RESUMO
The great majority of patients that are intolerant of wheat gluten protein due to celiac disease (CD) are human histocompatibility leukocyte antigen (HLA)-DQ2(+), and the remaining few normally express HLA-DQ8. These two class II molecules are chiefly responsible for the presentation of gluten peptides to the gluten-specific T cells that are found only in the gut of CD patients but not of controls. Interestingly, tissue transglutaminase (tTG)-mediated deamidation of gliadin plays an important role in recognition of this food antigen by intestinal T cells. Here we have used recombinant antigens to demonstrate that the intestinal T cell response to alpha-gliadin in adult CD is focused on two immunodominant, DQ2-restricted peptides that overlap by a seven-residue fragment of gliadin. We show that tTG converts a glutamine residue within this fragment into glutamic acid and that this process is critical for T cell recognition. Gluten-specific T cell lines from 16 different adult patients all responded to one or both of these deamidated peptides, indicating that these epitopes are highly relevant to disease pathology. Binding studies showed that the deamidated peptides displayed an increased affinity for DQ2, a molecule known to preferentially bind peptides containing negatively charged residues. Interestingly, the modified glutamine is accommodated in different pockets of DQ2 for the different epitopes. These results suggest modifications of anchor residues that lead to an improved affinity for major histocompatibility complex (MHC), and altered conformation of the peptide-MHC complex may be a critical factor leading to T cell responses to gliadin and the oral intolerance of gluten found in CD.
Assuntos
Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/metabolismo , Gliadina/farmacologia , Glutamina , Mucosa Intestinal/imunologia , Linfócitos T/imunologia , Transglutaminases/metabolismo , Adulto , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Criança , Sequência Consenso , Gliadina/química , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Humanos , Imunidade nas Mucosas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of the HLA-DQ2 (alpha1(*)0501, beta1(*)0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several size-variants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClalpha46 - 63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClalpha46 - 63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.
Assuntos
Doença Celíaca/imunologia , Antígenos HLA-DQ/metabolismo , Sequência de Aminoácidos , Doença Celíaca/genética , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Antígenos HLA-DQ/genética , Cadeias alfa de HLA-DQ , Cadeias beta de HLA-DQ , Humanos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(alpha 1*0501, beta 1*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(alpha 1*0201, beta 1*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I alpha chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. AT p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(alpha 1*0201, beta 1*0202) molecule. This most striking difference between the two DQ molecules is the presence of and additional anchor residue at p3 for the DQ(alpha 1*0201, beta 1*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(alpha 1*0501, beta 1*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(alpha 1*0501, beta 1*0201) and DQ(alpha 1*0201, beta 1*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(alpha 1*0501, beta 1*0201) molecule should be helpful in the identification of CD-inducing epitopes.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos HLA-DQ/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Sequência de Aminoácidos , Antígenos de Diferenciação de Linfócitos B/química , Antígenos HLA-DQ/química , Antígenos de Histocompatibilidade Classe II/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
In this study we describe the expression of two T cell receptor (TCR) gamma chains on the surface of a human T cell clone isolated from the peripheral blood. Each gamma chain was part of an independent and functional TCR. The dual receptor T cell clone (and all subclones derived from this clone) had stable expression of this phenotype. Immunoprecipitation studies revealed the expression of non-disulfide linked TCRs by this V gamma 4+V gamma 9+V delta 1+ T cell clone, which was in agreement with the finding that both V gamma gene transcripts were rearranged to C gamma 2-associated joining elements. Both gamma chains were derived from productive rearrangements of different (allelic) genes coding for a V gamma 4+ and a V gamma 9+ gamma-chain, and both were coupled to a V delta 1+ delta chain. Incubation of this V gamma 4+V gamma 9+V delta 1+ T cell clone with TCR gamma-chain-specific MoAbs rapidly induced an increase in intracellular Ca++, indicating that both gamma-chains are functional. Furthermore, this clone responded to stimulation with S. aureus derived superantigens. We suggest therefore that exogenous (super)antigens can trigger dual receptor T cells resulting in activation of these T cells.
