RESUMEN
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.
Asunto(s)
Mapeo Epitopo , Epítopos de Linfocito T/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Enfermedad Celíaca/inmunología , Línea Celular , Cromatografía en Gel , Mapeo Epitopo/métodos , Gliadina/química , Gliadina/inmunología , Glútenes/química , Antígenos HLA-DQ/química , Humanos , Péptidos/química , Péptidos/inmunología , Unión Proteica , Triticum/inmunologíaRESUMEN
Knowledge of the frequency of disease-driving CD4⺠T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten-reactive CD4⺠T cells, which recognize gluten peptides only in the context of the disease-associated HLA-DQ molecules, are key pathogenic players. By cloning CD4⺠T cells directly from intestinal biopsies of CD patients, we found that 0.5-1.8% of CD4⺠T cells were gluten reactive. About half of the gluten-reactive T cells were specific for either the immuno-dominant 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⺠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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/inmunología , Intestinos/inmunología , Biopsia , Enfermedad Celíaca/diagnóstico , Separación Celular , Células Clonales , Citometría de Flujo , Proteínas de Unión al GTP/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/metabolismo , Humanos , Epítopos Inmunodominantes/inmunología , Inmunoglobulina A/sangre , Intestinos/patología , Fragmentos de Péptidos/inmunología , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/inmunologíaRESUMEN
We describe the gluten T-cell response of a DR7DQ2/DR9DQ9 heterozygous celiac disease patient (CD555). Interestingly, this patient had T cells recognizing gluten in the context of human leukocyte antigen (HLA) molecules of both haplotypes. For the DR9DQ9 haplotype, DQ9 was identified as the antigen-presenting molecule. As DQ9 carries aspartate at DQ ß57 but is otherwise identical to DQ8 and not considered associated with celiac disease, we aimed to characterize this DQ9-restricted T-cell response in detail. By fractionation of pepsin-trypsin digested gliadin we identified an epitope stimulatory for several T-cell clones. This epitope was identical to an epitope (DQ8-glut-1) previously identified in DQ8 patients. In CD555, this was the dominant DQ9-restricted epitope, whereas no T-cell response was found toward two other DQ8-restricted epitopes. These findings correlated with peptide binding data demonstrating that this epitope bound better to DQ9 than the two other DQ8-restricted epitopes. Although glutamine to glutamate exchange at P9 improved binding of all three epitopes to DQ8, no such effect was observed for DQ9. The differential ability of DQ8 and DQ9 to harness a negatively charged anchor at P9 may result in fewer potential gluten epitopes in DQ9 patients. Our data further indicate that DQ9 is a susceptibility factor for celiac disease.
Asunto(s)
Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Epítopos de Linfocito T/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno/inmunología , Epítopos de Linfocito T/química , Femenino , Predisposición Genética a la Enfermedad , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Glútenes/química , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , RiesgoRESUMEN
BACKGROUND & AIMS: Celiac disease is a diet-induced, T cell-mediated enteropathy. The HLA variant DQ2.5 increases risk of the disease, and the homologous DQ2.2 confers a lower level of risk. As many as 5% of patients with celiac disease carry DQ2.2 without any other risk alleles. Epitopes commonly recognized by T cells of patients with HLA-DQ2.5 bind stably to DQ2.5 but unstably to DQ2.2. We investigated the response to gluten in patients with HLA-DQ2.2. METHODS: We generated intestinal T-cell lines and clones from 7 patients with HLA-DQ2.2 (but not DQ2.5) and characterized the responses of the cells to gluten. The epitope off-rate was evaluated by gel filtration and T cell-based assays. Peptide binding to DQ2.2 was studied with peptide substitutes and DQ2 mutants. RESULTS: Patients with DQ2.2 and no other risk alleles had gluten-reactive T cells that did not respond to the common DQ2.5-restricted T-cell epitopes. Instead, many of the T cells responded to a distinct epitope that was not recognized by those from patients with HLA-DQ2.5. This immunodominant epitope bound stably to DQ2.2. A serine residue at P3 was required for the stable binding. The effect of this residue related to a polymorphism at DQα22 that was previously shown to determine stable binding of peptides to DQ2.5. CONCLUSIONS: High levels of kinetic stability of peptide-major histocompatibility complexes are required to generate T-cell responses to gluten in celiac disease; the lower risk from DQ2.2 relates to constraints imposed on gluten peptides to stably bind this HLA molecule. These observations increase our understanding of the role of the major histocompatibility complex in determining T-cell responses in patients with celiac disease and are important for peptide-based vaccination strategies.