RESUMEN
Celiac disease is a human leukocyte antigen (HLA)-DQ2- and/or DQ8-associated T cell-mediated disorder that is induced by dietary gluten. Although it is established how gluten peptides bind HLA-DQ8 and HLA-DQ2, it is unclear how such peptide-HLA complexes are engaged by the T cell receptor (TCR), a recognition event that triggers disease pathology. We show that biased TCR usage (TRBV9(∗)01) underpins the recognition of HLA-DQ8-α-I-gliadin. The structure of a prototypical TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin complex shows that the TCR docks centrally above HLA-DQ8-α-I-gliadin, in which all complementarity-determining region-ß (CDRß) loops interact with the gliadin peptide. Mutagenesis at the TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin interface provides an energetic basis for the Vß bias. Moreover, CDR3 diversity accounts for TRBV9(∗)01(+) TCRs exhibiting differing reactivities toward the gliadin epitopes at various deamidation states. Accordingly, biased TCR usage is an important factor in the pathogenesis of DQ8-mediated celiac disease.
Asunto(s)
Enfermedad Celíaca/inmunología , Gliadina/inmunología , Antígenos HLA-DQ/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Epítopos de Linfocito T/inmunología , Antígenos HLA-DQ/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Dominios y Motivos de Interacción de Proteínas , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
Refractory celiac disease type II (RCDII) is a severe complication of celiac disease (CD) characterized by the presence of an enlarged clonal population of innate intraepithelial lymphocytes (IELs) lacking classical B-, T-, and natural killer (NK)-cell lineage markers (Lin-IELs) in the duodenum. In â¼50% of patients with RCDII, these Lin-IELs develop into a lymphoma for which no effective treatment is available. Current evidence indicates that the survival and expansion of these malignant Lin-IELs is driven by epithelial cell-derived IL-15. Like CD, RCDII is strongly associated with HLA-DQ2, suggesting the involvement of HLA-DQ2-restricted gluten-specific CD4+ T cells. We now show that gluten-specific CD4+ T cells isolated from CD duodenal biopsy specimens produce cytokines able to trigger proliferation of malignant Lin-IEL lines as powerfully as IL-15. Furthermore, we identify TNF, IL-2, and IL-21 as CD4+ T-cell cytokines that synergistically mediate this effect. Like IL-15, these cytokines were found to increase the phosphorylation of STAT5 and Akt and transcription of antiapoptotic mediator bcl-xL Several small-molecule inhibitors targeting the JAK/STAT pathway blocked proliferation elicited by IL-2 and IL-15, but only an inhibitor targeting the PI3K/Akt/mTOR pathway blocked proliferation induced by IL-15 as well as the CD4+ T-cell cytokines. Confirming and extending these findings, TNF, IL-2, and IL-21 also synergistically triggered the proliferation of freshly isolated Lin-IELs and CD3-CD56+ IELs (NK-IELs) from RCDII as well as non-RCDII duodenal biopsy specimens. These data provide evidence implicating CD4+ T-cell cytokines in the pathogenesis of RCDII. More broadly, they suggest that adaptive immune responses can contribute to innate IEL activation during mucosal inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Citocinas/farmacología , Linfocitos Intraepiteliales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Enfermedad Celíaca/genética , Enfermedad Celíaca/metabolismo , Proliferación Celular/genética , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Sinergismo Farmacológico , Duodeno/metabolismo , Humanos , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/farmacología , Interleucina-2/genética , Interleucina-2/metabolismo , Interleucina-2/farmacología , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/farmacología , Linfocitos Intraepiteliales/metabolismo , Proteínas Recombinantes/farmacología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In HLA-DQ8-associated celiac disease (CD), the pathogenic T cell response is directed toward an immunodominant α-gliadin-derived peptide (DQ8-glia-α1). However, our knowledge of TCR gene usage within the primary intestinal tissue of HLA-DQ8 (+) CD patients is limited. We identified two populations of HLA-DQ8-glia-α1 tetramer(+) CD4(+) T cells that were essentially undetectable in biopsy samples from patients on a gluten-free diet but expanded rapidly and specifically after antigenic stimulation. Distinguished by expression of TRBV9, both T cell populations displayed biased clonotypic repertoires and reacted similarly against HLA-DQ8-glia-α1. In particular, TRBV9 paired most often with TRAV26-2, whereas the majority of TRBV9(-) TCRs used TRBV6-1 with no clear TRAV gene preference. Strikingly, both tetramer(+)/TRBV9(+) and tetramer(+)/TRBV9(-) T cells possessed a non-germline-encoded arginine residue in their CDR3α and CDR3ß loops, respectively. Comparison of the crystal structures of three TRBV9(+) TCRs and a TRBV9(-) TCR revealed that, as a result of distinct TCR docking modes, the HLA-DQ8-glia-α1 contacts mediated by the CDR3-encoded arginine were almost identical between TRBV9(+) and TRBV9(-) TCRs. In all cases, this interaction centered on two hydrogen bonds with a specific serine residue in the bound peptide. Replacement of serine with alanine at this position abrogated TRBV9(+) and TRBV9(-) clonal T cell proliferation in response to HLA-DQ8-glia-α1. Gluten-specific memory CD4(+) T cells with structurally and functionally conserved TCRs therefore predominate in the disease-affected tissue of patients with HLA-DQ8-mediated CD.
