RESUMO
Most cancer vaccines target peptide antigens, necessitating personalization owing to the vast inter-individual diversity in major histocompatibility complex (MHC) molecules that present peptides to T cells. Furthermore, tumours frequently escape T cell-mediated immunity through mechanisms that interfere with peptide presentation1. Here we report a cancer vaccine that induces a coordinated attack by diverse T cell and natural killer (NK) cell populations. The vaccine targets the MICA and MICB (MICA/B) stress proteins expressed by many human cancers as a result of DNA damage2. MICA/B serve as ligands for the activating NKG2D receptor on T cells and NK cells, but tumours evade immune recognition by proteolytic MICA/B cleavage3,4. Vaccine-induced antibodies increase the density of MICA/B proteins on the surface of tumour cells by inhibiting proteolytic shedding, enhance presentation of tumour antigens by dendritic cells to T cells and augment the cytotoxic function of NK cells. Notably, this vaccine maintains efficacy against MHC class I-deficient tumours resistant to cytotoxic T cells through the coordinated action of NK cells and CD4+ T cells. The vaccine is also efficacious in a clinically important setting: immunization following surgical removal of primary, highly metastatic tumours inhibits the later outgrowth of metastases. This vaccine design enables protective immunity even against tumours with common escape mutations.
Assuntos
Síndromes Mielodisplásicas , Neoplasias , Dermatopatias Genéticas , Vacinas , Antígenos de Histocompatibilidade Classe I , Humanos , Células Matadoras Naturais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Neoplasias/prevenção & controleRESUMO
BACKGROUND: CD4+ T cells play a central role during the early stages of rheumatoid arthritis (RA), but to which extent they are required for the perpetuation of the disease is still not fully understood. The aim of the current study was to obtain conclusive evidence that T cells drive chronic relapsing arthritis. METHODS: We used the rat pristane-induced arthritis model, which accurately portrays the chronic relapsing-remitting disease course of RA, to examine the contribution of T cells to chronic arthritis. RESULTS: Rats subjected to whole-body irradiation and injected with CD4+ T cells from lymph nodes of pristane-injected donors developed chronic arthritis that lasted for more than 4 months, whereas T cells from the spleen only induced acute disease. Thymectomy in combination with irradiation enhanced the severity of arthritis, suggesting that sustained lymphopenia promotes T cell-driven chronic inflammation in this model. The ability of T cells to induce chronic arthritis correlated with their expression of Th17-associated transcripts, and while depletion of T cells in rats with chronic PIA led to transient, albeit significant, reduction in disease, neutralization of IL-17 resulted in almost complete and sustained remission. CONCLUSION: These findings show that, once activated, self-reactive T cells can sustain inflammatory responses for extended periods of time and suggest that such responses are promoted in the presence of IL-17.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/transplante , Doença Crônica , Feminino , Interleucina-17/imunologia , Masculino , RatosRESUMO
Polymorphisms in the MHC class II (MHCII) genes are strongly associated with rheumatoid arthritis, supporting the importance of autoreactive T helper (Th) cells for the development of this disease. Here, we used pristane-induced arthritis (PIA), induced by the non-antigenic hydrocarbon pristane, to study the impact of different MHCII alleles on T-cell activation and differentiation. In MHCII-congenic rats with disease-promoting MHCII alleles, pristane primarily induced activation of Th1 cells, whereas activated T cells were Th17 biased in rats with protective MHCII alleles. Neutralization of IFN-γ during T-cell activation abrogated the development of disease, suggesting that Th1 immunity is important for disease induction. Neutralization of IL-17, by contrast, suppressed arthritis only when performed in rats with established disease. Adoptive T-cell transfers showed that T cells acquired arthritogenic capacity earlier in strains with a prevailing Th1 response. Moreover, upon pristane injection, these strains exhibited more Ag-primed OX40+ and proliferating T cells of polyclonal origin. These data show that T cells are polarized upon the first encounter with peptide-MHCII complexes in an allele-dependent fashion. In PIA, the polyclonal expansion of autoreactive Th1 cells was necessary for the onset of arthritis, while IL-17 mediated immunity contributed to the progression to chronic disease.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Células Th1/imunologia , Células Th17/imunologia , Alelos , Animais , Anticorpos Bloqueadores/administração & dosagem , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Artrite Reumatoide/genética , Diferenciação Celular/genética , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Ativação Linfocitária/genética , Polimorfismo Genético , Ratos , Ratos Endogâmicos , TerpenosRESUMO
Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.
Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Antígenos HLA-DR/metabolismo , Peptídeos/análise , Sarcoidose/terapia , Adulto , Líquido da Lavagem Broncoalveolar/citologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Sarcoidose/imunologiaRESUMO
Genome-wide association studies have revealed many genetic loci associated with complex autoimmune diseases. In rheumatoid arthritis (RA), the MHC gene HLA-DRB1 is the strongest candidate predicting disease development. It has been suggested that other immune-regulating genes in the MHC contribute to the disease risk, but this contribution has been difficult to show because of the strong linkage disequilibrium within the MHC. We isolated genomic regions in the form of congenic fragments in rats to test whether there are additional susceptibility loci in the MHC. By both congenic mapping in inbred strains and SNP typing in wild rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in the MHC-III region, which regulates the onset, severity, and chronicity of arthritis. The Ltab-Ncr3 haplotype consists of five polymorphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb (lymphotoxin-ß), Lst1 (leukocyte-specific transcript 1), and Ncr3 (natural cytotoxicity-triggering receptor 3). Significant correlation in the expression of the Ltab-Ncr3 genes suggests that interaction of these genes may be important in keeping these genes clustered together as a conserved haplotype. We studied the arthritis association and the spliceo-transcriptome of four different Ltab-Ncr3 haplotypes and showed that higher Ltb and Ncr3 expression, lower Lst1 expression, and the expression of a shorter splice variant of Lst1 correlate with reduced arthritis severity in rats. Interestingly, patients with mild RA also showed higher NCR3 expression and lower LST1 expression than patients with severe RA. These data demonstrate the importance of a conserved haplotype in the regulation of complex diseases such as arthritis.
Assuntos
Artrite Reumatoide/genética , Antígenos de Histocompatibilidade/genética , Animais , Artrite Reumatoide/imunologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos , Antígenos de Histocompatibilidade/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Linfotoxina-alfa/genética , Linfotoxina-alfa/imunologia , Linfotoxina-beta/genética , Linfotoxina-beta/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Ratos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. METHODS: We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. RESULTS: Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. CONCLUSIONS: PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.
Assuntos
Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Imunossupressores/toxicidade , Terpenos/toxicidade , Animais , Feminino , Masculino , RatosRESUMO
Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the ß1-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response.
Assuntos
Colágeno Tipo II/química , Etilenos/química , Glicopeptídeos/química , Antígenos de Histocompatibilidade Classe II/química , Simulação de Dinâmica Molecular , Ligação de Hidrogênio , Estrutura Molecular , EstereoisomerismoRESUMO
Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade/genética , Sequência de Aminoácidos , Aminoácidos/genética , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Sítios de Ligação/genética , Peso Corporal/efeitos dos fármacos , Peso Corporal/imunologia , Modelos Animais de Doenças , Genótipo , Haplótipos/imunologia , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Genético/imunologia , Estrutura Terciária de Proteína , Ratos , Índice de Gravidade de Doença , Terpenos/imunologiaRESUMO
Citrullinated collagen II (CII) is a well-known autoantigen in rheumatoid arthritis (RA). However, the direct effects of CII citrullination on cell behavior have not been described. To study whether citrullination of CII could affect cellular functions, we measured the adhesion of 3 different cell types (human Saos2 osteosarcoma cells, human synovial fibroblasts, and rat mesenchymal stem cells) with impedance-based technology. The binding of different collagen receptor integrins to citrullinated collagen was studied by CHO cell lines, each overexpressing 1 of the 4 human collagen receptors on the cell surface, and with solid-phase binding assays, using the recombinant human integrin α1I, α2I, α10I, and α11I domains. Collagen citrullination decreased the adhesion of synovial fibroblasts â¼50% (P<0.05) and mesenchymal stem cells â¼40% (P<0.05) by specifically decreasing the binding of integrins α10ß1 and α11ß1 to arginine-containing motifs, such as GFOGER. In contrast, citrullination had only a minor effect on the function of α1ß1 and α2ß1 integrins, which have been reported to play a critical role in regulating leukocyte function. Molecular modeling was used to explain the detected functional differences at the structural level. Given that the integrins regulate cell metabolism, proliferation, and migration, we suggest that collagen citrullination modifies the pathogenesis of RA. Here, CII citrullination was shown to decrease the survival of mesenchymal stem cells.
