RESUMEN
BACKGROUND & AIMS: Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. METHODS: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). RESULTS: Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. CONCLUSIONS: A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Enfermedad Celíaca/inmunología , Duodeno/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Inmunidad Mucosa , Epítopos Inmunodominantes , Mucosa Intestinal/inmunología , Fragmentos de Péptidos/inmunología , Células Plasmáticas/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Estudios de Casos y Controles , Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/metabolismo , Línea Celular , Dieta Sin Gluten , Duodeno/metabolismo , Duodeno/patología , Proteínas de Unión al GTP/inmunología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Fenotipo , Células Plasmáticas/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/inmunologíaRESUMEN
Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3ß loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3ß loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3ß. This allowed prediction of expanded DQ2.5-glia-α2-reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Codón , Regiones Determinantes de Complementariedad/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Enfermedad Celíaca/inmunología , Células Clonales , Clonación Molecular , Epítopos de Linfocito T/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Humanos , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
Human leukocyte antigen (HLA)-DQ2.5 (DQA1*05/DQB1*02) is a class-II major histocompatibility complex protein associated with both type 1 diabetes and celiac disease. One unusual feature of DQ2.5 is its high class-II-associated invariant chain peptide (CLIP) content. Moreover, HLA-DQ2.5 preferentially binds the non-canonical CLIP2 over the canonical CLIP1. To better understand the structural basis of HLA-DQ2.5's unusual CLIP association characteristics, better insight into the HLA-DQ2.5·CLIP complex structures is required. To this end, we determined the X-ray crystal structure of the HLA-DQ2.5· CLIP1 and HLA-DQ2.5·CLIP2 complexes at 2.73 and 2.20 Å, respectively. We found that HLA-DQ2.5 has an unusually large P4 pocket and a positively charged peptide-binding groove that together promote preferential binding of CLIP2 over CLIP1. An α9-α22-α24-α31-ß86-ß90 hydrogen bond network located at the bottom of the peptide-binding groove, spanning from the P1 to P4 pockets, renders the residues in this region relatively immobile. This hydrogen bond network, along with a deletion mutation at α53, may lead to HLA-DM insensitivity in HLA-DQ2.5. A molecular dynamics simulation experiment reported here and recent biochemical studies by others support this hypothesis. The diminished HLA-DM sensitivity is the likely reason for the CLIP-rich phenotype of HLA-DQ2.5.
Asunto(s)
Antígenos HLA-DQ/química , Cadenas alfa de HLA-DQ/química , Cadenas beta de HLA-DQ/química , Simulación de Dinámica Molecular , Péptidos/química , Sitios de Unión , Antígenos HLA-DQ/genética , Cadenas alfa de HLA-DQ/genética , Cadenas beta de HLA-DQ/genética , Humanos , Péptidos/genéticaRESUMEN
This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Enfermedad Celíaca/inmunología , Epítopos de Linfocito T/inmunología , Glútenes/inmunología , Células Plasmáticas/inmunología , Secuencia de Aminoácidos , Linfocitos B/inmunología , Enfermedad Celíaca/metabolismo , Proteínas de Unión al GTP/inmunología , Gliadina/inmunología , Glútenes/química , Humanos , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Espectrometría de Masas , Fragmentos de Péptidos/inmunología , Células Plasmáticas/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteolisis , Transglutaminasas/inmunologíaRESUMEN
Celiac disease (CD) is an HLA-associated disorder characterized by a harmful T cell response to dietary gluten. It is not understood why most individuals who carry CD-associated HLA molecules, such as HLA-DQ2.5, do not develop CD despite continuous gluten exposure. In this study, we have used tetramers of HLA-DQ2.5 bound with immunodominant gluten epitopes to explore whether HLA-DQ2.5(+) healthy individuals mount a specific CD4(+) T cell response to gluten. We found that gluten tetramer-binding memory cells were rare in blood of healthy individuals. These cells showed lower tetramer-binding intensity and no signs of biased TCR usage compared with gluten tetramer-binding memory T cells from patients. After sorting and in vitro expansion, only 18% of the tetramer-binding memory cells from healthy subjects versus 79% in CD patients were gluten-reactive upon tetramer restaining. Further, T cell clones of tetramer-sorted memory cells of healthy individuals showed lower gluten-specific proliferative responses compared with those of CD patients, indicating that tetramer-binding memory cells in healthy control subjects may be cross-reactive T cells. In duodenal biopsy specimens of healthy control subjects, CD4(+) T cells were determined not to be gluten reactive. Finally, gluten tetramer-binding cells of healthy individuals did not coexpress regulatory T cell markers (Foxp3(+) CD25(+)) and cultured T cell clones did not express a cytokine profile that indicated immune-dampening properties. The results demonstrate that healthy HLA-DQ2.5(+) individuals do not mount a T cell response to immunodominant gluten epitopes of CD.