Asunto(s)
Enfermedad Celíaca/inmunología , Selección Clonal Mediada por Antígenos/inmunología , Gliadina/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Enfermedad Celíaca/genética , Enfermedad Celíaca/metabolismo , Línea Celular , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunofenotipificación , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica/inmunología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56(-)CD127(-), CD56(-)CD127(+), CD56(+)CD127(-) and CD56(+)CD127(+). Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127(-) branch expressing high levels of interleukin (IL) 2/15Rß but no IL-21R, and a CD127(+) branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56(-)CD127(-) and CD56(-)CD127(+) innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56(-)CD127(-) IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56(-)CD127(-) IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.
Asunto(s)
Complejo CD3/análisis , Enfermedad Celíaca , Duodeno/patología , Mucosa Intestinal , Péptidos y Proteínas de Señalización Intracelular/análisis , Subgrupos de Linfocitos T , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Diferenciación Celular/inmunología , Línea Celular , Citocinas/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-7/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , ARN Polimerasa I , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patologíaRESUMEN
OBJECTIVE: Refractory coeliac disease type II (RCDII) is a severe complication of coeliac disease (CD) characterised by aberrant intraepithelial lymphocytes (IELs) of unknown origin that display an atypical CD3(-)CD7(+)icCD3(+) phenotype. In approximately 40% of patients with RCDII these lymphocytes develop into an invasive lymphoma. In the current study we aimed to identify the physiological counterpart of these cells. DESIGN: RCDII cell lines were compared with T-cell receptor positive (TCR(+)) IEL (T-IEL) lines by microarray analysis, real-time quantitative PCR and flow cytometry. This information was used to identify cells with an RCDII-associated phenotype in duodenal biopsies from non-refractory individuals by multicolour flow cytometry. RESULTS: RCDII lines were transcriptionally distinct from T-IEL lines and expressed higher levels of multiple natural killer (NK) cell receptors. In addition to the CD3(-)CD7(+)icCD3(+) phenotype, the RCDII lines were distinguishable from other lymphocyte subsets by the absence of CD56, CD127 and CD34. Cells matching this surface lineage-negative (Lin(-)) CD7(+)CD127(-)CD34(-) phenotype expressed a functional interleukin-15 (IL-15) receptor and constituted a significant proportion of IELs in duodenal specimens of patients without CD, particularly children, and were also found in the thymus. In patients without CD, the Lin(-)CD7(+)CD127(-)CD34(-) subset was one of four subsets within the CD3(-)CD7(+)icCD3(+) population that could be distinguished on the basis of differential expression of CD56 and/or CD127. CONCLUSION: Our studies indicate that the CD3(-)CD7(+)icCD3(+) population is heterogeneous and reveal the existence of a Lin(-) subset that is distinct from T, B, NK and lymphoid tissue inducer cells. We speculate that this IL-15 responsive population represents the physiological counterpart of aberrant cells expanded in RCDII and transformed in RCDII-associated lymphoma.
