Volume 122, Issue 7, Pages 1729-1737 (June 2002) The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides  Willemijn Vader, Yvonne Kooy, Peter van Veelen, Arnoud de Ru, Diana Harris, Willemien Benckhuijsen, Salvador Peña, Luisa Mearin, Jan Wouter Drijfhout, Frits Koning  Gastroenterology  Volume 122, Issue 7, Pages 1729-1737 (June 2002) DOI: 10.1053/gast.2002.33606 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 GLU-specific responses of T-cell lines from pediatric CD patients. GLU recognition by small intestinal T-cell lines generated after initial GLU challenge with (A) GLU and (B) with tTG-GLU. The T-cell lines were tested against the GLU (hatched symbols) and tTG-GLU (closed symbols) preparations. (B) Three T-cell lines isolated from small intestinal biopsies of HLA-DQ2+ controls are shown. Responses were considered positive when the GLU-specific stimulation was 3 times above background: SI ≥ 3. The T-cell lines that were used for cloning are underlined. *Tested against GLU only. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Identification of the GLIA epitope GLIA-α20. The TCC JB20 responded to 5 of 50 peptide-pools, each containing 5 tTG treated GLIA and/or GLT peptides. A single sequence was present in the stimulatory pools but not in nonstimulatory pools, e.g., pool 32. A newly synthesized version of this peptide, termed GLIA-α20, was recognized by the TCC. Cpm represents incorporated 3H-thymidine values (the negative control was 312 ± 324 cpm). Some peptides were synthesized with a mixture of 2 amino acids at particular amino acid position. This is indicated as follows: U= P + L; X= P + Q; Y= P + S; Z= P + R. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Mass spectral analysis of deamidation of the GLIA-γ30 epitope. (A) Predicted ion fragment masses of the GLIA-γ30 epitope based on amino acid sequence. (B) Observed fragments of the GLIA-γ30 epitope, b-ions are indicated according to panel A. (C) Observed fragments of the GLIA-γ30 epitope after deamidation by tTG. The mass difference between the b-ions 228 and 413 in panel B corresponds to the sequence GQ. This mass difference has increased 1 dalton in panel C (b-ions 228 and 414), which corresponds to the sequence GE and indicates a Q to E conversion at position 4. Similarly, the Q at position 10 is converted into an E by tTG treatment as indicated by the 2 dalton shift of the b10-ion from 1049 to 1051. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Recognition of the novel DQ2 epitopes. The effect of deamidation on recognition of the new GLU epitopes by TCCs shows 3 major patterns. tTG treatment is required for T-cell recognition of the GLU epitopes GLT-17(46-60) by TCC NV17, GLT-56(40-59) by TCC MS156, and GLIA-α20(93-106) by TCC JB20. Equal or enhanced responses after specific deamidation by tTG are found for epitopes GLIA-γ30(222-236) (TCC SV30), and GLU-5 (TCC JP437 and NP27). In the third pattern, the T-cell reaction against the GLU-21 epitope by TCC SV21 is blocked by deamidation of the peptide. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Responses of 2 TCCs against homologue peptides. Response of TCCs TCC NV17 (hatched symbols) and TCC MS156 (closed symbols) to GLT/GLIA homologue peptides. TCC NV17 responds to all peptides except peptide 15, whereas TCC MS156 does not recognize peptides 12, 13, and 15. All peptides are GLT-derived sequences, whereas peptides 11 and 14 also match with GLIA sequences. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Overview of the T-cell responses against the known DQ2 epitopes. TCCs and lines of young and adult patients were tested against the DQ2 epitopes characterized in the present study and the previously published epitopes Glia-α2(62-75) PQPQLPYPQPQLPY, Glia-α9(57-68) QLQPFPQPQLPY, and Glia-γ1 (138-153) QPQQPQQSFPQQQRPF.13,15 The black boxes represent stimulation of the T cells by the epitope. The effect of deamidation of the epitope on the T-cell response is indicated in the individual boxes. tTG indicates requirement of deamidation for recognition, and no tTG indicates blocking of the response by deamidation of the peptide. The remaining responses are (largely) indifferent to deamidation. Gastroenterology 2002 122, 1729-1737DOI: (10.1053/gast.2002.33606) Copyright © 2002 American Gastroenterological Association Terms and Conditions