Direct cloning and tetramer staining to measure the frequency of intestinal gluten‐reactive T cells in celiac disease

Abstract

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 +...

T Cells in Celiac Disease

“…Finally, gluten:HLA-DQ tetramers have been used to stain single cell suspension of gut biopsies. It was determined that gluten specific T cells are present in biopsies of all untreated CD patients at a frequency of 0.1–1.2% of CD4 T cells, and, the results were concordant with results obtained by direct cloning of T cells (60). …”

Healthy HLA-DQ2.5+ Subjects Lack Regulatory and Memory T Cells Specific for Immunodominant Gluten Epitopes of Celiac Disease

“…We also found circulating gluten tetramer-binding T EM in healthy individuals, albeit at frequencies that were 100-fold lower than in CD patients, and there was no significant increase in the expression of gut-homing markers among these cells (16). In another recent study, we demonstrated that MHC tetramers can be used to identify gluten-reactive CD4 + T cells in lamina propria lymphocytes (LPLs) of CD patients (17).…”

“…These tetramers were produced and labeled with either allophycocyanin or PE as described previously (18). We used the same Abs, frequency calculations, and gating strategies as previously described (16,17) to identify or sort cells of interest on a LSR II, FACSAria I, or FACSAria II (BD Biosciences). Vb6.7 + T cells were identified with FITC OT-145 (Endogen).…”

Determinants of Gliadin-Specific T Cell Selection in Celiac Disease

“…Consequently, the emergence and/or expansion of such T cells is likely to be a crucial step toward disease development and may also be linked to differences in clinical presentation. It will therefore be important to compare and contrast naive and memory gluten-specific TCR repertoires, the latter at various stages of the disease, although this may be challenging given that gluten-specific CD4 + T cells become less abundant in the duodenum with dietary treatment (30).…”