Asunto(s)
Enfermedad Celíaca/inmunología , Antígenos HLA-DQ/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Línea Celular , Proliferación Celular , Selección Clonal Mediada por Antígenos , Citocinas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glútenes/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Memoria Inmunológica , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de LinfocitosRESUMEN
BACKGROUND: Celiac disease is a multifactorial and polygenic disease with autoimmune features. The disease is caused by an inappropriate immune response to gluten. Elimination of gluten from the diet leads to disease remission, which is the basis for today's treatment of the disease. There is an unmet need for new alternative treatments. KEY MESSAGES: Genetic findings point to adaptive immunity playing a key role in the pathogenesis of celiac disease. MHC is by far the single most important genetic factor in the disease. In addition, a number of non-MHC genes, the majority of which have functions related to T cells and B cells, also contribute to the genetic predisposition, but each of them has modest effect. The primary MHC association is with HLA-DQ2 and HLA-DQ8. These HLA molecules present gluten epitopes to CD4+ T cells which can be considered to be the master regulators of the immune reactions that lead to the disease. The epitopes which the T cells recognize are usually deamidated, and this deamidation is mediated by the enzyme transglutaminase 2 (TG2). Celiac disease patients have disease-specific antibodies. In addition to antibodies to gluten, these include autoantibodies to TG2. Antibodies to deamidated gluten are nearly as specific for celiac disease as the anti-TG2 antibodies. Both types of antibodies appear only to be produced in subjects who are HLA-DQ2 or HLA-DQ8 when they are consuming gluten. CONCLUSION: It is hardly coincidental that TG2 is implicated in T-cell epitope formation and at the same time a target for autoantibodies. Understanding this connection is one of the major challenges for obtaining a complete understanding of how gluten causes tissue destruction and remodeling of the mucosa in the small bowel.
Asunto(s)
Inmunidad Adaptativa , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Intestino Delgado/inmunología , Intestino Delgado/patología , Epítopos de Linfocito T/inmunología , Proteínas de Unión al GTP/inmunología , Humanos , Inmunoglobulina A/inmunología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/inmunologíaRESUMEN
Celiac disease is caused by intolerance to cereal gluten proteins, and HLA-DQ molecules are involved in the disease pathogenesis by presentation of gluten peptides to CD4(+) T cells. The α- or ß-chain sharing HLA molecules DQ2.5, DQ2.2, and DQ7.5 display different risks for the disease. It was recently demonstrated that T cells of DQ2.5 and DQ2.2 patients recognize distinct sets of gluten epitopes, suggesting that these two DQ2 variants select different peptides for display. To explore whether this is the case, we performed a comprehensive comparison of the endogenous self-peptides bound to HLA-DQ molecules of B-lymphoblastoid cell lines. Peptides were eluted from affinity-purified HLA molecules of nine cell lines and subjected to quadrupole orbitrap mass spectrometry and MaxQuant software analysis. Altogether, 12,712 endogenous peptides were identified at very different relative abundances. Hierarchical clustering of normalized quantitative data demonstrated significant differences in repertoires of peptides between the three DQ variant molecules. The neural network-based method, NNAlign, was used to identify peptide-binding motifs. The binding motifs of DQ2.5 and DQ7.5 concurred with previously established binding motifs. The binding motif of DQ2.2 was strikingly different from that of DQ2.5 with position P3 being a major anchor having a preference for threonine and serine. This is notable as three recently identified epitopes of gluten recognized by T cells of DQ2.2 celiac patients harbor serine at position P3. This study demonstrates that relative quantitative comparison of endogenous peptides sampled from our protein metabolism by HLA molecules provides clues to understand HLA association with disease.
