RESUMO
In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.
Assuntos
Doença Celíaca , Duodeno , Interleucina-7 , Mucosa Intestinal , Modelos Biológicos , Organoides , Humanos , Autoanticorpos/imunologia , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biópsia , Doença Celíaca/imunologia , Doença Celíaca/patologia , Doença Celíaca/metabolismo , Duodeno/imunologia , Duodeno/patologia , Duodeno/metabolismo , Epitopos/imunologia , Glutens/imunologia , Glutens/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/imunologia , Antígenos HLA-DQ/imunologia , Antígenos HLA-DQ/metabolismo , Interleucina-7/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Organoides/imunologia , Organoides/metabolismo , Organoides/patologia , Proteína 2 Glutamina gama-Glutamiltransferase/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND & AIMS: The treatment of celiac disease (CeD) with gluten-free diet (GFD) normalizes gut inflammation and disease-specific antibodies. CeD patients have HLA-restricted, gluten-specific T cells persisting in the blood and gut even after decades of GFD, which are reactivated and disease driving upon gluten exposure. Our aim was to examine the transition of activated gluten-specific T cells into a pool of persisting memory T cells concurrent with normalization of clinically relevant biomarkers during the first year of treatment. METHODS: We followed 17 CeD patients during their initial GFD year, leading to disease remission. We assessed activation and frequency of gluten-specific CD4+ blood and gut T cells with HLA-DQ2.5:gluten tetramers and flow cytometry, disease-specific serology, histology, and symptom scores. We assessed gluten-specific blood T cells within the first 3 weeks of GFD in 6 patients and serology in an additional 9 patients. RESULTS: Gluten-specific CD4+ T cells peaked in blood at day 14 while up-regulating Bcl-2 and down-regulating Ki-67 and then decreased in frequency within 10 weeks of GFD. CD38, ICOS, HLA-DR, and Ki-67 decreased in gluten-specific cells within 3 days. PD-1, CD39, and OX40 expression persisted even after 12 months. IgA-transglutaminase 2 decreased significantly within 4 weeks. CONCLUSIONS: GFD induces rapid changes in the phenotype and number of gluten-specific CD4+ blood T cells, including a peak of nonproliferating, nonapoptotic cells at day 14. Subsequent alterations in T-cell phenotype associate with the quiescent but chronic nature of treated CeD. The rapid changes affecting gluten-specific T cells and disease-specific antibodies offer opportunities for clinical trials aiming at developing nondietary treatments for patients with newly diagnosed CeD.
Assuntos
Linfócitos T CD4-Positivos , Doença Celíaca , Dieta Livre de Glúten , Glutens , Fenótipo , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Glutens/imunologia , Glutens/administração & dosagem , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos HLA-DQ/imunologia , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Ativação Linfocitária , Transglutaminases/imunologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células T de Memória/imunologia , Células T de Memória/metabolismo , Fatores de Tempo , Adulto Jovem , Resultado do Tratamento , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/metabolismoRESUMO
We compared the αß T-cell receptor repertoires of CD8+ αß intraepithelial lymphocytes from celiac disease patients and healthy subjects by single-cell sequencing. We demonstrate that the repertoires of untreated celiac disease patients were more polyclonal and more diverse than what was observed in both treated patients and healthy subjects.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença Celíaca/imunologia , Epitélio/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Biodiversidade , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Célula Única , TranscriptomaRESUMO
Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5-restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5-DQ2.5-glia-α1a and HLA-DQ2.5-DQ2.5-glia-ω1 tetramers and single-cell αß T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide-HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a- and DQ2.5-glia-ω1-reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5-peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide-HLA-II landscape in a human disease setting.
