T cells in peripheral blood after gluten challenge in coeliac disease

Abstract

Background: Current understanding of T cell epitopes in coeliac disease (CD) largely derives from intestinal T cell clones in vitro. T cell clones allow identification of gluten peptides that stimulate T cells but do not quantify their contribution to the overall gluten specific T cell response in individuals with CD when exposed to gluten in vivo.

Aims: To determine the contribution of a putative dominant T cell epitope to the overall gliadin T cell response in HLA-DQ2 CD in vivo.

Patients: HLA-DQ2+ individuals with CD and healthy controls.

Methods: Subjects consumed 20 g of gluten daily for three days. Interferon gamma (IFN-gamma) ELISPOT was performed using peripheral blood mononuclear cells (PBMC) to enumerate and characterise peptide and gliadin specific T cells before and after gluten challenge.

Results: In 50/59 CD subjects, irrespective of homo- or heterozygosity for HLA-DQ2, IFN-gamma ELISPOT responses for an optimal concentration of A-gliadin 57-73 Q-E65 were between 10 and 1500 per million PBMC, equivalent to a median 51% of the response for a "near optimal" concentration of deamidated gliadin. Whole deamidated gliadin and gliadin epitope specific T cells induced in peripheral blood expressed an intestinal homing integrin (alpha4beta7) and were HLA-DQ2 restricted. Peripheral blood T cells specific for A-gliadin 57-73 Q-E65 are rare in untreated CD but can be predictably induced two weeks after gluten exclusion.

Conclusion: In vivo gluten challenge is a simple safe method that allows relevant T cells to be analysed and quantified in peripheral blood by ELISPOT, and should permit comprehensive high throughput mapping of gluten T cell epitopes in large numbers of individuals with CD.

Figure 1

Peripheral blood mononuclear cell (PBMC) interferon γ (IFN-γ) ELISPOT dose-response relationship for individual coeliac disease subjects to A-gliadin p57–73 QE65 (n = 8) (A) and chymotrypsin digested gliadin with or without deamidation by tissue transglutaminase (tTG), and tTG alone (n = 5) (B). Results are expressed as per cent of an individual’s maximal response to any concentration of p57–73 QE65 or tTG-gliadin, respectively.

Figure 2

Interferon γ (IFN-γ) ELISPOT responses of peripheral blood mononuclear cells (PBMC) to p57–73 QE65 (25 μg/ml) (n = 9), tissue transglutaminase (tTG) deamidated gliadin (100 μg/ml) (n = 6), and purified protein derivative of Mycobacterium bovis (PPD 5 μg/ml) (n = 8) (median marked) after depletion using anti-integrin-β7 or -α^E, or anti-CD4 immunomagnetic beads, in HLA-DQ2+ coeliac disease subjects on a long term gluten free diet, six days after commencing a three day gluten challenge (A). Results are expressed as per cent of CD8 depleted PBMC responses to antigens; not every subject responded to each antigen. IFN-γ ELISPOT responses of PBMC to p57–73 QE65 (50 μg/ml) (n = 8), tTG deamidated gliadin (100 μg/ml) (n = 8), and PPD (5 μg/ml) (n = 5) (median marked) following preincubation for one hour with anti-HLA-DR (L243) or anti-HLA-DQ (SPvL3) antibody (B). Results are expressed as per cent of PBMC responses to antigens without preincubation with antibody.

Figure 3

Interferon γ (IFN-γ) ELISPOT responses (spot forming units (SFU)/10⁶ peripheral blood mononuclear cells (PBMC), median marked) to p57–73 QE65 (25 μg/ml) (A), tissue transglutaminase (tTG) deamidated gliadin (100 μg/ml) (B), and purified protein derivative of Mycobacterium bovis (PPD 5 μg/ml) (C) in patients with a new diagnosis of coeliac disease before adopting a gluten free diet (GFD) (“None”) (n = 11), and immediately before (Day 0) and on day 6 after a three day gluten challenge performed after following a strict GFD for one (n = 4), two (n = 5), or eight (n = 7) weeks.

Figure 4

Interferon γ (IFN-γ) ELISPOT responses of peripheral blood mononuclear cells (PBMC) from individual HLA-DQ2+ healthy subjects after a gluten free diet (GFD) for four weeks (n = 9) (A) and coeliac disease (CD) subjects on a long term GFD (n = 12) (D) to tissue transglutaminase (tTG) deamidated gliadin (500 μg/ml) before and on day 6 and day 10 after commencing a three day gluten challenge. IFN-γ ELISPOT responses of the same healthy (B) and CD (E) subjects to gliadin (500 μg/ml) with or without tTG deamidation (mean (SEM)). Individual IFN-γ ELISPOT responses to the p57–73 QE65 homologue, AG01 E9 (20 μg/ml) (see table 1 ▶ for sequence), in healthy (C) and CD (F) subjects. Individual IFN-γ ELISPOT responses of all HLA-DQ2+ CD subjects on a long term GFD (n = 59) to medium alone or p57–73 QE65 (25 or 50 μg/ml), AG01 E9 (20 μg/ml), or AG02 E9 (100 μg/ml) (see table 3 ▶ for sequences) (G), and according to HLA-DQ genotype (H). IFN-γ ELISPOT responses to tTG deamidated gliadin (500 μg/ml) on day 6 after a three day gluten challenge correlated with p57–73 QE65 in 17 individual CD subjects (I). SFU, spot forming units.