RESUMEN
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.
Asunto(s)
Enfermedad Celíaca , Duodeno , Interleucina-7 , Mucosa Intestinal , Modelos Biológicos , Organoides , Humanos , Autoanticuerpos/inmunología , Autoinmunidad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biopsia , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Enfermedad Celíaca/metabolismo , Duodeno/inmunología , Duodeno/patología , Duodeno/metabolismo , Epítopos/inmunología , Glútenes/inmunología , Glútenes/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Interleucina-7/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Organoides/inmunología , Organoides/metabolismo , Organoides/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The loading of processed peptides on to major histocompatibility complex II (MHC-II) molecules for recognition by T cells is vital to cell-mediated adaptive immunity. As part of this process, MHC-II associates with the invariant chain (Ii) during biosynthesis in the endoplasmic reticulum to prevent premature peptide loading and to serve as a scaffold for subsequent proteolytic processing into MHC-II-CLIP. Cryo-electron microscopy structures of full-length Human Leukocyte Antigen-DR (HLA-DR) and HLA-DQ complexes associated with Ii, resolved at 3.0 to 3.1 Å, elucidate the trimeric assembly of the HLA/Ii complex and define atomic-level interactions between HLA, Ii transmembrane domains, loop domains, and class II-associated invariant chain peptides (CLIP). Together with previous structures of MHC-II peptide loading intermediates DO and DM, our findings complete the structural path governing class II antigen presentation.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B , Microscopía por Crioelectrón , Antígenos de Histocompatibilidad Clase II , Humanos , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Antígenos HLA-DR/inmunología , Presentación de Antígeno , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DQ/inmunología , Modelos Moleculares , Retículo Endoplásmico/metabolismo , Conformación Proteica , Unión ProteicaRESUMEN
Understanding how MHC class II (MHC-II) binding peptides with differing lengths exhibit specific interaction at the core and extended sites within the large MHC-II pocket is a very important aspect of immunological research for designing peptides. Certain efforts were made to generate peptide conformations amenable for MHC-II binding and calculate the binding energy of such complex formation but not directed toward developing a relationship between the peptide conformation in MHC-II structures and the binding affinity (BA) (IC50 ). We present here a machine-learning approach to calculate the BA of the peptides within the MHC-II pocket for HLA-DRA1, HLA-DRB1, HLA-DP, and HLA-DQ allotypes. Instead of generating ensembles of peptide conformations conventionally, the biased mode of conformations was created by considering the peptides in the crystal structures of pMHC-II complexes as the templates, followed by site-directed peptide docking. The structural interaction fingerprints generated from such docked pMHC-II structures along with the Moran autocorrelation descriptors were trained using a random forest regressor specific to each MHC-II peptide lengths (9-19). The entire workflow is automated using Linux shell and Perl scripts to promote the utilization of MHC2AffyPred program to any characterized MHC-II allotypes and is made for free access at https://github.com/SiddhiJani/MHC2AffyPred. The MHC2AffyPred attained better performance (correlation coefficient [CC] of .612-.898) than MHCII3D (.03-.594) and NetMHCIIpan-3.2 (.289-.692) programs in the HLA-DRA1, HLA-DRB1 types. Similarly, the MHC2AffyPred program achieved CC between .91 and .98 for HLA-DP and HLA-DQ peptides (13-mer to 17-mer). Further, a case study on MHC-II binding 15-mer peptides of severe acute respiratory syndrome coronavirus-2 showed very close competency in computing the IC50 values compared to the sequence-based NetMHCIIpan v3.2 and v4.0 programs with a correlation of .998 and .570, respectively.