Asunto(s)
Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Duodeno/inmunología , Duodeno/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Linfocitos/inmunología , Linfocitos/patología , Antígenos CD/inmunología , Biomarcadores/análisis , Biopsia , Línea Celular , Células Cultivadas , Citometría de Flujo , Humanos , Interleucina-15/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de Matrices TisularesRESUMEN
In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Enfermedad Celíaca/inmunología , Citotoxicidad Inmunológica/inmunología , Células Epiteliales/inmunología , Linfocitos/inmunología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Células CACO-2 , Enfermedad Celíaca/metabolismo , Enfermedad Celíaca/patología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Células Epiteliales/metabolismo , Citometría de Flujo , Células HT29 , Humanos , Cadenas alfa de Integrinas/inmunología , Cadenas alfa de Integrinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células K562 , Linfocitos/metabolismoRESUMEN
Because susceptibility to celiac disease is associated strongly with HLA-DQ2 (DQA1*05/DQB1*02) and weakly with HLA-DQ8 (DQA1*03/DQB1*03), a subset of patients carries both HLA-DQ2 and HLA-DQ8. As a result, these patients may express two types of mixed HLA-DQ2/8 transdimers (encoded by DQA1*05/DQB1*03 and DQA1*03/DQB1*02) in addition to HLA-DQ2 and HLA-DQ8. Using T cells from a celiac disease patient expressing HLA-DQ8trans (encoded by DQA*0501/DQB*0302), but neither HLA-DQ2 nor HLA-DQ8, we demonstrate that this transdimer is expressed on the cell surface and can present multiple gluten peptides to T cell clones isolated from the duodenum of this patient. Furthermore, T cell clones derived from this patient and HLA-DQ2/8 heterozygous celiac disease patients respond to gluten peptides presented by HLA-DQ8trans, as well as HLA-DQ8, in a similar fashion. Finally, one gluten peptide is recognized better when presented by HLA-DQ8trans, which correlates with preferential binding of this peptide to HLA-DQ8trans. These results implicate HLA-DQ8trans in celiac disease pathogenesis and demonstrate extensive T cell cross-reactivity between HLA-DQ8 and HLA-DQ8trans. Because type 1 diabetes is strongly associated with the presence of HLA-DQ8trans, our findings may bear relevance to this disease as well.
Asunto(s)
Epítopos de Linfocito T/inmunología , Gliadina/inmunología , Gliadina/metabolismo , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Cadenas beta de HLA-DQ/metabolismo , Subgrupos de Linfocitos T/inmunología , Presentación de Antígeno/inmunología , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/metabolismo , Línea Celular , Línea Celular Transformada , Células Clonales , Reacciones Cruzadas/inmunología , Epítopos de Linfocito T/metabolismo , Células HEK293 , Antígenos HLA-DQ/química , Cadenas beta de HLA-DQ/química , Humanos , Multimerización de Proteína , Subgrupos de Linfocitos T/metabolismoRESUMEN
Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA.
Asunto(s)
Anemia Aplásica , Pancitopenia , Humanos , Médula Ósea , Linfocitos T CD8-positivos/patología , Ciclosporina/uso terapéutico , Suero Antilinfocítico/uso terapéuticoRESUMEN
BACKGROUND & AIMS: Patients with celiac disease have permanent intolerance to gluten. Because of the high frequency of this disorder (approximately 1 in 100 individuals), we investigated whether oral tolerance to gluten differs from that to other food proteins. METHODS: Using transgenic mice that express human HLA-DQ2 and a gliadin-specific, humanized T-cell receptor, we compared gluten-specific T-cell responses with tolerogenic mucosal T-cell responses to the model food protein ovalbumin. RESULTS: Consistent with previous findings, the ovalbumin-specific response occurred in the mesenteric lymph nodes and induced Foxp3(+) regulatory T cells. In contrast, ingestion of deamidated gliadin induced T-cell proliferation predominantly in the spleen but little in mesenteric lymph nodes. The gliadin-reactive T cells had an effector-like phenotype and secreted large amounts of interferon gamma but also secreted interleukin-10. Despite their effector-like phenotype, gliadin-reactive T cells had regulatory functions, because transfer of the cells suppressed a gliadin-induced, delayed-type hypersensitivity response. CONCLUSIONS: Ingestion of deamidated gliadin induces differentiation of tolerogenic, type 1 regulatory T cells in spleens of HLA-DQ2 transgenic mice. These data indicate that under homeostatic conditions, the T-cell response to deamidated gliadin is tolerance, which is not conditioned by the mucosal immune system but instead requires interleukin-10 induction by antigen presentation in the spleen.