Assuntos
Apresentação de Antígeno , Epitopos de Linfócito T/imunologia , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Feminino , Humanos , MasculinoRESUMO
BACKGROUND & AIMS: Celiac disease is characterized by HLA-DQ2/8-restricted responses of CD4+ T cells to cereal gluten proteins. A diagnosis of celiac disease based on serologic and histologic evidence requires patients to be on gluten-containing diets. The growing number of individuals adhering to a gluten-free diet (GFD) without exclusion of celiac disease complicates its detection. HLA-DQ-gluten tetramers can be used to detect gluten-specific T cells in blood of patients with celiac disease, even if they are on a GFD. We investigated whether an HLA-DQ-gluten tetramer-based assay accurately identifies patients with celiac disease. METHODS: We produced HLA-DQ-gluten tetramers and added them to peripheral blood mononuclear cells isolated from 143 HLA-DQ2.5+ subjects (62 subjects with celiac disease on a GFD, 19 subjects without celiac disease on a GFD [due to self-reported gluten sensitivity], 10 subjects with celiac disease on a gluten-containing diet, and 52 presumed healthy individuals [controls]). T cells that bound HLA-DQ-gluten tetramers were quantified by flow cytometry. Laboratory tests and flow cytometry gating analyses were performed by researchers blinded to sample type, except for samples from subjects with celiac disease on a gluten-containing diet. Test precision analyses were performed using samples from 10 subjects. RESULTS: For the HLA-DQ-gluten tetramer-based assay, we combined flow-cytometry variables in a multiple regression model that identified individuals with celiac disease on a GFD with an area under the receiver operating characteristic curve value of 0.96 (95% confidence interval [CI] 0.89-1.00) vs subjects without celiac disease on a GFD. The assay detected individuals with celiac disease on a gluten-containing diet vs controls with an area under the receiver operating characteristic curve value of 0.95 (95% CI 0.90-1.00). Optimized cutoff values identified subjects with celiac disease on a GFD with 97% sensitivity (95% CI 0.92-1.00) and 95% specificity (95% CI 0.84-1.00) vs subjects without celiac disease on a GFD. The values identified subjects with celiac disease on a gluten-containing diet with 100% sensitivity (95% CI 1.00-1.00]) and 90% specificity (95% CI 0.83-0.98) vs controls. In an analysis of 4 controls with positive results from the HLA-DQ-gluten tetramer test, 2 had unrecognized celiac disease and the remaining 2 had T cells that proliferated in response to gluten antigen in vitro. CONCLUSIONS: An HLA-DQ-gluten tetramer-based assays that detects gluten-reactive T cells identifies patients with and without celiac disease with a high level of accuracy, regardless of whether the individuals are on a GFD. This test would allow individuals with suspected celiac disease to avoid gluten challenge and duodenal biopsy, but requires validation in a larger study. Clinicaltrials.gov no: NCT02442219.
Assuntos
Doença Celíaca/diagnóstico , Citometria de Fluxo , Glutens/imunologia , Antígenos HLA-DQ/sangue , Linfócitos T/imunologia , Área Sob a Curva , Biomarcadores/sangue , Biópsia , Estudos de Casos e Controles , Doença Celíaca/sangue , Doença Celíaca/dietoterapia , Doença Celíaca/imunologia , Linhagem Celular , Proliferação de Células , Dieta Livre de Glúten , Duodeno/imunologia , Duodeno/patologia , Humanos , Ativação Linfocitária , Valor Preditivo dos Testes , Curva ROC , Reprodutibilidade dos Testes , Testes SorológicosRESUMO
Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.
Assuntos
Biomarcadores/análise , Doença Celíaca/complicações , Enteropatias/diagnóstico , Intestino Delgado/metabolismo , Espectrometria de Massas/métodos , Proteoma/análise , Adulto , Dieta Livre de Glúten , Feminino , Humanos , Enteropatias/etiologia , Enteropatias/metabolismo , Intestino Delgado/patologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Doença Celíaca/imunologia , Antígenos HLA-DQ/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linhagem Celular , Proliferação de Células , Seleção Clonal Mediada por Antígeno , Citocinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glutens/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Memória Imunológica , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação LinfocitáriaRESUMO
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.
Assuntos
Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Glutens/imunologia , Antígenos HLA-DQ/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Doença Celíaca/imunologia , Linhagem Celular , Cromatografia em Gel , Mapeamento de Epitopos/métodos , Gliadina/química , Gliadina/imunologia , Glutens/química , Antígenos HLA-DQ/química , Humanos , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Triticum/imunologiaRESUMO
CD4⺠T cells that recognize dietary gluten antigens presented by the disease-associated HLA-DQ2 or DQ8 molecules are central players in coeliac disease. Unbiased sequencing of the human TCRα variable (TRAV) and humanTCRß variable (TRBV) genes of 68 HLA-DQ2.5-glia-α2-specific T cells from coeliac disease patients confirmed previous reports of over-usage of the TRBV7-2 gene segment, a conserved Arg residue in the complementarity-determining region (CDR) 3ß loop and prevalent usage of the canonical ASSxRxTDTQY CDR3ß loop among T cells with this specificity. In 30 clones that had the canonical TCRß chain, we found a strict usage of the TRAV26-1 gene segment in the TCRα chain. There was variable usage of the TRAJ genes and diverse CDR3α sequences with no apparent conserved motifs. This study extends previous reports on biased TCR usage in both HLA-DQ2.5- and DQ8-restricted gluten-specific TCRs and provides data for further studies on TRAV and TRBV pairing.