Asunto(s)
COVID-19 , Humanos , Cadenas HLA-DRB1/metabolismo , Péptidos/química , Antígenos HLA-DP/química , Antígenos HLA-DP/metabolismo , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Aprendizaje Automático , Unión ProteicaRESUMEN
Severe aplastic anemia (SAA) is a bone marrow failure disorder caused by autoimmune dysfunction. The presentation by dendritic cells (DCs) is the key step in initiating the immune response against unknown antigens in SAA patients. In the previous phase, we found that compared to healthy controls, patients with SAA had an increased proportion of circulating myeloid/conventional dendritic cells (mDCs/cDCs) with enhanced phagocytosis, more secretion of Th1-type cytokines (IL-2, TNF-α, IFN-γ) in the bone marrow, and a reduced proportion of Treg cells. In this study, we found that cDCs sorted from SAA patients had higher expression level of HLA-DQ, co-stimulatory molecules CD86, PTK and ERK1/2 than the remission SAA patients and healthy controls. Moreover, downregulation of HLA-DQ protein levels on cDCs derived from SAA patients resulted in reduced phagocytosis rate and CD86 expression of cDCs. When the cDCs above were co-cultured with CD4+ cells from the same patients, reduced secretion of Th1 type of lymphocyte cytokines was observed. Analysis of clinically relevant data suggests that HLA-DQ expression levels were closely related to disease severity and immune status of patients. These findings show that the role of HLA-DQ in the immunopathogenesis of SAA is potentially important and worth further study.
Asunto(s)
Anemia Aplásica , Humanos , Médula Ósea/patología , Factor de Necrosis Tumoral alfa , Antígenos HLA-DQ/metabolismoRESUMEN
The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5-mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.
Asunto(s)
Enfermedad Celíaca , Antígenos HLA-DQ , Receptores de Antígenos de Linfocitos T , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/metabolismo , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/metabolismo , Línea Celular , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Glútenes/química , Glútenes/inmunología , Glútenes/metabolismo , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Immunogenicity of gliadin peptides in celiac disease (CD) is majorly determined by the pattern of molecular interactions with HLA-DQ and T-cell receptors (TCR). Investigation of the interactions between immune-dominant gliadin peptides, DQ protein, and TCR are warranted to unravel the basis of immunogenicity and variability contributed by the genetic polymorphisms. Homology modeling of HLA and TCR done using Swiss Model and iTASSER, respectively. Molecular interactions of eight common deamidated immune-dominant gliadin with HLA-DQ allotypes and specific TCR gene pairs were evaluated. Docking of the three structures was performed with ClusPro2.0 and ProDiGY was used to predict binding energies. Effects of known allelic polymorphisms and reported susceptibility SNPs were predicted on protein-protein interactions. CD susceptible allele, HLA-DQ2.5 was shown to have considerable binding affinity to 33-mer gliadin (ΔG = - 13.9; Kd = 1.5E - 10) in the presence of TRAV26/TRBV7. Higher binding affinity was predicted (ΔG = - 14.3, Kd = 8.9E - 11) when TRBV28 was replaced with TRBV20 paired with TRAV4 suggesting its role in CD predisposition. SNP rs12722069 at HLA-DQ8 that codes Arg76α forms three H-bonds with Glu12 and two H-bonds with Asn13 of DQ2 restricted gliadin in the presence of TRAV8-3/TRBV6. None of the HLA-DQ polymorphisms was found to be in linkage disequilibrium with reported CD susceptibility markers. Haplotypic presentations of rs12722069-G, rs1130392-C, rs3188043-C and rs4193-A with CD reported SNPs were observed in sub-ethnic groups. Highly polymorphic sites of HLA alleles and TCR variable regions could be utilized for better risk prediction models in CD. Therapeutic strategies by identifying inhibitors or blockers targeting specific gliadin:HLA-DQ:TCR binding sites could be investigated.
Asunto(s)
Enfermedad Celíaca , Humanos , Enfermedad Celíaca/genética , Enfermedad Celíaca/metabolismo , Gliadina/genética , Gliadina/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Polimorfismo Genético , Péptidos/metabolismoRESUMEN
BACKGROUND: Genetic ancestry plays a role in asthma health disparities. OBJECTIVE: Our aim was to evaluate the impact of ancestry on and identify genetic variants associated with asthma, total serum IgE level, and lung function. METHODS: A total of 436 Peruvian children (aged 9-19 years) with asthma and 291 without asthma were genotyped by using the Illumina Multi-Ethnic Global Array. Genome-wide proportions of indigenous ancestry populations from continental America (NAT) and European ancestry from the Iberian populations in Spain (IBS) were estimated by using ADMIXTURE. We assessed the relationship between ancestry and the phenotypes and performed a genome-wide association study. RESULTS: The mean ancestry proportions were 84.7% NAT (case patients, 84.2%; controls, 85.4%) and 15.3% IBS (15.8%; 14.6%). With adjustment for asthma, NAT was associated with higher total serum IgE levels (P < .001) and IBS was associated with lower total serum IgE levels (P < .001). NAT was associated with higher FEV1 percent predicted values (P < .001), whereas IBS was associated with lower FEV1 values in the controls but not in the case patients. The HLA-DR/DQ region on chromosome 6 (Chr6) was strongly associated with total serum IgE (rs3135348; P = 3.438 × 10-10) and was independent of an association with the haplotype HLA-DQA1â¼HLA-DQB1:04.01â¼04.02 (P = 1.55 × 10-05). For lung function, we identified a locus (rs4410198; P = 5.536 × 10-11) mapping to Chr19, near a cluster of zinc finger interacting genes that colocalizes to the long noncoding RNA CTD-2537I9.5. This novel locus was replicated in an independent sample of pediatric case patients with asthma with similar admixture from Brazil (P = .005). CONCLUSION: This study confirms the role of HLA in atopy, and identifies a novel locus mapping to a long noncoding RNA for lung function that may be specific to children with NAT.
