RESUMO
The association between carriage of the human leucocyte antigen (HLA)-B*51 allele and development of Behçet's disease (BD) has been known since the early 1970s, but the exact mechanisms responsible for its role in pathogenesis remain much-debated. In an effort to explain the disease process, it has been suggested that BD constitutes one of a newly termed group of diseases, the 'MHC-I-opathies'. Other MHC-I-opathies include ankylosing spondylitis and HLA-B*27-associated spondyloarthropathies and HLA-C*0602-associated skin psoriasis. Recent work analysing the peptidome of HLA-B*51 suggests that altered peptide presentation by HLA-B*51 is vital to the disease process. In this review, we argue that immune receptor interactions with HLA-B*51 or the HLA-B*51-peptide complex could lead to development of inflammation in BD. The evidence for CD8+ T cell involvement is weak, and based on emerging studies it seems more likely that natural killer (NK) or other cell interactions, perhaps mediated by leucocyte immunoglobulin-like receptor (LILR) or killer immunoglobulin-like receptor (KIR) receptors, are culpable in pathogenesis. HLA misfolding leading directly to inflammation is another hypothesis for BD pathogenesis that deserves greater investigation. Ultimately, greater understanding of HLA-B*51's unique role in BD will probably lead to improved development of therapeutic strategies.
Assuntos
Síndrome de Behçet/etiologia , Antígeno HLA-B51/genética , Antígeno HLA-B51/imunologia , Alelos , Apresentação de Antígeno/imunologia , Autoimunidade/genética , Autoimunidade/imunologia , Síndrome de Behçet/metabolismo , Citocinas/metabolismo , Suscetibilidade a Doenças , Epitopos/química , Epitopos/imunologia , Predisposição Genética para Doença , Antígeno HLA-B51/química , Antígeno HLA-B51/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Microbiota , Peptídeos/química , Peptídeos/imunologia , Dobramento de Proteína , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A+GM-CSF+ double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF+CD4+ cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF+ and IL-17A+GM-CSF+ double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Espondilartrite/genética , Transcriptoma , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Estudo de Associação Genômica Ampla , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Espondilartrite/metabolismo , Adulto JovemRESUMO
OBJECTIVE: To determine the microRNA (miR) signature in ankylosing spondylitis (AS) T helper (Th)17 cells. METHODS: Interleukin (IL)-17A-producing CD4+ T cells from patients with AS and healthy controls were FACS-sorted for miR sequencing and qPCR validation. miR-10b function was determined by miR mimic expression followed by cytokine measurement, transcriptome analysis, qPCR and luciferase assays. RESULTS: AS Th17 cells exhibited a miR signature characterised by upregulation of miR-155-5p, miR-210-3p and miR-10b. miR-10b has not been described previously in Th17 cells and was selected for further characterisation. miR-10b is transiently induced in in vitro differentiated Th17 cells. Transcriptome, qPCR and luciferase assays suggest that MAP3K7 is targeted by miR-10b. Both miR-10b overexpression and MAP3K7 silencing inhibited production of IL-17A by both total CD4 and differentiating Th17 cells. CONCLUSIONS: AS Th17 cells have a specific miR signature and upregulate miR-10b in vitro. Our data suggest that miR-10b is upregulated by proinflammatory cytokines and may act as a feedback loop to suppress IL-17A by targeting MAP3K7. miR-10b is a potential therapeutic candidate to suppress pathogenic Th17 cell function in patients with AS.