Assuntos
Doença Celíaca/imunologia , Epitopos de Linfócito T/imunologia , Glutens/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Regiões Determinantes de Complementaridade/imunologia , Antígenos HLA-DQ/imunologia , HumanosRESUMO
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.
Assuntos
Imunidade Adaptativa , Doença Celíaca/imunologia , Doença Celíaca/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Epitopos de Linfócito T/imunologia , Proteínas de Ligação ao GTP/imunologia , Humanos , Imunoglobulina A/imunologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/imunologiaRESUMO
Autoantibodies against the enzyme transglutaminase 2 (TG2) are characteristic of celiac disease (CeD), and TG2-specific immunoglobulin (Ig) A plasma cells are abundant in gut biopsies of patients. Here, we describe the corresponding population of autoreactive B cells in blood. Circulating TG2-specific IgA cells are present in untreated patients on a gluten-containing diet but not in controls. They are clonally related to TG2-specific small intestinal plasma cells, and they express gut-homing molecules, indicating that they are plasma cell precursors. Unlike other IgA-switched cells, the TG2-specific cells are negative for CD27, placing them in the double-negative (IgD-CD27-) category. They have a plasmablast or activated memory B cell phenotype, and they harbor fewer variable region mutations than other IgA cells. Based on their similarity to naive B cells, we propose that autoreactive IgA cells in CeD are generated mainly through chronic recruitment of naive B cells via an extrafollicular response involving gluten-specific CD4+ T cells.
Assuntos
Linfócitos B , Doença Celíaca , Proteínas de Ligação ao GTP , Imunoglobulina A , Plasmócitos , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases , Doença Celíaca/imunologia , Doença Celíaca/patologia , Humanos , Transglutaminases/imunologia , Transglutaminases/metabolismo , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina A/sangue , Linfócitos B/imunologia , Linfócitos B/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/sangue , Adulto , Masculino , Feminino , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Glutens/imunologiaRESUMO
CD4+ T cells specific for cereal gluten proteins are key players in celiac disease (CeD) pathogenesis. While several CeD-relevant gluten T cell epitopes have been identified, epitopes recognized by a substantial proportion of gluten-reactive T cells remain unknown. The identification of such CeD-driving gluten epitopes is important for the food industry and in clinical settings. Here, we have combined the knowledge of a distinct phenotype of gluten-reactive T cells and key features of known gluten epitopes for the discovery of unknown epitopes. We tested 42 wheat gluten-reactive T cell clones, isolated on the basis of their distinct phenotype and with no reactivity to known epitopes, against a panel of synthetic peptides bioinformatically identified from a wheat gluten protein database. We were able to assign reactivity to 10 T cell clones and identified a 9-nucleotide oligomer core region of five previously uncharacterized gliadin/glutenin epitopes. This work represents an advance in the effort to identify CeD-driving gluten epitopes.
Assuntos
Doença Celíaca , Humanos , Doença Celíaca/metabolismo , Epitopos de Linfócito T , Glutens , Gliadina/genética , Gliadina/metabolismo , Peptídeos/metabolismoRESUMO
The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4+ T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method's efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5+ CeD patients, Ttet+ /Tphe+ , Ttet- /Tphe+ , and Ttet- /Tphe- cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet+ /Tphe+ TCCs, 65.3% of Ttet- /Tphe+ TCCs and 0% of Ttet- /Tphe- TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4+ T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4+ T cells in autoimmune conditions.
Assuntos
Doença Celíaca , Autoimunidade , Linfócitos T CD4-Positivos/patologia , Doença Celíaca/genética , Doença Celíaca/patologia , Humanos , Fenótipo , Linfócitos T/patologiaRESUMO
In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.
Assuntos
Doenças Autoimunes , COVID-19 , Animais , Linfócitos T CD8-Positivos , Humanos , Camundongos , Receptores KIR , Linfócitos T ReguladoresRESUMO
Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3ß, and paired CDR3α:CDR3ß motif candidates. Among these, a previously known conserved CDR3ß R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3ß motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRß sequences of which 145, 102 and 78 belonged to TCRα, TCRß and paired TCRαß sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent.