Asunto(s)
Asma/genética , Genotipo , Inmunoglobulina E/metabolismo , Pueblos Indígenas , Pulmón/metabolismo , Adolescente , Américas , Asma/epidemiología , Niño , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Antígenos HLA-DQ/metabolismo , Humanos , Pulmón/inmunología , Masculino , Perú/epidemiología , Polimorfismo de Nucleótido Simple , ARN Largo no Codificante/genética , España , Adulto JovenRESUMEN
Binding prediction tools are commonly used to identify peptides presented on MHC class II molecules. Recently, a wealth of data in the form of naturally eluted ligands has become available and discrepancies between ligand elution data and binding predictions have been reported. Quantitative metrics for such comparisons are currently lacking. In this study, we assessed how efficiently MHC class II binding predictions can identify naturally eluted peptides, and investigated instances with discrepancies between the two methods in detail. We found that, in general, MHC class II eluted ligands are predicted to bind to their reported restriction element with high affinity. But, for several studies reporting an increased number of ligands that were not predicted to bind, we found that the reported MHC restriction was ambiguous. Additional analyses determined that most of the ligands predicted to not bind, are predicted to bind other co-expressed MHC class II molecules. For selected alleles, we addressed discrepancies between elution data and binding predictions by experimental measurements and found that predicted and measured affinities correlate well. For DQA1*05:01/DQB1*02:01 (DQ2.5) however, binding predictions did miss several peptides that were determined experimentally to be binders. For these peptides and several known DQ2.5 binders, we determined key residues for conferring DQ2.5 binding capacity, which revealed that DQ2.5 utilizes two different binding motifs, of which only one is predicted effectively. These findings have important implications for the interpretation of ligand elution data and for the improvement of MHC class II binding predictions.
Asunto(s)
Antígenos HLA-DQ/metabolismo , Alelos , Sitios de Unión/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ligandos , Péptidos/metabolismo , Unión Proteica/fisiologíaRESUMEN
Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls' DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.
Asunto(s)
Actinas/metabolismo , Enfermedad Celíaca/metabolismo , Forma de la Célula , Células Dendríticas/inmunología , Monocitos/metabolismo , Enfermedad Celíaca/patología , Adhesión Celular , Niño , Preescolar , Células Dendríticas/patología , Femenino , Fibronectinas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Antígenos HLA-DQ/metabolismo , Humanos , Masculino , Monocitos/patología , Fosfoproteínas/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The autoimmune condition, Celiac Disease (CeD), displays broad clinical symptoms due to gluten exposure. Its genetic association with DQ variants in the human leukocyte antigen (HLA) system has been recognised. Monocyte-derived mature dendritic cells (MoDCs) present gluten peptides through HLA-DQ and co-stimulatory molecules to T lymphocytes, eliciting a cytokine-rich microenvironment. Having access to CeD associated families prevalent in the Czech Republic, this study utilised an in vitro model to investigate their differential monocyte profile. The higher monocyte yields isolated from PBMCs of CeD patients versus control individuals also reflected the greater proportion of dendritic cells derived from these sources following lipopolysaccharide (LPS)/ peptic-tryptic-gliadin (PTG) fragment stimulation. Cell surface markers of CeD monocytes and MoDCs were subsequently profiled. This foremost study identified a novel bio-profile characterised by elevated CD64 and reduced CD33 levels, unique to CD14++ monocytes of CeD patients. Normalisation to LPS stimulation revealed the increased sensitivity of CeD-MoDCs to PTG, as shown by CD86 and HLA-DQ flow cytometric readouts. Enhanced CD86 and HLA-DQ expression in CeD-MoDCs were revealed by confocal microscopy. Analysis highlighted their dominance at the CeD-MoDC membrane in comparison to controls, reflective of superior antigen presentation ability. In conclusion, this investigative study deciphered the monocytes and MoDCs of CeD patients with the identification of a novel bio-profile marker of potential diagnostic value for clinical interpretation. Herein, the characterisation of CD86 and HLA-DQ as activators to stimulants, along with robust membrane assembly reflective of efficient antigen presentation, offers CeD targeted therapeutic avenues worth further exploration.