Asunto(s)
Gliadina/inmunología , Tolerancia Inmunológica/inmunología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Animales , Quimiocina CCL2/metabolismo , Factores de Transcripción Forkhead/inmunología , Gliadina/farmacología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ganglios Linfáticos/inmunología , Mesenterio , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ovalbúmina/inmunología , Bazo/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The performance of Gluten-Tec (EuroProxima, Arnhem, The Netherlands) was tested through an interlaboratory study in accordance with AOAC guidelines. Gluten-Tec is a competitive ELISA that detects an immunostimulatory epitope of a-gliadin in dietary food for celiacs. Fifteen laboratories, representing 14 different countries, announced their interest in taking part in this study. Of the 12 laboratories that sent the results within the established timeframe, two submitted inappropriate standard curves and were excluded from the statistical analysis. Four different food matrixes (rice-based baby food, maize bread, chocolate cake mix, and beer) were selected for preparing the test samples. Two gliadin extraction procedures were used: the conventional 60% ethanol, and a new method based on the reducing reagent dithiothreitol. The 38 samples (19 blind duplicates) tested in this study were prepared by diluting the different extracts in order to cover a wide range of gliadin levels. Both sample extraction and dilution were performed by EuroProxima; the present interlaboratory study was focused only on testing the ELISA part of the Gluten-Tec kit protocol. Repeatability values (within-laboratory variance), expressed as RSD(r) ranged from 6.2 to 25.7%, while reproducibility values (interlaboratory variance), expressed as RSD(R), ranged from 10.6 to 45.9%. Both statistical parameters were in the acceptable range of ELISAs under these conditions, and the method will be presented to the Codex Alimentarius as a preferred method for gluten analysis.
Asunto(s)
Enfermedad Celíaca/inducido químicamente , Ensayo de Inmunoadsorción Enzimática/métodos , Péptidos/análisis , Alérgenos/análisis , Cerveza/análisis , Cromatografía Líquida de Alta Presión , Proteínas en la Dieta/análisis , Análisis de los Alimentos , Hipersensibilidad a los Alimentos/inmunología , Gliadina/análisis , Gliadina/aislamiento & purificación , Glútenes/análisis , Humanos , Indicadores y Reactivos , Lactante , Alimentos Infantiles , Límite de Detección , Juego de Reactivos para Diagnóstico , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND & AIMS: Refractory celiac disease type II (RCDII) is a rare indolent lymphoma in the small intestine characterized by a clonally expanded intraepithelial intracellular CD3+surfaceCD3-CD7+CD56- aberrant cell population. However, RCDII pathogenesis is ill-defined. Here, we aimed at single-cell characterization of the innate and adaptive immune system in RCDII. METHODS: Paired small intestinal and blood samples from 12 RCDII patients and 6 healthy controls were assessed by single-cell mass cytometry with a 39-cell surface marker antibody panel, designed to capture heterogeneity of the innate and adaptive immune system. A second single-cell mass cytometry panel that included transcription factors and immune checkpoints was used for analysis of paired samples from 5 RCDII patients. Single-cell RNA sequencing analysis was performed on duodenal samples from 2 RCDII patients. Finally, we developed a 40-marker imaging mass cytometry antibody panel to evaluate cell-cell interactions in duodenal biopsy specimens of RCDII patients. RESULTS: We provide evidence for intertumoral and intratumoral cell heterogeneity within the duodenal and peripheral aberrant cell population present in RCDII. Phenotypic discrepancy was observed between peripheral and duodenal aberrant cells. In addition, we observed that part of the aberrant cell population proliferated and observed co-localization of aberrant cells with CD163+ antigen-presenting cells (APCs) in situ. In addition, we observed phenotypic discrepancy between peripheral and duodenal aberrant cells. CONCLUSIONS: Novel high-dimensional single-cell technologies show substantial intertumoral and intratumoral heterogeneity in the aberrant cell population in RCDII. This may underlie variability in refractory disease status between patients and responsiveness to therapy, pointing to the need for personalized therapy in RCDII based on patient-specific immune profiles.
