No abstract

Tetramers of MHC-peptide complexes are used for detection and characterization of antigen-specific T cell responses, but they require knowledge about both antigenic peptide and the MHC restriction element. The successful application of these reagents in human diseases involving CD4 ؉ T cells is limited. Celiac disease, an intestinal inflammation driven by mucosal CD4 ؉ T cells recognizing wheat gluten peptides in the context of disease-associated HLA-DQ molecules, is an ideal model to test the potential clinical use of these reagents. We investigated whether gluten-specific T cells can be detected in the peripheral blood of celiac disease patients using DQ2 tetramers. Nine DQ2 ؉ patients and six control individuals on a gluten-free diet were recruited to the study. Participants consumed 160 g of gluten-containing bread daily for 3 days. After bread-challenge, gluten-specific T cells were detectable in the peripheral blood of celiac patients but not controls both directly by tetramer staining and indirectly by enzyme-linked immunospot. These T cells expressed the ␤7 integrin indicative of gut-homing properties. Most of the cells had a memory phenotype, but many other phenotypic markers showed a heterogeneous pattern. Tetramer staining of gluten-specific T cells has the potential to be used for diagnosis of celiac disease. tetramers T he development of multimeric MHC-peptide complexes has revolutionized the analysis of antigen-specific T cell responses. Tetramers are such reagents consisting of four soluble recombinant MHC molecules, each loaded with a single peptide and bound to a streptavidin molecule that is coupled with a fluorogenic marker (1). Multivalent engagement of the MHCpeptide complexes leads to a stable binding of the tetramer to T cell receptors on the T cell surface, allowing direct visualization of T cells with a defined specificity.MHC class I tetramer technology has greatly facilitated our understanding of CD8 ϩ T cell responses in viral infections and cancer (2). The benefit of tetramers for characterization and diagnosis of human autoimmune and infectious diseases has, however, been modest (3-6). This particularly relates to MHC class II tetramers used for the characterization of antigen-specific CD4 ϩ T cells. Only a few studies of relevance to autoimmunity exist (7-9). MHC class II tetramers are more difficult to produce than MCH class I tetramers (10, 11), and CD4 ϩ T cells of a given specificity appear to be present at much lower frequencies than their CD8 ϩ counterparts (12, 13). Several criteria have to be met to be able to detect antigen-reactive T cells with MHC II tetramers: both the peptide epitope and HLA-restriction element have to be identified, and sufficient frequency and relative high avidity of reactive T cells are needed (14).Celiac disease, a chronic inf lammatory disorder of the small intestine precipitated by ingestion of cereal gluten proteins, presents as an ideal model to test the potential clinical use of MHC class II tetramers. The disorder is driven by intestinal glu...

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

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.