Assuntos
Adesão Celular/fisiologia , Citrulina/química , Colágeno Tipo II/química , Integrinas/fisiologia , Motivos de Aminoácidos , Aminoacilação , Animais , Arginina/química , Neoplasias Ósseas/patologia , Células CHO , Linhagem Celular Tumoral , Células Cultivadas , Galinhas , Colágeno Tipo II/farmacologia , Cricetinae , Cricetulus , Fibroblastos/patologia , Humanos , Integrinas/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoartrite/patologia , Osteossarcoma/patologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Membrana Sinovial/patologia , TransfecçãoRESUMO
Genetic variation in the major histocompatibility complex (MHC) affects CD4â¶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4â¶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4â¶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of â¼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Complexo Principal de Histocompatibilidade/genética , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Alelos , Animais , Apresentação de Antígeno , Diferenciação Celular/genética , Linhagem da Célula , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Ratos , Recombinação Genética , Seleção GenéticaRESUMO
OBJECTIVE: To investigate type II collagen (CII) as a joint-specific target of the anti-citrullinated protein antibody (ACPA) response in rheumatoid arthritis (RA). METHODS: Potential citrullinated neoepitopes were identified by high-resolution tandem mass spectrometry (MS/MS) of in vitro peptidylarginine deiminase 2 (PAD-2)-treated CII, and the relationship between citrullination and CII conformation was investigated by circular dichroism and conformation-dependent antibodies. Based on the MS analyses, synthetic peptides were designed and analyzed for serum IgG reactivity in the Epidemiological Investigation of RA (EIRA) case-control cohort of 1,949 RA patients and 278 healthy controls. Peptide-specific antibodies were purified from RA patient serum and used to stain RA cartilage specimens. RESULTS: We described the conformation-dependent citrullination pattern of CII after PAD-2 treatment at room temperature and 37°C and showed that CII could be citrullinated in its native triple-helical conformation. Screening of Arg and Cit pairs of synthetic peptides revealed new citrullinated B cell epitopes on CII. Antibodies directed to 2 proximal epitopes close to the C-terminus of the CII triple helix were recognized by autoantibodies in 21% and 17% of RA patients, respectively. Affinity-purified antibodies from RA sera directed to these 2 epitopes, but not antibodies directed to citrullinated α-enolase peptide 1, bound to RA cartilage. CONCLUSION: These findings suggest that cartilage-directed anticitrulline immunity contributes to the induction of joint inflammation in RA.
Assuntos
Artrite Reumatoide/metabolismo , Autoanticorpos/metabolismo , Cartilagem Articular/metabolismo , Citrulina/imunologia , Colágeno Tipo II/metabolismo , Animais , Estudos de Casos e Controles , Colágeno Tipo II/efeitos dos fármacos , Epitopos/imunologia , Humanos , Hidrolases/farmacologia , Imunoglobulina G/metabolismo , Técnicas In Vitro , Desiminases de Arginina em Proteínas , Ratos , Espectrometria de Massas em TandemRESUMO
INTRODUCTION: Collagen-induced arthritis (CIA) has traditionally been performed in MHC class II A(q)-expressing mice, whereas most genetically modified mice are on the C57BL/6 background (expressing the b haplotype of the major histocompatibility complex (MHC) class II region). However, C57BL/6 mice develop arthritis after immunisation with chicken-derived collagen type II (CII), but arthritis susceptibility has been variable, and the immune specificity has not been clarified. OBJECTIVE: To establish a CIA model on the C57BL/6 background with a more predictable and defined immune response to CII. RESULTS: Both chicken and rat CII were arthritogenic in C57BL/6 mice provided they were introduced with high doses of Mycobacterium tuberculosis adjuvant. However, contaminating pepsin was strongly immunogenic and was essential for arthritis development. H-2(b)-restricted T cell epitopes on chicken or rat CII could not be identified, but expression of A(q) on the C57BL/6 background induced T cell response to the CII260-270 epitope, and also prolonged the arthritis to be more chronic. CONCLUSIONS: The putative (auto)antigen and its arthritogenic determinants in C57BL/6 mice remains undisclosed, questioning the value of the model for addressing T cell-driven pathological pathways in arthritis. To circumvent this impediment, we recommend MHC class II congenic C57BL/6N.Q mice, expressing A(q), with which T cell determinants have been thoroughly characterised.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Genes MHC da Classe II/genética , Linfócitos T/imunologia , Animais , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Galinhas , Epitopos de Linfócito T/imunologia , Haplótipos , Imunização , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Mycobacterium/imunologia , RatosRESUMO
OBJECTIVE: Chronic inflammation of the peripheral joints is a hallmark of rheumatoid arthritis (RA). The autoantibody response in RA has been shown to be directed mainly to ubiquitous antigens, whereas the response to cartilage proteins has been less extensively investigated. This study was undertaken to characterize the immune response in pristane-induced arthritis (PIA) in the rat to the cartilage-specific proteins type II collagen (CII) and type XI collagen (CXI) and to genetically fine-map their underlying major histocompatibility complex (MHC) associations. METHODS: The genetic control of CII and CXI immunity was mapped using intra-MHC-recombinant inbred strains immunized with the respective collagens. Reactivity with CII and CXI was tested in acute and chronic PIA and in 356 HLA-typed patients with recently diagnosed RA. RESULTS: Mapping of arthritis susceptibility within the MHC region revealed a 144-223-kb locus containing <12 genes, including paralogs for HLA-DQ and HLA-DR. Susceptibility to CII and CXI was linked to haplotypes RT1(av1) (DA) and RT1(f) (DA.1F), respectively. After injection of pristane, rats of both strains developed weak T cell and IgG responses to CII, but not to CXI. In chronic arthritis, however, collagen reactivity was stronger, specific for CXI, and restricted to rats with RT1(f) MHC. Among RA patients, 12% exhibited a specific IgG response to CXI, 6% to CII, and 6% to both collagens. CONCLUSION: These findings demonstrate a shift in cartilage recognition in early and chronic arthritis in the rat, suggesting that CXI autoreactivity contributes to the perpetuation of chronic disease. The results provide evidence of the importance of joint antigens in arthritis development.