Assuntos
Interleucina-17/biossíntese , MAP Quinase Quinase Quinases/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Células Th17/metabolismo , Regulação para Cima , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Feminino , Inativação Gênica , Humanos , Interleucina-6/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Espondilite Anquilosante , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adulto JovemRESUMO
Ankylosing spondylitis (AS), the most common form of spondyloarthropathy, is a chronic, progressive multi-system inflammatory disorder characteristically affecting the sacroiliac joints and axial skeleton. Although the exact mechanisms underlying the pathogenesis of AS remain to be elucidated, the presence of human leucocyte antigen (HLA)-B27 is known to markedly increase its risk of development. Current treatments include non-steroidal anti-inflammatory drugs (NSAIDs) and tumour necrosis factor (TNF) blockers. In recent years, the interleukin (IL)-23/IL-17 pathway has been shown to have significance in the pathogenesis of AS and treatment modalities targeting this pathway have been shown to be beneficial in various other inflammatory conditions. This review provides an overview of the IL-23/IL-17 pathway in the pathogenesis of AS and summarizes new potential treatments for AS and related inflammatory diseases.
Assuntos
Imunoterapia , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Espondilite Anquilosante/imunologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Antígeno HLA-B27/genética , Humanos , Terapia de Alvo Molecular , Transdução de Sinais , Espondilite Anquilosante/terapiaRESUMO
OBJECTIVES: Major histocompatibility complex class I (MHC-I) proteins exist at the cell surface in antigen presenting forms and as beta2m-independent free heavy chains (FHCs). FHCs have been implicated in spondyloarthritis, but little is known about their expression in healthy individuals. We studied FHC expression on various human cell types, comparing spondyloarthropathy patients with healthy and rheumatoid arthritis (RA) patient controls. METHODS: MHC-I expression was analysed by flow cytometry. FHC levels were normalized for overall MHC-I to generate a relative expression level. Relative FHC levels were analysed for peripheral blood and trophoblast samples from healthy volunteers, RA and spondyloarthropathy patients. Macrophages and dendritic cells were cultured in vitro to analyse changes following activation. Peripheral blood leucocytes from patients with ankylosing spondylitis (AS) and RA were treated with inflammatory stimuli and subsequent alterations in their relative FHC levels were analysed. RESULTS: We found consistent patterns of differential relative FHC expression across lymphocyte subpopulations and particularly high expression on extravillous trophoblast. FHCs were present at higher levels in a reactive arthritis (ReA) population than in healthy controls and RA patients; differences not merely due to the presence of Human Leucocyte Antigen (HLA) B27. Treatment of leucocytes from arthritic patients with bacterial lipopolysaccharide resulted in significant up-regulation of FHC compared with an HLA B27+ control population. CONCLUSIONS: Our findings define normal levels and tissue expression of FHCs, and support the hypothesis that disregulation of heavy chain expression may play a pathogenic role in spondyloarthropathy.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Espondiloartropatias/imunologia , Adulto , Artrite Reativa/imunologia , Artrite Reumatoide/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Antígeno HLA-B27/metabolismo , Humanos , Leucócitos/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Proibitinas , Trofoblastos/imunologiaRESUMO
The strong association of the HLA class 1 allele HLA B27 with ankylosing spondylitis (AS) has been recognized for over 25 yr, however the pathogenic mechanism linking HLA B27 with AS and other spondyloarthropathies remains a mystery. We now know that the principal natural function of HLA B27 is an immunologic one, namely to bind antigenic peptides and then present them to T lymphocytes. I have shown that HLA B27 functions as an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals. A working molecular model of how T cells recognize HLA B27 has been generated and tested. Evidence that T cells have a role in spondyloarthritis has also been found. First, expanded populations of T lymphocytes were found in both the blood and synovial fluid of patients with reactive arthritis (ReA). Secondly, a strong cytotoxic T-cell response to an HLA B27-restricted peptide epitope from Chlamydia trachomatis was found in a patient with ReA. This peptide, derived from a bacterium known to trigger ReA, is thus a candidate 'arthritogenic' peptide. We have also found evidence that HLA B27 has an unusual cell biology compared with other HLA molecules. HLA B27 demonstrates an unusual ability to form heavy chain homodimers in vitro. Dimerization is dependent upon disulphide bonding through an unpaired cysteine at position 67. Remarkably these dimers lack beta2 microglobulin, previously thought to be an essential component of all mature MHC class 1 molecules. HLA B27 homodimer formation has also been demonstrated in certain cell lines in vivo, and preliminary data suggest that significant numbers of T cells from patients with spondyloarthropathy express a ligand for HLA B27 homodimers. These findings have extended our understanding of the beneficial immunologic function of HLA B27, and have also led us to propose the testable new hypothesis that HLA B27 heavy chain dimerization may be involved in the pathogenesis of spondyloarthritis.