Assuntos
Motivos de Aminoácidos/genética , Linfócitos T CD4-Positivos/metabolismo , Glutens/genética , Receptores de Antígenos de Linfócitos T/genética , Doença Celíaca/genética , Regiões Determinantes de Complementaridade/genética , Epitopos de Linfócito T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Antígenos HLA-DQ/genética , Humanos , Epitopos Imunodominantes/genética , Ativação Linfocitária/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Gluten-specific CD4+ T cells being drivers of celiac disease (CeD) are obvious targets for immunotherapy. Little is known about how cell markers harnessed for T-cell-directed therapy can change with time and upon activation in CeD and other autoimmune conditions. In-depth characterization of gluten-specific CD4+ T cells and CeD-associated (CD38+ and CD103+ ) CD8+ and γδ+ T cells in blood of treated CeD patients undergoing a 3 day gluten challenge is reported. The phenotypic profile of gluten-specific cells changes profoundly with gluten exposure and the cells adopt the profile of gluten-specific cells in untreated disease (CD147+ , CD70+ , programmed cell death protein 1 (PD-1)+ , inducible T-cell costimulator (ICOS)+ , CD28+ , CD95+ , CD38+ , and CD161+ ), yet with some markers being unique for day 6 cells (C-X-C chemokine receptor type 6 (CXCR6), CD132, and CD147) and with integrin α4ß7, C-C motif chemokine receptor 9 (CCR9), and CXCR3 being expressed stably at baseline and day 6. Among gluten-specific CD4+ T cells, 52% are CXCR5+ at baseline, perhaps indicative of germinal-center reactions, while on day 6 all are CXCR5- . Strikingly, the phenotypic profile of gluten-specific CD4+ T cells on day 6 largely overlaps with that of CeD-associated (CD38+ and CD103+ ) CD8+ and γδ+ T cells. The antigen-induced shift in phenotype of CD4+ T cells being shared with other disease-associated T cells is relevant for development of T-cell-directed therapies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Celíaca/terapia , Glutens/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença Celíaca/imunologia , Glutens/química , Antígenos HLA-DQ/química , Antígenos HLA-DQ/imunologia , Humanos , Imunoterapia , Cadeias alfa de Integrinas/metabolismo , Linfócitos Intraepiteliais/citologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Fenótipo , Multimerização ProteicaRESUMO
Gut intraepithelial γδ and CD8+ αß T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αß T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αß T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αß TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αß T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αß T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αß T-cell clonotypes that may not be specific for the gluten antigen.
Assuntos
Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença Celíaca/etiologia , Evolução Clonal/imunologia , Cadeias alfa de Integrinas/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Evolução Clonal/genética , Glutens/imunologia , Humanos , Imuno-Histoquímica , Imunofenotipagem , Contagem de Linfócitos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Previous reports show that Ly49 + CD8 + T cells can suppress autoimmunity in mouse models of autoimmune diseases. Here we find a markedly increased frequency of CD8 + T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various autoimmune diseases. Moreover, KIR + CD8 + T cells can efficiently eliminate pathogenic gliadin-specific CD4 + T cells from Celiac disease (CeD) patients' leukocytes in vitro . Furthermore, we observe elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Expanded KIR + CD8 + T cells from these different diseases display shared phenotypes and similar T cell receptor sequences. These results characterize a regulatory CD8 + T cell subset in humans, broadly active in both autoimmune and infectious diseases, which we hypothesize functions to control self-reactive or otherwise pathogenic T cells. ONE-SENTENCE SUMMARY: Here we identified KIR + CD8 + T cells as a regulatory CD8 + T cell subset in humans that suppresses self-reactive or otherwise pathogenic CD4 + T cells.
RESUMO
To develop better vaccines and more targeted treatments for cancer and autoimmune disorders, the disease-specific T cells and their cognate antigens need to be better characterized. For more than two decades, peptide-major histocompatibility complex (pMHC) tetramers and flow cytometry have been the gold standard for detection of CD8+ and CD4+ T cells specific to antigens in the context of MHC class I and class II, respectively. Nonetheless, more recent studies combining such reagents with mass cytometry, that is, cytometry by time of flight (CyTOF), have offered far more comprehensive profiling of antigen-specific T-cell responses. In addition, mass cytometry has enabled ex vivo screening of CD8+ T-cell reactivities against hundreds of MHC class I restricted candidate epitopes. MHC class II molecules, on the other hand, have been challenging to combine with mass cytometry as they are more complex and bind with lower affinities to cognate T-cell receptors than MHC class I molecules. In this review, I discuss how techniques originally developed to improve the staining capacity of pMHC tetramers in flow cytometry led to the successful combination of such reagents with mass cytometry. Especially, I will highlight very recent advances facilitating the combination with pMHC class II tetramers. Together, these mass cytometry-based studies can help develop more targeted treatments for cancer and autoimmune disorders.