Asunto(s)
Presentación de Antígeno/inmunología , Enfermedad Celíaca/inmunología , Células Dendríticas/inmunología , Gliadina/efectos adversos , Adolescente , Adulto , Anciano , Antígenos CD/metabolismo , Enfermedades Autoinmunes/inmunología , Biomarcadores/metabolismo , Enfermedad Celíaca/epidemiología , Membrana Celular/metabolismo , República Checa/epidemiología , Susceptibilidad a Enfermedades , Familia , Femenino , Antígenos HLA-DQ/metabolismo , Humanos , Lipopolisacáridos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Linaje , Linfocitos T Citotóxicos/inmunología , Adulto JovenRESUMEN
Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.
Asunto(s)
Enfermedad Celíaca/tratamiento farmacológico , Toxina del Cólera/administración & dosificación , Citocinas/metabolismo , Gliadina/administración & dosificación , Antígenos HLA-DQ/genética , Transglutaminasas/metabolismo , Administración Intranasal , Animales , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Toxina del Cólera/inmunología , Citocinas/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Regulación de la Expresión Génica , Gliadina/química , Gliadina/genética , Gliadina/inmunología , Antígenos HLA-DQ/metabolismo , Inmunización , Epítopos Inmunodominantes/inmunología , Ratones , Ratones Transgénicos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunologíaRESUMEN
AIMS/HYPOTHESIS: Heterogeneity in individuals with type 1 diabetes has become more generally appreciated, but has not yet been extensively and systematically characterised. Here, we aimed to characterise type 1 diabetes heterogeneity by creating immunological, genetic and clinical profiles for individuals with juvenile-onset type 1 diabetes in a cross-sectional study. METHODS: Participants were HLA-genotyped to determine HLA-DR-DQ risk, and SNP-genotyped to generate a non-HLA genetic risk score (GRS) based on 93 type 1 diabetes-associated SNP variants outside the MHC region. Islet autoimmunity was assessed as T cell proliferation upon stimulation with the beta cell antigens GAD65, islet antigen-2 (IA-2), preproinsulin (PPI) and defective ribosomal product of the insulin gene (INS-DRIP). Clinical parameters were collected retrospectively. RESULTS: Of 80 individuals, 67 had proliferation responses to one or more islet antigens, with vast differences in the extent of proliferation. Based on the multitude and amplitude of the proliferation responses, individuals were clustered into non-, intermediate and high responders. High responders could not be characterised entirely by enrichment for the highest risk HLA-DR3-DQ2/DR4-DQ8 genotype. However, high responders did have a significantly higher non-HLA GRS. Clinically, high T cell responses to beta cell antigens did not reflect in worsened glycaemic control, increased complications, development of associated autoimmunity or younger age at disease onset. The number of beta cell antigens that an individual responded to increased with disease duration, pointing to chronic islet autoimmunity and epitope spreading. CONCLUSIONS/INTERPRETATION: Collectively, these data provide new insights into type 1 diabetes disease heterogeneity and highlight the importance of stratifying patients on the basis of their genetic and autoimmune signatures for immunotherapy and personalised disease management.