Assuntos
Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Doenças Autoimunes/etiologia , Sequência de Bases , Células Clonais , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta/genética , Superantígenos/imunologiaRESUMO
It has previously been shown that murine tissue derived T-cells expressing the gamma delta T-cell receptor can respond to autologous (stressed) cells implying the recognition of an autoantigen. Here we report that a large proportion of human synovial tissue and peripheral blood derived V delta 1+ gamma delta T-cell clones proliferate in response to stimulation with autologous and allogeneic EBV-transformed B-lymphoblastoid cell lines (LCL). In contrast, V delta 1- gamma/delta and alpha/beta TCR+ T-cell clones isolated from the same tissue samples did not display proliferation towards the LCL. The proliferative response of these V delta 1+ clones was dependent on contact between responder and stimulator cells and could be blocked by a MoAb to LFA-1 and by antibodies to the gamma delta TCR/CD3 complex. Because the responses of these clones to LCL cells appear to be independent of the gamma-chain co-expressed with the V delta 1-chain these resemble a superantigen response. The capacity of this subset of V delta 1+ T-cell clones to proliferate after stimulation with LCL may imply the recognition of an endogenous epitope. Moreover, since so far we have been able to isolate only LCL reactive gamma delta T-cell clones from synovial tissue and peripheral blood of reactive arthritis patients and not from peripheral blood of healthy individuals, the frequency of such 'autoreactive' gamma delta cells may be higher in these patients.
Assuntos
Linfócitos B/imunologia , Transformação Celular Viral/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Anticorpos Monoclonais , Artrite Reativa/imunologia , Autoantígenos/imunologia , Adesão Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Antígenos HLA/imunologia , Herpesvirus Humano 4 , Humanos , Imunofenotipagem , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Cells expressing the V delta 1+ gene segment are a minor gamma delta T cell population in human peripheral blood but predominate in epithelial and (inflamed) tissues. The characteristic dendritic-like morphology of these gamma delta T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of V delta 1+ gamma delta T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) gamma chain co-expressed with the V delta 1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of V delta 1+ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive V delta 1+ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the V delta 1+ T cells. Finally, we investigated the diversity of the responding V delta 1+ gamma delta T cell clones by sequence analysis of the TcR delta junctional regions. No restricted V-D-J sequences were found among the LCL-responsive V delta 1+ T cell clones, arguing strongly against a mono- or oligoclonal V delta 1+ gamma delta T cell response to LCL. These findings may explain the presence of polyclonally activated V delta 1+ T cells in inflamed tissues where activated B cells are often present.
Assuntos
Adenosina Trifosfatases , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Sequência de Aminoácidos , Antígenos CD/fisiologia , Apirase , Linfócitos B/imunologia , Antígeno B7-1/fisiologia , Sequência de Bases , Transformação Celular Viral , Células Clonais , Primers do DNA/química , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T , Herpesvirus Humano 4 , Humanos , Técnicas In Vitro , Ativação Linfocitária , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T gama-delta/químicaRESUMO
The association of celiac disease (CD) with HLA-DQ2 and HLA-DQ8 is indicative of preferential mucosal T cell recognition of gluten fragments bound to either DQ allele. We have recently identified two gluten-derived, HLA-DQ8-restricted T cell stimulatory peptides, one each from gliadin and glutenin, recognized by specific T cell clones derived from the small intestine of CD patients. We have now performed molecular modeling and examined the fine specificity of these peptides in complex with HLA-DQ8. There is only one binding register for both peptides, with glutamine residues at the p1 and p9 anchor positions. Both T cell clones recognize substituted peptides at p1 and p9, but poorly so at p2-p8, especially the gliadin-specific clone. Contrasting patterns of recognition of p9Gln --> Glu peptide variants (both predicted as better DQ8 binders by modeling) were observed: enhancement of recognition for the gliadin peptide, yet complete absence thereof for the glutenin peptide. The double-substituted gliadin peptide variant p1/9Gln --> Glu, which can also arise by pepsin/acid/transglutaminase treatment, shows a considerable increase in sensitivity of recognition, consistent with better binding of this peptide to DQ8, as predicted by energy minimization. Surprisingly, the two native peptides are also recognized by their respective T cell clones in the context of the related molecule HLA-DQ9 (beta57Asp(+)). The p1/9Gln --> Glu gliadin peptide variant is likewise recognized, albeit with a 10-fold lower sensitivity, the first reported p9Glu binding in a beta57Asp(+) MHC II allele. Our results have important implications for the pathogenesis of autoimmune disease and the possible manipulation of aberrant responses thereof.
