1. Volume 149, Issue 6, Pages 1541-1552.e2 (November 2015)
Consistency in Polyclonal T-cell Responses to Gluten Between Children and Adults With Celiac Disease 
Melinda Y. Hardy, Adam Girardin, Catherine Pizzey, Donald J. Cameron, Katherine A. Watson, Stefania Picascia, Renata Auricchio, Luigi Greco, Carmen Gianfrani, Nicole L. La Gruta, Robert P. Anderson, Jason A. Tye-Din 
Gastroenterology 
Volume 149, Issue 6, Pages e2 (November 2015)
DOI: /j.gastro
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2. Figure 1
Wheat-derived T-cell epitope hierarchy in pediatric celiac disease. Numbers are raw spot-forming units (SFU) from IFN-γ ELISpot assays performed on peripheral blood mononuclear cells (PBMC) from wheat-challenged celiac disease (CD) patients. Color coding represents responses above cutoff as a percentage of maximal peptide SFU (red, >70%; orange, 41–70%; yellow, 21–40%; green, 11–20%; and blue, 6–10%). Assay cutoffs are shown in purple, negative protein results in gray, and protein responses greater then peptide responses in yellow. Only peptides that reached >70% in at least 1 individual are shown. H, HLA-DQ2.5 homozygous individuals. Defined or predicted peptide sequence cores are in bold; ND, not done.
Gastroenterology  , e2DOI: ( /j.gastro )
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3. Figure 2
Wheat challenge induces gluten-specific T-cell responses in children with CD. Pediatric CD volunteers undertook 3-day oral wheat challenge, and T-cell responses to wheat-derived proteins and peptides were assessed by IFN-γ ELISpot. (A) Responses to deamidated gliadin, peptide W02 containing DQ2.5-glia-α1a/α2, and peptide W03 containing DQ2.5-glia-ω1/ω2 prior to (D0) and after (D6) wheat challenge (background subtracted) are shown. Gluten-specific responses were observed on D6 only and were significantly higher than D0 responses (P < .05, Kruskal-Wallis), whereas no differences were seen for tetanus toxoid. (B) Peptides W02 and W03 were tested in native and deamidated forms. Deamidation enhanced the response to peptide in all ages (n = 4, 3–5 years of age; n = 7, 6–10 years of age; n = 8, 11–18 years of age), some statistically significant (P < .05, Kruskal-Wallis). (C) Polyclonal responses to peptides described by Vader et al15 as immunogenic to TCLs isolated from children. Line depicts response cutoff. (D) IFN-γ ELISpot responses of n = 9 Italian pediatric CD volunteers to deamidated gliadin, W02, and W03 (background subtracted).
Gastroenterology  , e2DOI: ( /j.gastro )
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4. Figure 3
Effects of age, HLA-DQ2.5 zygosity, and time since diagnosis on gluten peptide T-cell responses. Peptides W02 and W03 were assessed in a dose-ranging study in pediatric and adult CD volunteers following oral wheat challenge. EC50 values were calculated and compared: (A) between age groups, (B) between HLA-DQ2.5 homozygous (n = 8) and heterozygous individuals (n = 10–13), and (E) between volunteers with diagnoses less than 2 years prior to gluten challenge (n = 8) or more than 2 years (n = 6–8). (C) ELISpot response magnitude is shown in homozygous (n = 7) and heterozygous (n = 22) individuals. (D) ELISpot response magnitude was categorized by age (n = 7, 3–5 years of age; n = 10, 6–10 years of age; and n = 10, 11–18 years of age). Median and interquartile ranges are shown (*P < .05, Kruskal-Wallis or Mann-Whitney U test).
Gastroenterology  , e2DOI: ( /j.gastro )
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5. Figure 4
Cross-reactive T-cell responses in children with celiac disease after wheat challenge. Numbers are raw spot-forming units (SFU) from IFN-γ ELISpot assays performed on peripheral blood mononuclear cells (PBMC) from wheat-challenged CD patients. Color coding represents significant responses as percentages of maximal peptide SFU (red, >70%; orange, 41–70%; yellow, 21–40%; green, 11–20%; and blue, 6–10%). Defined or predicted peptide sequence cores are in bold.
Gastroenterology  , e2DOI: ( /j.gastro )
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6. Figure 5
T-cell clone promiscuity. T-cell clones specific to DQ2.5-glia-α1a/α2 or DQ2.5-glia-ω1/ω2 were tested against wheat, barley, and rye peptide libraries by IFN-γ ELISpots. Positive responses are shaded as follows: wheat, blue; barley, orange; and rye, green. Peptide sequences are not shown. Nonreactive peptides were removed.
Gastroenterology  , e2DOI: ( /j.gastro )
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7. Figure 6
Similar TCR bias occurred in gluten-specific T cells from children and adults with CD. (A) Single IFN-γ–producing DQ2.5-glia-α1a/α2-specific CD4+ T cells were isolated by fluorescence-activated cell sorting for multiplex PCR. Sequencing analysis of the TCR Vα (TRAV) and Vβ chains (TRBV) expressed by this population was performed in 3 wheat-challenged volunteers with CD (subjects C14, C27, and A1). The compositions of the TRAV and TRBV usage are shown in separate pie charts, with individual chains represented by different colors. The most commonly used chains are listed within the pie charts. (B) Simpson’s diversity index for Vα and Vβ CDR3 sequences were calculated for the same individuals as in A.
Gastroenterology  , e2DOI: ( /j.gastro )
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8. Supplementary Figure 1
Poor response to tetanus toxoid predicts lack of response to gluten peptides. Volunteers were separated into responders (n = 31) or non-responders (n = 9) based on the IFN-γ ELISpot response to gluten after oral wheat challenge. Responses to the positive control antigen tetanus toxoid were compared on D6 following wheat challenge. Line depicts response cut-off. Median response with interquartile range is shown.
Gastroenterology  , e2DOI: ( /j.gastro )
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