Asunto(s)
Autoinmunidad/fisiología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Adolescente , Adulto , Autoinmunidad/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Niño , Preescolar , Estudios Transversales , Diabetes Mellitus Tipo 1/genética , Femenino , Genotipo , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Insulina/metabolismo , Masculino , Análisis de Componente Principal , Precursores de Proteínas/metabolismo , Estudios Retrospectivos , Linfocitos T/metabolismo , Adulto JovenRESUMEN
Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-d-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23-DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Antígenos HLA-DQ/metabolismo , Células Secretoras de Insulina/inmunología , Péptidos/antagonistas & inhibidores , Animales , Presentación de Antígeno/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Femenino , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/aislamiento & purificación , Humanos , Células Secretoras de Insulina/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Simulación de Dinámica Molecular , Péptidos/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Newly expressed proteins in genetically modified (GM) crops are subject to celiac disease risk assessment according to EFSA guidelines. Amino acid identity matches between short peptides (9aa) and known celiac restricted epitopes are required to be further evaluated through peptide modeling; however, validated methods and criteria are not yet available. In this investigation, several structures of HLA-DQ2.5/peptide/TCR (T-cell receptor) complexes were analyzed and two template-based peptide molding software packages were evaluated using various peptides including ones not associated with celiac disease. Structural characterization indicates that residues at P(position)1, P2, P5, P8, and P9 in the 9aa restricted epitopes also contribute to the binding of celiac peptides to the HLA-DQ2.5 antigen in addition to the presence of the motif Q/EX1PX2 starting at P4 or P6. The recognition of the HLA-DQ2.5/peptide complex by TCR is through specific interactions between the residues in the restricted epitopes and some loop structures in the TCR. The template-based software package GalaxyPepDock seems to be suitable for the application of peptide modeling when an estimated accuracy value of >0.95 combined with >160 interaction similarity score are used as a threshold for biologically meaningful in silico binding. Nevertheless, caution should be exercised when applying peptide modeling to celiac disease risk assessment until methods are rigorously validated and further evaluated to demonstrate its value in the risk assessment of newly expressed proteins in GM crops.
Asunto(s)
Enfermedad Celíaca , Antígenos HLA-DQ/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Receptores de Antígenos de Linfocitos T/metabolismo , Medición de Riesgo/métodos , Epítopos , Inocuidad de los Alimentos , Humanos , Proteínas de Plantas/genéticaRESUMEN
BACKGROUND: Regulatory T cells (Tregs) inhibit the activation of cluster of differentiation (CD) 4+ , CD8+ T cells and the antigen-presenting process of antigen-presenting cells, and may play an important role in acquired severe aplastic anemia (SAA). METHODS: Flow cytometry was used to measure CD4+ CD25+ CD127dim Tregs, cytotoxic T lymphocyte antigen 4 (CTLA-4) expression on Tregs, and human leukocyte antigen (HLA)-DQ expression on myeloid dendritic cells (mDCs). The correlations of CTLA-4 and HLA-DQ with immune status and clinical indicators and the changes in these indicators after immunosuppressive therapy (IST) were analyzed. RESULTS: In SAA patients, the number of Tregs and their CTLA-4 expression were low but recovered after IST; the HLA-DQ expression on mDCs was high but decreased after IST. The CTLA-4 expression on Tregs and the HLA-DQ expression on mDCs showed a negative correlation. The CTLA-4 on Tregs was positively but HLA-DQ on mDCs negatively correlated with the number of Tregs, natural killer (NK) cell number, and CD4+ T/CD8+ T ratio. CTLA-4 was positively but HLA-DQ negatively correlated with the percentage of granulocytoid and erythroid cells in bone marrow, white blood cell count in PB, absolute neutrophil count in PB, and the percentage of reticulocytes in PB. CONCLUSIONS: CTLA-4/HLA-DQ may be key in the regulation of Tregs on mDCs in SAA patients. Our findings should be helpful for further investigation of the mechanism of immune pathogenesis in SAA patients. Studies on the regulators of Treg and CTLA-4 activity will be valuable for SAA therapeutic target research and disease monitoring.