Asunto(s)
Enfermedad Celíaca , Biomarcadores , Enfermedad Celíaca/genética , Duodeno/patología , Humanos , Intestino Delgado/patología , Análisis de la Célula IndividualRESUMEN
Celiac Disease (CeD) is a complex immune disorder involving villous atrophy in the small intestine that is triggered by gluten intake. Current CeD diagnosis is based on late-stage pathophysiological parameters such as detection of specific antibodies in blood and histochemical detection of villus atrophy and lymphocyte infiltration in intestinal biopsies. To date, no early onset biomarkers are available that would help prevent widespread villous atrophy and severe symptoms and co-morbidities. To search for novel CeD biomarkers, we used single-cell RNA sequencing (scRNAseq) to investigate PBMC samples from 11 children before and after seroconversion for CeD and 10 control individuals matched for age, sex and HLA-genotype. We generated scRNAseq profiles of 9559 cells and identified the expected major cellular lineages. Cell proportions remained stable across the different timepoints and health conditions, but we observed differences in gene expression profiles in specific cell types when comparing patient samples before and after disease development and comparing patients with controls. Based on the time when transcripts were differentially expressed, we could classify the deregulated genes as biomarkers for active CeD or as potential pre-diagnostic markers. Pathway analysis showed that active CeD biomarkers display a transcriptional profile associated with antigen activation in CD4+ T cells, whereas NK cells express a subset of biomarker genes even before CeD diagnosis. Intersection of biomarker genes with CeD-associated genetic risk loci pinpointed genetic factors that might play a role in CeD onset. Investigation of potential cellular interaction pathways of PBMC cell subpopulations highlighted the importance of TNF pathways in CeD. Altogether, our results pinpoint genes and pathways that are altered prior to and during CeD onset, thereby identifying novel potential biomarkers for CeD diagnosis in blood.
Asunto(s)
Enfermedad Celíaca , Atrofia , Biomarcadores , Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/genética , Niño , Humanos , Leucocitos Mononucleares/metabolismo , Análisis de Secuencia de ARN , SeroconversiónRESUMEN
Chronic intestinal inflammation underlies inflammatory bowel disease (IBD). Previous studies indicated alterations in the cellular immune system; however, it has been challenging to interrogate the role of all immune cell subsets simultaneously. Therefore, we aimed to identify immune cell types associated with inflammation in IBD using high-dimensional mass cytometry. We analyzed 188 intestinal biopsies and paired blood samples of newly-diagnosed, treatment-naive patients (n=42) and controls (n=26) in two independent cohorts. We applied mass cytometry (36-antibody panel) to resolve single cells and analyzed the data with unbiased Hierarchical-SNE. In addition, imaging-mass cytometry (IMC) was performed to reveal the spatial distribution of the immune subsets in the tissue. We identified 44 distinct immune subsets. Correlation network analysis identified a network of inflammation-associated subsets, including HLA-DR+CD38+ EM CD4+ T cells, T regulatory-like cells, PD1+ EM CD8+ T cells, neutrophils, CD27+ TCRγδ cells and NK cells. All disease-associated subsets were validated in a second cohort. This network was abundant in a subset of patients, independent of IBD subtype, severity or intestinal location. Putative disease-associated CD4+ T cells were detectable in blood. Finally, imaging-mass cytometry revealed the spatial colocalization of neutrophils, memory CD4+ T cells and myeloid cells in the inflamed intestine. Our study indicates that a cellular network of both innate and adaptive immune cells colocalizes in inflamed biopsies from a subset of patients. These results contribute to dissecting disease heterogeneity and may guide the development of targeted therapeutics in IBD.