Assuntos
Antígeno HLA-B27/genética , Espondilite Anquilosante/imunologia , Distinções e Prêmios , Antígeno HLA-B27/química , Antígeno HLA-B27/fisiologia , História do Século XXI , Humanos , Modelos Moleculares , Proibitinas , Conformação Proteica , Valores de Referência , Reumatologia/históriaRESUMO
The association of HLA-B27 with ankylosing spondylitis and reactive arthritis is the strongest one known between an MHC class I Ag and a disease. We have searched the proteome of the bacterium Chlamydia trachomatis for HLA-B27 binding peptides that are stimulatory for CD8(+) cells both in a model of HLA-B27 transgenic mice and in patients. This was done by combining two biomathematical computer programs, the first of which predicts HLA-B27 peptide binding epitopes, and the second the probability of HLA-B27 peptide generation by the proteasome system. After preselection, immunodominant peptides were identified by Ag-specific flow cytometry. Using this approach we have identified for the first time nine peptides derived from different C. trachomatis proteins that are stimulatory for CD8(+) T cells. Eight of these nine murine-derived peptides were recognized by cytotoxic T cells. The same strategy was used to identify B27-restricted chlamydial peptides in three patients with reactive arthritis. Eleven peptides were found to be stimulatory for patient-derived CD8(+) T cells, of which eight overlapped those found in mice. Additionally, we applied the tetramer technology, showing that a B27/chlamydial peptide containing one of the chlamydial peptides stained CD8(+) T cells in patients with Chlamydia-induced arthritis. This comprehensive approach offers the possibility of clarifying the pathogenesis of B27-associated diseases.
Assuntos
Proteínas de Bactérias/imunologia , Chlamydia trachomatis/imunologia , Antígeno HLA-B27/imunologia , Proteoma/imunologia , Animais , Artrite Reativa/etiologia , Artrite Reativa/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Chlamydia/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Antígeno HLA-B27/genética , Humanos , Camundongos , Camundongos Transgênicos , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismoAssuntos
Antígeno HLA-B27/química , Animais , Animais Geneticamente Modificados , Apresentação de Antígeno , Artrite Psoriásica/genética , Artrite Psoriásica/imunologia , Artrite Reativa/genética , Artrite Reativa/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Cistina/química , Dimerização , Fibrilinas , Predisposição Genética para Doença , Antígeno HLA-B27/genética , Antígeno HLA-B27/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária , Síndrome de Marfan/metabolismo , Camundongos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/metabolismo , Modelos Imunológicos , Especificidade de Órgãos , Conformação Proteica , Ratos , Articulação Sacroilíaca/imunologia , Articulação Sacroilíaca/patologia , Espondilite Anquilosante/genética , Espondilite Anquilosante/imunologia , Estresse Mecânico , Fator de Crescimento Transformador beta/fisiologia , Uveíte Anterior/genética , Uveíte Anterior/imunologiaRESUMO
HLA-B27 has a striking association with inflammatory arthritis. We show that free HLA-B27 heavy chains can form a disulfide-bonded homodimer, dependent on residue Cys67 in their extracellular alpha 1 domain. Despite the absence of beta 2-microglobulin, HLA-B27 heavy chain homodimers (termed HC-B27) were stabilized by a known peptide epitope. HC-B27 complexes were recognized by the conformation-specific Ab W6/32, but not the ME1 Ab. Surface labeling and immunoprecipitation demonstrated the presence of similar W6/32-reactive free heavy chains at the surface of HLA-B27-transfected T2 cells. HC-B27 homodimer formation might explain the ability of HLA-B27 to induce spondyloarthropathy in beta 2-microglobulin-deficient mice.