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Anemia Aplásica/inmunología , Antígeno CTLA-4/metabolismo , Células Dendríticas/inmunología , Antígenos HLA-DQ/metabolismo , Inmunidad Celular , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Anciano , Niño , Femenino , Humanos , Células Asesinas Naturales/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Gastrointestinal disturbances, nutritional deficiencies, and food intolerances are frequently observed in children with neurodevelopmental disorders (NDD). To reveal possible association of celiac disease risk variants (HLA-DQ), lactose intolerance associated variant (LCT-13910C>T) as well as variant associated with vitamin D function (VDR FokI) with NDD, polymerase chain reaction-based methodology was used. Additionally, intestinal peptide permeability was estimated in NDD patients and healthy children by measuring the level of peptides in urine using high-performance liquid chromatography. Levels of opioid peptides, casomorphin 8, and gluten exorphin C were significantly elevated in urine samples of NDD patients (P = 0.004 and P = 0.005, respectively), but no association of genetic risk variants for celiac disease and lactose intolerance with NDD was found. Our results indicate that increased intestinal peptide permeability observed in analyzed NDD patients is not associated with genetic predictors of celiac disease or lactose intolerance. We have also found that FF genotype of VDR FokI and lower serum levels of vitamin D (25-OH) showed association with childhood autism (CHA), a subgroup of NDD. We hypothesize that vitamin D might be important for the development of CHA.
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Enfermedad Celíaca/genética , Intolerancia a la Lactosa/genética , Trastornos del Neurodesarrollo/orina , Péptidos/orina , Receptores de Calcitriol/sangre , Vitamina D/sangre , Estudios de Casos y Controles , Enfermedad Celíaca/sangre , Enfermedad Celíaca/orina , Niño , Preescolar , Femenino , Variación Genética , Técnicas de Genotipaje , Antígenos HLA-DQ/metabolismo , Humanos , Intolerancia a la Lactosa/sangre , Intolerancia a la Lactosa/orina , Masculino , Trastornos del Neurodesarrollo/sangre , Trastornos del Neurodesarrollo/genética , Péptidos/farmacocinética , Receptores de Calcitriol/genética , Factores de Riesgo , UrinálisisRESUMEN
HLA-DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII-bound peptides in human embryonic kidney 293T cells expressing HLA-DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM-resistant, DM-dependent, or DM-sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ-peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T-cell epitopes to DM editing.
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Diabetes Mellitus Tipo 1/inmunología , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/metabolismo , Secuencias de Aminoácidos/genética , Presentación de Antígeno , Antígenos/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Simulación por Computador , Epítopos de Linfocito T/genética , Células HEK293 , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica , Estabilidad Proteica , Espectrometría de Masas en TándemRESUMEN
ß-cell autoantibodies against insulin (IAA), GAD65 (GADA) and IA-2 (IA-2A) precede onset of childhood type 1 diabetes (T1D). Incidence of the first appearing ß-cell autoantibodies peaks at a young age and is patterned by T1D-associated genes, suggesting an early environmental influence. Here, we tested if gestational infections and interactions with child's human leukocyte antigen (HLA) and non-HLA genes affected the appearance of the first ß-cell autoantibody. Singletons of mothers without diabetes (n = 7472) with T1D-associated HLA-DR-DQ genotypes were prospectively followed quarterly through the first 4 years of life, then semiannually until age 6 years, using standardized autoantibody analyses. Maternal infections during pregnancy were assessed via questionnaire 3-4.5 months post-delivery. Polymorphisms in twelve non-HLA genes associated with the first appearing ß-cell autoantibodies were included in a Cox regression analysis. IAA predominated as the first appearing ß-cell autoantibody in younger children (n = 226, median age at seroconversion 1.8 years) and GADA (n = 212; 3.2 years) in children aged ≥2 years. Gestational infections were not associated with the first appearing ß-cell autoantibodies overall. However, gestational respiratory infections (G-RI) showed a consistent protective influence on IAA (HR 0.64, 95% CI 0.45-0.91) among CTLA4-(AG, GG) children (G-RI*CTLA4 interaction, p = 0.002). The predominant associations of HLA-DR-DQ 4-8/8-4 with IAA and HLA-DR-DQ 3-2/3-2 with GADA were not observed if a G-RI was reported (G-RI*HLA-DR-DQ interaction, p = 0.03). The role of G-RI may depend on offspring HLA and CTLA-4 alleles and supports a bidirectional trigger for IAA or GADA as a first appearing ß-cell autoantibody in early life.