Asunto(s)
Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Linfocitos T CD8-positivos , Humanos , Inflamación , Intestinos/patologíaRESUMEN
Celiac disease is an auto-immune disease in which an immune response to dietary gluten leads to inflammation and subsequent atrophy of small intestinal villi, causing severe bowel discomfort and malabsorption of nutrients. The major instigating factor for the immune response in celiac disease is the activation of gluten-specific CD4+ T cells expressing T cell receptors that recognize gluten peptides presented in the context of HLA-DQ2 and DQ8. Here we provide an in-depth characterization of 28 gluten-specific T cell clones. We assess their transcriptional and epigenetic response to T cell receptor stimulation and link this to genetic factors associated with celiac disease. Gluten-specific T cells have a distinct transcriptional profile that mostly resembles that of Th1 cells but also express cytokines characteristic of other types of T-helper cells. This transcriptional response appears not to be regulated by changes in chromatin state, but rather by early upregulation of transcription factors and non-coding RNAs that likely orchestrate the subsequent activation of genes that play a role in immune pathways. Finally, integration of chromatin and transcription factor binding profiles suggest that genes activated by T cell receptor stimulation of glutenspecific T cells may be impacted by genetic variation at several genetic loci associated with celiac disease.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Celíaca/inducido químicamente , Enfermedad Celíaca/patología , Citocinas/inmunología , Citocinas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glútenes/administración & dosificación , Glútenes/inmunología , Humanos , Receptores de Antígenos de Linfocitos T/genética , TranscriptomaRESUMEN
Enteropathy-associated T-cell lymphoma, an often fatal complication of celiac disease, can result from expansion of aberrant intraepithelial lymphocytes in refractory celiac disease type II (RCD II). Aberrant intraepithelial lymphocytes and lymphoma cells are intracellularly CD3epsilon(+) but lack expression of the T-cell receptor (TCR)-CD3 complex on the cell surface. It is unknown what causes the loss of TCR-CD3 expression. We report the isolation of a cell line from an RCD II patient with the characteristic phenotype of enteropathy-associated T-cell lymphoma. We demonstrate that in this cell line the TCR-alpha and -beta chains as well as the CD3gamma, CD3delta, CD3epsilon, and zeta-chains are present intracellularly and that assembly of the CD3gammaepsilon, CD3deltaepsilon, and zetazeta-dimers is normal. However, dimerization of the TCR chains and proper assembly of the TCR-CD3 complex are defective. On introduction of exogenous TCR-beta chains, but not of TCR-alpha chains, assembly and functional cell surface expression of the TCR-CD3 complex were restored. Defective synthesis of both TCR chains was found to underlie loss of TCR expression in similar cell lines isolated from 2 additional patients. (Pre)malignant transformation in RCD II thus correlates with defective synthesis or defective association of the TCR chains, resulting in loss of surface TCR-CD3 expression.
Asunto(s)
Complejo CD3 , Enfermedad Celíaca/complicaciones , Linfoma de Células T/etiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/deficiencia , Receptores de Antígenos de Linfocitos T , Biopsia , Línea Celular Tumoral , Transformación Celular Neoplásica , Dimerización , Humanos , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Multimerización de ProteínaRESUMEN
The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD.
Asunto(s)
Bacterias/inmunología , Enfermedad Celíaca/inmunología , Gliadina/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Triticum/inmunología , Bacterias/química , Enfermedad Celíaca/microbiología , Línea Celular , Células Cultivadas , Reacciones Cruzadas , Cristalografía por Rayos X , Epítopos/inmunología , Gliadina/química , Glútenes/inmunología , Antígenos HLA-DQ/química , Antígenos HLA-DQ/inmunología , Humanos , Modelos Moleculares , Imitación Molecular , Péptidos/química , Receptores de Antígenos de Linfocitos T/química , Linfocitos T/inmunología , Triticum/químicaRESUMEN
Celiac disease (CeD) is a complex T cell-mediated enteropathy induced by gluten. Although genome-wide association studies have identified numerous genomic regions associated with CeD, it is difficult to accurately pinpoint which genes in these loci are most likely to cause CeD. We used four different in silico approaches-Mendelian randomization inverse variance weighting, COLOC, LD overlap, and DEPICT-to integrate information gathered from a large transcriptomics dataset. This identified 118 prioritized genes across 50 CeD-associated regions. Co-expression and pathway analysis of these genes indicated an association with adaptive and innate cytokine signaling and T cell activation pathways. Fifty-one of these genes are targets of known drug compounds or likely druggable genes, suggesting that our methods can be used to pinpoint potential therapeutic targets. In addition, we detected 172 gene combinations that were affected by our CeD-prioritized genes in trans. Notably, 41 of these trans-mediated genes appear to be under control of one master regulator, TRAF-type zinc finger domain containing 1 (TRAFD1), and were found to be involved in interferon (IFN)γ signaling and MHC I antigen processing/presentation. Finally, we performed in vitro experiments in a human monocytic cell line that validated the role of TRAFD1 as an immune regulator acting in trans. Our strategy confirmed the role of adaptive immunity in CeD and revealed a genetic link between CeD and IFNγ signaling as well as with MHC I antigen processing, both major players of immune activation and CeD pathogenesis.