Asunto(s)
Enfermedad Celíaca/inmunología , Antígenos HLA-DQ/metabolismo , Secuencia de Aminoácidos , Presentación de Antígeno , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/genética , Enfermedad Celíaca/metabolismo , Línea Celular Transformada , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Glútenes/química , Glútenes/genética , Glútenes/inmunología , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Herpesvirus Humano 4 , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Mapeo Peptídico , Péptidos/química , Péptidos/inmunología , Dominios y Motivos de Interacción de Proteínas , ProteómicaRESUMEN
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
BACKGROUND: Diagnosing coeliac disease (CD) can be challenging, despite highly specific autoantibodies and typical mucosal changes in the small intestine. The T-cell response to gluten is a hallmark of the disease that has been hitherto unexploited in clinical work-up. OBJECTIVES: We aimed to develop a new method that directly visualizes and characterizes gluten-reactive CD4+ T cells in blood, independently of gluten challenge, and to explore its diagnostic potential. METHODS: We performed bead-enrichment of DQ2.5-glia-α1a and DQ2.5-glia-α2 tetramer+ cells in the blood of control individuals, treated (TCD) and untreated patients (UCD). We visualized these cells by flow cytometry, sorted them and cloned them. We assessed their specificity by antigen stimulation and re-staining with tetramers. RESULTS: We detected significantly more gliadin-tetramer+ CD4+ effector memory T cells (TEM) in UCD and TCD patients, compared to controls. Significantly more gliadin-tetramer+ TEM in the CD patients than in controls expressed the gut-homing marker integrin-ß7. CONCLUSION: Quantification of gut-homing, gluten-specific TEM in peripheral blood, visualized with human leukocyte antigen (HLA) -tetramers, may be used to distinguish CD patients from healthy individuals. Easy access to gluten-reactive blood T cells from diseased and healthy individuals may lead to new insights on the disease-driving CD4+ T cells in CD.
RESUMEN
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
OBJECTIVES: Investigation of uncertain celiac disease (CD) in patients already on a gluten-free diet (GFD) is difficult. We evaluated HLA-DQ2-gliadin tetramers for detection of gluten-specific T cells in peripheral blood and histological changes in the duodenum after a short gluten challenge as a diagnostic tool. METHODS: HLA-DQ2+ individuals on a GFD for at least 4 weeks were investigated; 35 with uncertain diagnosis, 13 CD patients, and 2 disease controls. All participants had a challenge with four slices of gluten-containing white bread, daily for 3 days (d1-d3). An esophagogastroduodenoscopy with biopsy sampling was done on d0 and d4. Biopsies were scored according to revised Marsh criteria. Peripheral blood CD4+ T cells were isolated, stained with HLA-DQ2-gliadin peptide tetramers, and analyzed by flow cytometry on d0 and d6. RESULTS: After challenge, a positive tetramer test was seen in 11/13 CD patients. Four of these subjects also showed typical histological changes on challenge. Of the 35 patients with uncertain diagnosis, 3 were diagnosed with CD. Two of these three patients had both positive tetramer staining and histological changes in biopsies after challenge. CONCLUSIONS: Tetramer staining for gluten-specific T cells is a sensitive method in detecting an immune response in CD patients after a short gluten challenge. The prevalence of CD in the group with self-prescribed GFD was about 10%.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/sangre , Enfermedad Celíaca/diagnóstico , Tracto Gastrointestinal/inmunología , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Adolescente , Adulto , Anciano , Biopsia , Pan , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Técnicas de Diagnóstico del Sistema Digestivo , Endoscopía del Sistema Digestivo , Femenino , Citometría de Flujo , Antígenos HLA-DQ/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
New tools are needed for managing celiac sprue, a lifelong immune disease of the small intestine. Ongoing drug trials are also prompting a search for noninvasive biomarkers of gluten-induced intestinal change. We have synthesized and characterized noninflammatory gluten peptide analogs in which key Gln residues are replaced by Asn or His. Like their proinflammatory counterparts, these biomarkers are resistant to gastrointestinal proteases, susceptible to glutenases, and permeable across enterocyte barriers. Unlike gluten peptides, however, they are not appreciably recognized by transglutaminase, HLA-DQ2, or disease-specific T cells. In vitro and animal studies show that the biomarkers can detect intestinal permeability changes as well as glutenase-catalyzed gastric detoxification of gluten. Accordingly, controlled clinical studies are warranted to evaluate the use of these peptides as probes for abnormal intestinal permeability in celiac patients and for glutenase efficacy in clinical trials and practice.
