1. T-Cell Response to Gluten in Patients With HLA-DQ2
T-Cell Response to Gluten in Patients With HLA-DQ2.2 Reveals Requirement of Peptide-MHC Stability in Celiac Disease 
Michael Bodd, Chu–Young Kim, Knut E.A. Lundin, Ludvig M. Sollid 
Gastroenterology 
Volume 142, Issue 3, Pages (March 2012)
DOI: /j.gastro
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2. Figure 1
Identification of the DQ2.2-glut-L-1 epitope recognized by TCC 627.A from a DQ2.2 patient. (A) T-cell proliferation of TCC 627.A obtained from a DQ2.2 patient to synthetic peptides representing commonly recognized DQ2.5-restricted epitopes (left panel), chymotrypsin-digested gluten treated with TG2 (Gluten TG2), or medium. The right panel shows proliferation by stimulation with gluten TG2 in the presence of blocking antibodies against HLA-DQ and HLA-DR. (B) Proliferation of the TCCs against native (open symbols) and TG2-treated (filled symbols) native DQ2.2-glut-L-1 peptide (QQPPFSQQQQPVLPQ) and DQ2.2-glut-L-1 analogues (QQQQPPFSQQQQSPFSQQQQ and QQPPFSQQQQQPLPQ). Predicted binding cores of 9 residues are underlined. In all assays, irradiated DQ2.2 EBV-transformed B cells were used as antigen-presenting cells. Proliferation was assessed as [3H]thymidine incorporation, measured in counts per minute (CPM) (mean of triplicates; error bars represent SEM).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

3. Figure 2
Comparing the specificity of the antigluten T-cell response in DQ2.2 and DQ2.5 patients with celiac disease. Proliferation of intestinal gluten-reactive TCLs stimulated with irradiated EBV-transformed B cells, gluten peptides, or chymotrypsin-digested gluten either untreated (Gluten) or treated with TG2 (Gluten TG2). Native DQ2.2-glut-L-1 epitope represented by a peptide with the sequence QQPPFSQQQQPVLPQ was treated with TG2. The left panel shows one representative gluten-reactive TCL from each of 4 DQ2.2+ (DQ2.5−) patients, and the right panel shows testing of 8 representative TCLs from a total of 17 TCLs tested, each from different DQ2.5+ (DQ2.2−) patients. For TCLs from DQ2.5+ (DQ2.2−) patients, DQ2.5 EBV-transformed B cells were used as antigen-presenting cells, whereas for TCLs from DQ2.2+ (DQ2.5−) patients, DQ2.2 EBV-transformed B cells were used. Proliferation was assessed as [3H]thymidine incorporation, measured in counts per minute (CPM) (mean of triplicates; error bars represent SEM).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

4. Figure 3
The DQ2.2-glut-L-1 epitope shows sustained binding to DQ2.2. (A) Thrombin-treated water-soluble recombinant DQ2.2 molecules were loaded with fluorescein isothiocyanate–labeled DQ2.2-glut-L-1 peptide (FITC-miniPEG-QPPFSEQEQPVLP) for 3 days. Dissociation of labeled DQ2.2-glut-L-1 peptide in the presence of unlabeled competitive high-affinity binder was measured. Experiments were performed in the presence or absence of HLA-DM as indicated. (B) Proliferation responses of 2 TCCs stimulated with irradiated DQ2.2 or DQ2.5 EBV-transformed B cells loaded for 2 hours with peptide. The antigen-presenting cells were washed and incubated for various time points followed by addition of T cells. TCC was stimulated with a peptide harboring the DQ2.2-glut-L-1 epitope (Ac-QQPPFSEQEQPVLPQ), and TCC was stimulated with a peptide harboring the DQ2.5-glia-α-2 epitope (PQPELPYPQPQL). Proliferation was assessed by [3H]thymidine incorporation, measured in counts per minute (CPM) (mean of duplicates). Panel B is representative of 2 separate experiments, and 2 different DQ2.2-glut-L-1 –restricted TCCs were tested in each experiment. All peptides in panel B were used at 10 μM, except for DQ2.5 with DQ2.2-glut-L-1 (500 μM). Error bars represent SEM.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Binding of peptides to DQ2.5 and DQ2.2 molecules. Peptide binding of peptides harboring the DQ2.2-glut-L-1 epitope (Ac-QQPPFSEQEQPVLPQ) and P3 substitutes of this epitope was measured in a competitive peptide binding assay using the EBV-transformed B-cell lines #9023 and #9050 as the source of HLA-DQ2.5 and HLA-DQ2.2 molecules, respectively. Median inhibitory concentrations were established by measuring the inhibitory effect of binding of the competitor peptide on the binding of an indicator peptide. Three independent 4-fold titration experiments were performed for each molecule. The results shown are from one representative experiment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

6. Figure 5
Serine at P3 is unfavored in the presence of a tyrosine at α-22 of DQ2. Thrombin-treated water-soluble DQ2.5, DQ2.2, DQ2.2 Fα22Y, or DQ2.5 Yα22F molecules were loaded with fluorescein isothiocyanate–labeled peptides for 3 days in the presence of HLA-DM. The left panel shows dissociation of labeled DQ2.2-glut-L-1 peptide in the presence of unlabeled competitive high-affinity binder and HLA-DM. The right panel shows fluorescence intensity in arbitrary units (AU) after loading of fluorescently labeled DQ2.2-glut-L-1 peptide and the P3 alanine and the P3 glycine substitutes. Measurement of off-rate of the DQ2.2-glut-L-1 peptide on DQ2.2 is from the same experiment as in Figure 3. The results are from one experiment.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions

7. Figure 6
Visualization of peptide-MHC interaction around the P3 residue. (A) X-ray crystal structure of the DQ2.5:DQ2.5-glia-α-1a (PFPQPELPY) complex. (B) Model of the DQ2.2:DQ2.2-glut-L-1 (PFSEQEQPV) complex. HLA is represented with Corey–Pauling–Koltun spheres. Residue α22 and α24 are colored according to atom type (C, orange; N, blue; O, red), and surrounding residues are colored entirely in gray. The bound peptide is shown as a stick model (C, yellow; N, blue; O, red). For each complex, the potential hydrogen-bond interaction is denoted as blue dashes in the inset.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2012 AGA Institute Terms and Conditions