We have analyzed the production of the effector cytokines interleukin (IL)-17, IL-21, and IL-22 in gluten-reactive CD4(+) T cells of celiac disease patients, either cultured from small intestinal biopsies or isolated from peripheral blood after an oral gluten challenge. Combining intracellular cytokine staining with DQ2-α-II gliadin peptide tetramer staining of intestinal polyclonal T-cell lines, we found that gluten-specific T cells produced interferon-γ (IFN-γ) and IL-21, but not IL-17 or IL-22, even if other T cells of the same lines produced these cytokines. Similarly, in DQ2-α-II-specific T cells in peripheral blood of gluten-challenged patients, very few stained for intracellular IL-17, whereas many cells stained for IFN-γ. We conclude that gluten-reactive T cells produce IL-21 and IFN-γ, but not IL-17. Their production of IL-21 suggests a role for this cytokine in the pathogenesis of celiac disease.

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Knowledge of the frequency of disease-driving CD4 + T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten-reactive CD4+ T cells, which recognize gluten peptides only in the context of the disease-associated HLA-DQ molecules, are key pathogenic players. By cloning CD4 + T cells directly from intestinal biopsies of CD patients, we found that 0.5-1.8% of CD4 + T cells were gluten reactive. About half of the gluten-reactive T cells were specific for either the immunodominant DQ2.5-glia-α1a or DQ2.5-glia-α2 epitopes, suggesting that direct visualization of gluten-specific T cells could be possible. Assessed by flow cytometry, tetramer-positive T cells were present in 10/10 untreated CD patients with a frequency of 0.1-1.2% of CD4 + T cells. Gluten-specific T cells were also detectable in most treated CD patients (7/10). Moreover, the frequency of gluten-specific T cells correlated with the degree of histological damage in the gut mucosa as scored by Marsh-grading, and also with serum IgA anti-transglutaminase 2 antibody levels. Tetramer staining of gluten-reactive T cells in biopsy material is a useful tool for future studies of such cells in CD and could also potentially serve as a diagnostic supplement in selected cases. Keywords: Celiac disease r Frequency r Gluten-specific T cells r TetramerAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCeliac disease (CD) is a chronic inflammatory disease of the small intestine triggered by gluten proteins from wheat, barley, and ryeCorrespondence: Dr. Melinda Ráki e-mail: melinda.raki@rr-research.no [1]. The only available treatment for CD is a lifelong gluten-free diet. CD is a multifactorial, polygenic disease, and the HLA locus is by far the single most important genetic risk factor [2]. The great majority of CD patients express the HLA class II molecule * These authors contributed equally to this work. Many of these T cells recognize either the α gliadin epitope DQ2.5-gliaα1a or DQ2.5-glia-α2 [9,10]. Importantly, deamidation of specific glutamine residues to glutamate by the enzyme transglutaminase 2 (TG2) increases the T-cell stimulatory capacity of most gluten epitopes [10][11][12]. CD provides a unique opportunity to study CD4 + T cells recognizing the disease-driving antigen (gluten) at the site of inflammation. The development of MHC-peptide multimers has greatly facilitated the detection of antigen-specific T cells [13]. Glutenspecific T cells can be visualized in the peripheral blood of CD patients undergoing a short gluten challenge by using DQ2.5-glia-α1a and DQ2.5-glia-α2 tetramers [14]. However, isolation of gluten-reactive T cells from the intestinal lesion still requires long term in vitro culturing. It is becoming increasingly evident that the phenotype of CD4 + T cells is plastic [15]. Studying freshly isolated T cells will therefore be important to accurately characterize these T cells.The frequency of antigen-specific CD4 +...

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Even though MHC class II is a dominant susceptibility factor for many diseases, culprit T cell epitopes presented by disease-associated MHC molecules remain largely elusive. T cells of celiac disease lesions recognize cereal gluten epitopes presented by the disease-associated HLA molecules DQ2.5, DQ2.2, or DQ8. Employing celiac disease and complex gluten Ag digests as a model, we tested the feasibility of using DQ2.5 and DQ2.2 as an affinity matrix for identification of disease-relevant T cell epitopes. Known gluten T cell epitope peptides were enriched by DQ2.5, whereas a different set of peptides was enriched by DQ2.2. Of 86 DQ2.2-enriched peptides, four core sequences dominated. One of these core sequences is a previously known epitope and two others are novel epitopes. The study provides insight into the selection of gluten epitopes by DQ2.2. Furthermore, the approach presented is relevant for epitope identification in other MHC class II–associated disorders.