RESUMEN
Wheat gluten confers superior baking quality to wheat based products but elicits a pro-inflammatory immune response in patients with celiac disease. Transamidation of gluten by microbial transglutaminase (mTG) and tissue transglutaminase (tTG) reduces the immunogenicity of gluten; however, little information is available on the minimal modification sufficient to eliminate gliadin immunogenicity nor has the effectiveness of transamidation been studied with T-cell clones from patients. Here we demonstrate that mTG can efficiently couple three different acyl-acceptor molecules, l-lysine, glycine ethyl ester, and hydroxylamine, to gliadin peptides and protein. While all three acyl-acceptor molecules were cross-linked to the same Q-residues, not all modifications were equally effective in silencing T-cell reactivity. Finally, we observed that tTG can partially reverse the mTG-catalyzed transamidation by its isopeptidase activity. These results set the stage to determine the impact of these modifications on the baking quality of gluten proteins and in vivo immunogenicity of such food products.
Asunto(s)
Gliadina/química , Gliadina/inmunología , Streptomyces/enzimología , Transglutaminasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Glútenes/química , Glútenes/inmunología , Humanos , Estructura Molecular , Streptomyces/genética , Linfocitos T/inmunología , Ácido Tranexámico/inmunología , Transglutaminasas/genéticaRESUMEN
In HLA-DQ8-associated celiac disease, TRAV26-2+-TRBV9+ and TRAV8-3+-TRBV6+ T cells recognize the immunodominant DQ8-glia-α1 epitope, whereupon a non-germline-encoded arginine residue played a key role in binding HLA-DQ8-glia-α1. Whether distinct T cell receptor (TCR) recognition modes exist for gliadin epitopes remains unclear. TCR repertoire analysis revealed populations of HLA-DQ8-glia-α1 and HLA-DQ8.5-glia-γ1 restricted TRAV20+-TRBV9+ T cells that did not possess a non-germline-encoded arginine residue. The crystal structures of a TRAV20+-TRBV9+ TCR-HLA-DQ8-glia-α1 complex and two TRAV20+-TRBV9+ TCR-HLA-DQ8.5-glia-γ1 complexes were determined. This revealed that the differential specificity toward DQ8-glia-α1 and DQ8.5-glia-γ1 was governed by CDR3ß-loop-mediated interactions. Surprisingly, a germline-encoded arginine residue within the CDR1α loop of the TRAV20+ TCR substituted for the role of the non-germline-encoded arginine in the TRAV26-2+-TRBV9+ and TRAV8-3+-TRBV6+ TCRs. Thus, in celiac disease, the responding TCR repertoire is driven by a common mechanism that selects for structural elements within the TCR that have convergent binding solutions in HLA-DQ8-gliadin recognition.
Asunto(s)
Enfermedad Celíaca/inmunología , Gliadina/inmunología , Antígenos HLA-DQ/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Presentación de Antígeno , Células Cultivadas , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Humanos , Epítopos Inmunodominantes , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
In patients with celiac disease, but not in healthy controls, gluten-specific CD4 T cells are present in the small intestinal lamina propria. Specific stimulation of these T cells due to gluten consumption leads to the release of pro-inflammatory cytokines, in particular IFNγ and IL-21. This leads to tissue damage, the typical morphological alterations like the flattening of the intestinal epithelium, and a variety of disease-associated symptoms including malnutrition, diarrhea, stomach ache, and failure to thrive. Removal of gluten from the diet eliminates the trigger for these CD4 T cells and leads to recovery. These CD4 T cells thus play a crucial role in the disease pathogenesis. Here we describe how such T cells can be isolated and characterized.