQ2-a-II peptide introduces a negative charge in the residue re- epitopes presented by disease-associated HLA-DQ2 (DQA1*05:01/ siding in pocket 4 of the HLA-DQ2 peptide binding groove, DQB1*02:01) or HLA-DQ8 (DQA1*03/DQB1*03:02) molecules a pocket where negatively charged residues are favored (16, 17). (6, 7). These gluten-reactive CD4+ T cells appear to be essential for Consequently, the binding affinity of the DQ2-a-II peptide to the development of the disease, and they can be readily cultured HLA-DQ2 increases by up to 10-fold after deamidation (9). from intestinal lesions of diseased patients but not control subjects However, this is clearly less than the increase in T cell responses (6, 8). Over the years, a number of gluten T cell epitopes, each elicited by deamidation, suggesting that TCR recognition may recognized by distinct T cell clones derived from celiac disease also be involved in the improved recognition of the deamidated patients, have been identified (9–14). For most of these epitopes, gluten peptides. posttranslational conversion of Gln residues to Glu by the enzyme In the current study, we found a biased usage of TCR Vb6.7 chain transglutaminase 2 is required for efficient T cell recognition (15). encoded by the TRBV7-2 gene segment among TCRs reactive to the DQ2-a-II gliadin epitope. Based on this knowledge, we per- *Department of Immunology, Centre for Immune Regulation, University of Oslo, formed single-cell TCR cloning of a large number of Vb6.7-posi- 0027 Oslo, Norway; †Department of Immunology, Centre for Immune Regulation, tive, DQ2-aII tetramer-positive CD4+ T cells sorted from periph- ‡ Oslo University Hospital, Rikshospitalet, 0027 Oslo, Norway; Department of Mo- eral blood of gluten-challenged celiac patients. Analysis of the lecular Biosciences, Centre for Immune Regulation, University of Oslo, 0316 Oslo, Norway; and xDepartment of Medicine, Oslo University Hospital, Rikshospitalet, TCR sequences revealed in vivo clonal expansion, convergent re- 0027 Oslo, Norway combination, semipublic response, and the notable conservation of 1M.R. and K.S.G. contributed equally to this work. a non-germline-encoded Arg residue in the CDR3b loop. Se- Received for publication May 25, 2011. Accepted for publication July 7, 2011. quencing and functional data support the notion that the deamidated This work was supported by the Research Council of Norway and the South-Eastern residue in the DQ2-a-II peptide poses constraints on the TCR Norway Regional Health Authority. structure in which the conserved Arg residue is a critical element. Address correspondence and reprint requests to Dr. Shuo-Wang Qiao, Department of Immunology, Centre for Immune Regulation, Rikshospitalet, 20 Sognsvannsveien, Materials and Methods N-0027 Oslo, Norway. E-mail address: [email protected] T cell culture, tetramer staining, and FACS sorting The online version of this article contains supplemental material. Abbreviations used in this article: IMGT, international ImMunoGeneTics information Biological material was obtained from celiac disease patients according to system; scTCR, single-chain TCR; RU, resonance unit; SPR, surface plasmon reso- protocols approved by the regional ethics committee, and subjects donating nance; TCC, T cell clone. material gave written informed consent. Polyclonal T cell lines and T cell clones (TCCs) were established from intestinal biopsies of celiac disease Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 patients as previously described (18). T cells were expanded with anti- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101526 The Journal of Immunology 3065 CD3/anti-CD28 beads (Invitrogen) for at least 7 d before mRNA isolation cultured in RPMI 1640 plus 10% FCS. We reached .90% transduction and TCR cloning. efficiency, which was maintained throughout the experiments. Tetramerized recombinant HLA-DQ2 tethered with gluten-derived In Ag stimulation assays, 50,000 DQ2.2 (IHW09050) or DQ2.5 (patient peptides containing the T cell epitopes DQ2-a-I (QLQPFPQPELPY, CD114) homozygous EBV-transformed cells were incubated with native underlined 9mer core sequence), DQ2-a-II (PQPELPYPQPE), and the or deamidated gliadin 33mer peptides containing the DQ2-a-II epitope control peptide CLIP2 (MATPLLMQALPMGAL) were generated as (33merQ, LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF; 33merE, previously described (19, 20). Likewise, the DQ2-g-II tetramer was gen- LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF) and native or deam- erated with the same protocol where the DQ2-g-II peptide QGIIQPEQ- idated 14-mer DQ2-a-II peptides (aII.Q-PQPQLPYPQPQLPY; aII.E- PAQL was covalently coupled to HLA-DQ2. Circulating tetramer-positive PQPELPYPQPELPY) at 37˚C for 1–4 h before the addition of 25,000 T cells in peripheral blood of celiac patients 6 d after a 3-d gluten chal- TCR-transduced hybridoma cells. In some experiments, 10 mg/ml anti- lenge were stained according to a previously established protocol (21). HLA class II blocking Abs SPV-L3 (anti-DQ), B7/21 (anti-DP), or Single, Vb6.7-positive (FITC–OT145 from Endogen), DQ2-aII tetramer- B8.11 (anti-DR) was added. In anti-CD3 stimulation assays, wells were positive cells were sorted by a FACSAria sorter (BD) into 96-well PCR coated overnight with 0.2 to 5 mg/ml hamster anti-mouse CD3ε Ab (clone plates (Bio-Rad) with 10 ml ice-cold lysis-reverse-transcription buffer 145.2C11, a gift from B. Bogen, Oslo, Norway) and washed before the containing 50 mM Tris-HCl pH 8.2, 75 mM KCl, 3 mM MgCl2, 0.5% addition of 10,000 TCR-transduced hybridoma cells. In Troybody ex- Nonidet P-40, 2 mM deoxyribonucleotide triphosphate 10 mM DTT, periments, recombinant anti–HLA-DP Abs in which 33merQ or 33merE 800 nM TRBC_rev primer 59-TTCACCCACCAGCTCAGCTCC-39, 800 peptides were inserted into loops between b-strands in constant domains nM TRAC_rev primer 59-AGTCAGATTTGTTGCTCCAGGCC-39,12U (25) were used as Ags, and 10,000 DQ2+ monocyte-derived dendritic cells RNasin (Promega), and 30 U SuperScript II reverse transcriptase (Invi- were used as APCs.
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