Assuntos
Artrite/etiologia , Artrite/imunologia , Colágeno Tipo XI/imunologia , Antígenos de Histocompatibilidade Classe II/sangue , Complexo Principal de Histocompatibilidade/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Animais , Artrite/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Doença Crônica , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ratos , Ratos EndogâmicosRESUMO
The structural basis for antigen presentation by class II major histocompatibility complex (MHC) proteins to CD4(+) T-cells is important for understanding and possibly treating autoimmune diseases. In the work described in this paper, (E)-alkene and ethylene amide-bond isosteres were used to investigate the effect of removing hydrogen-bonding possibilities from the CII259-270 glycopeptide, which is bound by the arthritis-associated murine A(q) class II MHC protein. The isostere-modified glycopeptides showed varying and unexpectedly large losses of A(q) binding that could be linked to the dynamics of the system. Molecular dynamics (MD) simulations revealed that the backbone of CII259-270 and the A(q) protein are able to form up to 11 hydrogen bonds, but fewer than this number are present at any one time. Most of the strong hydrogen-bond interactions were formed by the N-terminal part of the glycopeptide, i.e., in the region where the isosteric replacements were made. The structural dynamics also revealed that hydrogen bonds were strongly coupled to each other; the loss of one hydrogen-bond interaction had a profound effect on the entire hydrogen-bonding network. The A(q) binding data revealed that an ethylene isostere glycopeptide unexpectedly bound more strongly to A(q) than the corresponding (E)-alkene, which is in contrast to the trend observed for the other isosteres. Analysis of the MD trajectories revealed that the complex conformation of this ethylene isostere was structurally different and had an altered molecular interaction pattern compared to the other A(q)/glycopeptide complexes. The introduced amide-bond isosteres also affected the interactions of the glycopeptide/A(q) complexes with T-cell receptors. The dynamic variation of the patterns and strengths of the hydrogen-bond interactions in the class II MHC system is of critical importance for the class II MHC/peptide/TCR signaling system.
Assuntos
Alcenos/química , Linfócitos T CD4-Positivos/imunologia , Etilenos/química , Glicopeptídeos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Animais , Apresentação de Antígeno , Linhagem Celular , Glicopeptídeos/química , Antígenos de Histocompatibilidade Classe II/química , Hibridomas , Ligação de Hidrogênio , Camundongos , Estrutura Secundária de ProteínaRESUMO
The discovery of antibodies specific for citrullinated protein epitopes [anti-citrullinated protein antibodies (ACPAs)] is a hallmark for the diagnosis and prognosis of rheumatoid arthritis (RA) and will also be a useful tool for understanding the fundamental pathologic processes. There are several essential questions pertaining to ACPA that remain to be explored, such as understanding the early specificity of the underlying T-cell recognition, whether the production of ACPA is a primary or secondary process, and in the event of such antibodies being arthritogenic, whether they could possibly regulate the disease development. To answer these questions, animal models are needed, but unfortunately ACPA is not a prominent feature of any of the classical animal models of RA. However, we showed recently that ACPA can be isolated from animals susceptible to collagen-induced arthritis that are specific for citrullinated type II collagen (CII). The citrulline specificity could be visualized, and the specificity is determined primarily by a direct interaction with citrulline. We also demonstrated that these antibodies are specific for the citrullinated epitopes and are pathogenic in vivo. A new hypothesis to explain how inflammation in RA can be directed to cartilaginous joints and be self-perpetuating is suggested, which involves recognition of post-translational modifications (glycosylation and citrullination) on CII by T and B cells that can have both arthritogenic and regulatory consequences.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Autoimunidade , Citrulina/imunologia , Proteínas da Matriz Extracelular/imunologia , Processamento de Proteína Pós-Traducional , Animais , Especificidade de Anticorpos , Artrite Experimental/imunologia , Artrite Reumatoide/genética , Artrite Reumatoide/terapia , Autoimunidade/genética , Colágeno Tipo II/imunologia , Progressão da Doença , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Humanos , Mediadores da Inflamação/imunologia , Conformação ProteicaRESUMO
Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversed-phase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.