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Antígeno CTLA-4/metabolismo , Células Secretoras de Insulina/inmunología , Efectos Tardíos de la Exposición Prenatal/inmunología , Infecciones del Sistema Respiratorio/inmunología , Autoanticuerpos/metabolismo , Femenino , Edad Gestacional , Glutamato Descarboxilasa/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Lactante , Insulina/inmunología , Masculino , Polimorfismo Genético , Embarazo , Efectos Tardíos de la Exposición Prenatal/epidemiología , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/inmunología , Infecciones del Sistema Respiratorio/epidemiologíaRESUMEN
Formation of microthrombi is a hallmark of acquired thrombotic thrombocytopenic purpura. These microthrombi originate from insufficient processing of ultra large von Willebrand factor multimers by ADAMTS13 due to the development of anti-ADAMTS13 autoantibodies. Several studies have identified the major histocompatibility complex class II alleles HLA-DRB1*11, HLA-DQB1*03 and HLA-DQB1*02:02 as risk factors for acquired thrombotic thrombocytopenic purpura development. Previous research in our department indicated that ADAMTS13 CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR are presented on HLA-DRB1*11 and HLA-DRB1*03, respectively. Here, we describe the repertoire of ADAMTS13 peptides presented on HLA-DQ. In parallel, the repertoire of ADAMTS13-derived peptides presented on HLA-DR was monitored. Using HLA-DR- and HLA-DQ-specific antibodies, we purified HLA/peptide complexes from ADAMTS13-pulsed monocyte-derived dendritic cells. Using this approach, we identified ADAMTS13-derived peptides presented on HLA-DR for all 9 samples analyzed; ADAMTS13-derived peptides presented on HLA-DQ were identified in 4 out of 9 samples. We were able to confirm the presentation of the CUB2 domain-derived peptides FINVAPHAR and LIRDTHSLR on HLA-DR. In total, 12 different core-peptide sequences were identified on HLA-DR and 8 on HLA-DQ. For HLA-DR11, several potential new core-peptides were found; 4 novel core-peptides were exclusively identified on HLA-DQ. Furthermore, an in silico analysis was performed using the EpiMatrix and JanusMatrix tools to evaluate the eluted peptides, in the context of HLA-DR, for putative effector or regulatory T-cell responses at the population level. The results from this study provide a basis for the identification of immuno-dominant epitopes on ADAMTS13 involved in the onset of acquired thrombotic thrombocytopenic purpura.
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Proteína ADAMTS13/química , Proteína ADAMTS13/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DR/inmunología , Espectrometría de Masas , Péptidos/química , Péptidos/inmunología , Proteína ADAMTS13/metabolismo , Animales , Presentación de Antígeno , Células Dendríticas , Mapeo Epitopo/métodos , Genotipo , Células HEK293 , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Espectrometría de Masas/métodos , Ratones , Péptidos/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: Celiac disease (CD) occurs in subjects positive for HLA-DQ2 and/or DQ8 gene loci at any age following ingestion of gluten-containing food. An increased permeability of the mucosa allows interactions between gliadin macromolecules and genetic factors. It has been observed that Helicobacter pylori has the ability to modulate the integrity of the duodenal epithelium. We aimed to determine whether H. pylori infection may enhance the occurrence of CD in genetically susceptible subjects. MATERIALS AND METHODS: This was a prospective observational study. Patients undergoing upper endoscopy for any reason and positive for HLA-DQ2 and/or DQ8 haplotypes with or without CD were included. H. pylori infection was defined as a positive gastric histopathology and/or 13C-urea breath test. Prevalence of infection was compared between enrolled subjects with and without CD. Multiple logistic regression analysis, adjusting odds ratios for patient age, gender, smoking habit, residency, body mass index, and assumption of nonsteroidal anti-inflammatory drugs (NSAIDs) and proton-pump inhibitors (PPIs) were performed. RESULTS: A total of 397 genetically susceptible individuals (mean age: 37.7 ± 15.3 years; 86% women) were enrolled between October 2014 and October 2017. There were 265 (68%) patients with a diagnosis of CD. Overall, the prevalence of H. pylori infection was 33% and was similar in patients with and without CD (32% vs 36%). Adjustment for all covariates did not reveal any significant association, although adjusted odds ratio (OR) for CD was higher in female (OR = 1.302), in patients H. pylori positive (OR = 1.277), followed by use of NSAIDs (OR = 1.126), respectively. The use of PPIs appeared to be mildly protective against CD (OR = 0.644). CONCLUSION: Our study did not reveal any significant relationship between H. pylori and CD risk, even taking into account other confounders. More importantly, our findings do not support a "protective" role of H. pylori infection against CD, as previously reported. Therefore, there are no reasons to avoid eradication of H. pylori also in subject genetically susceptible for CD.