Asunto(s)
Enfermedad Celíaca/metabolismo , Glútenes/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Enfermedad Celíaca/inmunología , Glútenes/química , Glútenes/farmacología , Humanos , Marcaje Isotópico , Masculino , Datos de Secuencia Molecular , Péptidos/química , Ratas , Ratas Wistar , Linfocitos T/inmunología , Transglutaminasas/inmunología , Transglutaminasas/metabolismoRESUMEN
Celiac disease driven by an antigluten T cell response is strongly associated with the histocompatibility antigen HLA-DQ2.5 but is barely associated with HLA-DQ2.2. Yet these molecules have very similar peptide-binding motifs and both present gluten T cell epitopes. We found that DQ2.5(+) antigen-presenting cells (APCs) had greater stability of bound peptides and protracted gluten presentation relative to that of DQ2.2(+) cells. The improved ability of DQ2.5 to retain its peptide cargo can be ascribed to a polymorphism of DQalpha22 whereby DQ2.5 (tyrosine) can establish a hydrogen bond to the peptide main chain but DQ2.2 (phenylalanine) cannot. Our findings suggest that the kinetic stability of complexes of peptide and major histocompatibility complex (MHC) is of importance for the association of HLA with disease.
Asunto(s)
Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Enfermedad Celíaca/genética , Glútenes/inmunología , Antígenos HLA-DQ/química , Enfermedad Celíaca/inmunología , Línea Celular , Citometría de Flujo , Predisposición Genética a la Enfermedad , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Humanos , Enlace de Hidrógeno , Proteínas Asociadas a Microtúbulos/inmunología , Proteínas Asociadas a Microtúbulos/metabolismo , Péptidos/química , Péptidos/inmunología , Péptidos/metabolismo , Polimorfismo Genético , Unión Proteica/inmunología , Factores de Riesgo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
We here describe that soluble HLA-DQ2 (sDQ2) molecules, when expressed in Drosophila melanogaster S2 insect cells without a covalently tethered peptide, associate tightly with the D. melanogaster calcium binding protein DCB-45. The interaction between the proteins is stable in S2 cell culture and during affinity purification, which is done at high salt concentrations and pH 11.5. After affinity purification, the sDQ2/DCB-45 complex exists in substantial quantities next to a small amount of free heterodimeric sDQ2 and large amounts of aggregated sDQ2 free of DCB-45. Motivated by the stable complex formation and our interest in the development of reagents which inhibit HLA-DQ2 peptide binding, we have further characterized the sDQ2/DCB-45 interaction. Several lines of evidence indicate that an N-terminal fragment of DCB-45 is involved in the interaction with the peptide binding groove of sDQ2. Further mapping of this fragment of 54 residues identified a pentadecapeptide with high affinity for sDQ2 which may serve as a lead compound for the design of HLA-DQ2 blockers.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Drosophila/química , Antígenos HLA-DQ/química , Proteínas Nucleares/química , Mapeo de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Cromatografía de Afinidad , Dimerización , Drosophila melanogaster , Genes Sintéticos , Vectores Genéticos/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/aislamiento & purificación , Antígenos HLA-DQ/metabolismo , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Humanos , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Transducción GenéticaRESUMEN
Atypical invariant chain (Ii) CLIP fragments (CLIP2) have been found in association with HLA-DQ2 (DQ2) purified from cell lysates. We mapped the binding register of CLIP2 (Ii 96-104) to DQ2 and found proline at the P1 position, in contrast to the canonical CLIP1 (Ii 83-101) register with methionine at P1. CLIP1/2 peptides are the predominant peptide species, even for DQ2 from HLA-DM (DM)-expressing cells. We hypothesized that DQ2-CLIP1/2 might be poor substrates for DM. We measured DM-mediated exchange of CLIP and other peptides for high-affinity indicator peptides and found it is inefficient for DQ2. DM-DQ-binding and DM chaperone effects on conformation and levels of DQ are also reduced for DQ2, compared with DQ1. We suggest that the unusual interaction of DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/inmunología , Enfermedades Autoinmunes/inmunología , Antígenos HLA-D/inmunología , Antígenos HLA-DQ/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Chaperonas Moleculares/inmunología , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Diferenciación de Linfocitos B/metabolismo , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Línea Celular Transformada , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mapeo Peptídico/métodos , Unión Proteica/genética , Unión Proteica/inmunologíaRESUMEN