Assuntos
Antígeno HLA-B27/química , Microglobulina beta-2/química , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Cisteína/química , Dimerização , Dissulfetos/química , Dissulfetos/metabolismo , Antígeno HLA-B27/genética , Antígeno HLA-B27/imunologia , Antígeno HLA-B27/metabolismo , Humanos , Testes de Precipitina , Conformação Proteica , Transfecção/imunologia , Microglobulina beta-2/isolamento & purificação , Microglobulina beta-2/metabolismoRESUMO
Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.
Assuntos
Apresentação de Antígeno , Antígenos Virais/imunologia , Regiões Determinantes de Complementaridade , Antígeno HLA-B27/imunologia , Cadeias alfa de Imunoglobulina/imunologia , Vírus da Influenza A/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ratos , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
The 3' end of the T cell receptor V beta 7.1 gene contains the five nucleotides CAAGA between the broadly conserved consensus sequence of nucleotides TGC/T GCC AGC AGC (which encode cysteine, alanine, serine and serine at positions 92-95 of the beta chain) and the heptamer that signals rearrangement. These nucleotides are frequently preserved during gene rearrangement, resulting in the common presence of glutamine at position 96 and of aspartate or glutamate at position 97 of the V beta 7.1 chain CDR3 loop in peripheral blood lymphocytes. There is selection of V beta 7.1 and of the V beta 7.1 gene-encoded glutamate at position 97 of the beta chain CDR3 loop in the cytotoxic T lymphocyte response to the HLA B2705-restricted influenza A nucleoprotein epitope SRYWAIRTR. Our results indicate that selection of V beta 7.1 gene-encoded amino acid residues on CDR3 loops may be one factor driving selection of V beta 7.1 in this response.
Assuntos
Imunoglobulinas/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Linfócitos B/imunologia , Sequência de Bases , Sítios de Ligação/genética , Regulação da Expressão Gênica/imunologia , Humanos , Dados de Sequência Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the "footprint" of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are "flag" residues with TcR-accessible side chains.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Antígeno HLA-B27/imunologia , Proteínas de Ligação a RNA , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Antígenos Virais/imunologia , Sítios de Ligação , Citotoxicidade Imunológica , Epitopos , Humanos , Imunidade Celular , Técnicas In Vitro , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Nucleoproteínas/imunologia , Peptídeos/imunologia , Proteínas do Core Viral/imunologiaRESUMO
Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Sequência de Aminoácidos , Animais , Humanos , Hibridomas , Leucemia Basofílica Aguda , Ativação Linfocitária , Dados de Sequência Molecular , Testes de Precipitina , Ratos , Receptores de Antígenos de Linfócitos T alfa-beta/química , Proteínas Recombinantes/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
Eight HLA B27-restricted influenza A virus nucleoprotein 383-391-specific cytotoxic T lymphocyte (CTL) clones were obtained from three unrelated donors following natural infection. T cell receptor (TcR) usage was studied using the "anchored" polymerase chain reaction. TcR alpha-chain usage was restricted with three predominant V alpha (V alpha 12.1, 14.1, 22) and two predominant J alpha segments. beta-chain variable-region usage was also conserved, with V beta 7 being used by five clones despite contributing less than 2% of peripheral blood lymphocyte V beta sequences of one individual studied. The TcR beta-chain junctional region was highly conserved even between CTL clones from unrelated individuals, with a negatively charged amino acid, contributed to by N-region addition, encoded at position 97 in all but two clones. This study shows that peptide-specific HLA B27-restricted CTL following influenza virus infection use very similar TcR and, when considered with previous studies, suggests a pattern of TcR conservation for major histocompatibility complex class I-restricted responses. No difference in TcR usage was detected between one healthy donor and two with HLA B27-associated arthritis.