Assuntos
Doenças Autoimunes/imunologia , Doença Celíaca/imunologia , Epitopos/imunologia , Genes MHC da Classe II , Gliadina/imunologia , Glutens/análogos & derivados , Glutens/imunologia , Antígenos HLA-DQ/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Apresentação de Antígeno , Autoantígenos/imunologia , Doenças Autoimunes/genética , Sítios de Ligação , Doença Celíaca/genética , Epitopos/química , Predisposição Genética para Doença , Gliadina/química , Glutens/química , Antígenos HLA-DQ/química , Antígenos HLA-DQ/imunologia , Mucosa Intestinal/imunologia , Ativação Linfocitária , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Polimorfismo Genético , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The majority of human peripheral gamma delta T cells express the V gamma 9 gene in combination with the V delta 2 gene. The diversity of this subset of gamma delta T cells is limited by a preferential usage of the J gamma P gene segment and a highly distinctive junctional motif of the T-cell receptor (TCR) delta chain. We and others have observed that peripheral blood derived V gamma 9+V delta 2+ gamma delta T cells of healthy individuals are activated after stimulation with interleukin-2 (IL-2) in vitro, but only a small percentage of gamma delta T cells subsequently proliferates. To assess whether the proliferating, IL-2-responsive gamma delta T cells represent a selective group of T cells, we have analysed TCR junctional features of IL-2-responsive gamma delta T cells. Out of 30 individuals studied, nine were identified as IL-2-responders and three as IL-2-hyperresponders. The TCR V(D)J gene usage from IL-2 stimulated peripheral blood lymphocytes of these IL-2-(hyper)responsive individuals was analysed. The results showed that in most individuals gamma delta T cells polyclonally expanded after stimulation with IL-2. In two IL-2-hyperresponder individuals, however, a monoclonal expansion of a particular V gamma 9+V delta 2+ gamma delta T cell was found. In one of these individuals, this V gamma 9+V delta 2+ T-cell clone expressed a very rare gamma delta TCR type because of the presence of an Ala within the junctional region at a conserved position relative to V delta framework residues (delta 97), which is very infrequently used by peripheral blood V gamma 9+V delta 2+ cells. This particular clonotype could also be detected in unstimulated PBL samples taken from that individual, and made up for 30% of the total peripheral gamma delta T-cell pool. These data indicate that in general IL-2-responsive V gamma 9+V delta 2+ gamma delta T cells represent a polyclonal population, reflecting in vivo stimulation with multiple antigens or superantigens. In contrast, monoclonal expansions of gamma delta T cells after stimulation with IL-2 can also occur, which may be related to an in vivo stimulation by one particular antigen, rendering this gamma delta T-cell type dominant in the peripheral blood.
Assuntos
Interleucina-2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Bases , Southern Blotting , Células Cultivadas , Genes MHC da Classe II , Humanos , Imunidade Celular/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
Gluten ingestion causes coeliac disease in susceptible individuals. Gluten is a heterogeneous mixture of glutenin and gliadin, the latter of which is considered responsible for disease induction. By combining high-performance liquid chromatography purification steps of gluten with a T cell bioassay and mass spectral analyses, we have identified a glutenin peptide (glt04 707-742) that activates T cells from the small intestine of a coeliac disease patient and results in the secretion of large amounts of IFN-gamma. The minimal T cell stimulatory core of the peptide (residues 724-734) is repetitively present in glutenin molecules. Moreover, it was observed that a large number of naturally occurring variants of this peptide are recognized by the T cells. These data suggest that the large heterogeneity of glutenin proteins dramatically increases the number of available T cell epitopes. Together, the results provide new insight into the nature of the gluten antigens that lead to coeliac disease and suggest that glutenin, next to gliadin-derived antigens, may be involved in the disease process.
Assuntos
Glutens/análogos & derivados , Imunidade nas Mucosas/imunologia , Linfócitos T/imunologia , Triticum/imunologia , Sequência de Aminoácidos , Plexo Celíaco/imunologia , Células Clonais/imunologia , Epitopos de Linfócito T/análise , Epitopos de Linfócito T/imunologia , Glutens/química , Glutens/genética , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Cadeias beta de HLA-DQ , Humanos , Intestino Delgado/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos , Mapeamento de PeptídeosRESUMO
Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.