Celiac disease is a prevalent disorder of the small intestine that is caused by an inflammatory reaction to dietary gluten in genetically susceptible individuals. More than 90% of patients express the HLA-DQ2 molecule, whereas DQ8 is carried by most of the remaining patients. DQ2- and DQ8-mediated presentation of gluten peptides to CD4+ T cells is a key event in the pathogenesis of the disease. The association of celiac disease with these human leukocyte antigen (HLA) molecules is explained by a preferential binding of gluten peptides to these HLA molecules, although the actual data on this in the literature are scarce. The objective of this study was to test this hypothesis. A panel of peptides representing DQ2-restricted gluten T-cell epitopes was tested for binding to various HLA class II molecules using various experimental approaches. The results demonstrate that the gluten T-cell epitopes mainly bind to the DQ2 molecule.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Glútenes/inmunología , Antígenos HLA-D/inmunología , Presentación de Antígeno/inmunología , Sitios de Unión , Unión Competitiva , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Celíaca/inmunología , Epítopos de Linfocito T/metabolismo , Predisposición Genética a la Enfermedad , Glútenes/química , Glútenes/metabolismo , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Humanos , Enlace de Hidrógeno , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Ensayo de Unión RadioliganteRESUMEN
Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells. Blocking the peptide-binding site of DQ2 may be a way to treat celiac disease. In this study, two types of peptide analogues, modeled after natural gluten antigens, were studied as DQ2 blockers. (a) Cyclic peptides. Cyclic peptides containing the DQ2-alphaI gliadin epitope LQPFPQPELPY were synthesized with flanking cysteine residues introduced and subsequently crosslinked via a disulfide bond. Alternatively, cyclic peptides were prepared with stable polyethylene glycol bridges across internal lysine residues of modified antigenic peptides such as KQPFPEKELPY and LQLQPFPQPEKPYPQPEKPY. The effect of cyclization as well as the length of the spacer in the cyclic peptides on DQ2 binding and T cell recognition was analyzed. Inhibition of peptide-DQ2 recognition by the T cell receptor was observed in T cell proliferation assays. (b) Dimeric peptides. Previously we developed a new type of peptide blocker with much enhanced affinity for DQ2 by dimerizing LQLQPFPQPEKPYPQPELPY through the lysine side chains. Herein, the effect of linker length on both DQ2 binding and T cell inhibition was investigated. One dimeric peptide analogue with an intermediate linker length was found to be especially effective at inhibiting DQ2 mediated antigen presentation. The implications of these findings for the treatment of celiac disease are discussed.
Asunto(s)
Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Glútenes/química , Antígenos HLA-DQ/química , Antígenos HLA-DQ/inmunología , Péptidos/química , Péptidos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclización , Dimerización , Disulfuros/química , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Estructura Molecular , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
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 beta(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.
Asunto(s)
Enfermedad Celíaca/sangre , Tracto Gastrointestinal/inmunología , Glútenes/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Pan , Estudios de Casos y Controles , Enfermedad Celíaca/terapia , Diferenciación Celular , Antígenos HLA-DQ/química , Antígenos HLA-DQ/inmunología , Humanos , Interferón gamma/biosíntesis , Leucocitos Mononucleares/inmunología , Persona de Mediana Edad , Fenotipo , Estructura Cuaternaria de Proteína , Linfocitos T/citologíaRESUMEN
Human leukocyte antigen DQ2 is a class II major histocompatibility complex protein that plays a critical role in the pathogenesis of Celiac Sprue by binding to epitopes derived from dietary gluten and triggering the inflammatory response of disease-specific T cells. Inhibition of DQ2-mediated antigen presentation in the small intestinal mucosa of Celiac Sprue patients therefore represents a potentially attractive mode of therapy for this widespread but unmet medical need. Starting from a pro-inflammatory, proteolytically resistant, 33-residue peptide, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, we embarked upon a systematic effort to dissect the relationships between peptide structure and DQ2 affinity and to translate these insights into prototypical DQ2 blocking agents. Three structural determinants within the first 20 residues of this 33-mer peptide, including a PQPELPYPQ epitope, its N-terminal flanking sequence, and a downstream Glu residue, were found to be important for DQ2 binding. Guided by the X-ray crystal structure of DQ2, the L11 and L18 residues in the truncated 20-mer analogue were replaced with sterically bulky groups so as to retain high DQ2 affinity but abrogate T cell recognition. A dimeric ligand, synthesized by regiospecific coupling of the 20-mer peptide with a bifunctional linker, was identified as an especially potent DQ2 binding agent. Two such ligands were able to attenuate the proliferation of disease-specific T cell lines in response to gluten antigens and, therefore, represent prototypical examples of pharmacologically suitable DQ2 blocking agents for the potential treatment of Celiac Sprue.