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1.
Nature ; 612(7941): 771-777, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36477533

RESUMO

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.


Assuntos
Autoimunidade , Antígenos HLA-B , Peptídeos , Receptores de Antígenos de Linfócitos T , Humanos , Autoantígenos/química , Autoantígenos/imunologia , Autoantígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos HLA-B/imunologia , Antígenos HLA-B/metabolismo , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Líquido Sinovial/imunologia , Espondilite Anquilosante/imunologia , Uveíte Anterior/imunologia , Biblioteca de Peptídeos , Reações Cruzadas , Motivos de Aminoácidos
2.
Immunology ; 155(2): 273-282, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29772075

RESUMO

CD6 is a type I T-cell surface receptor that modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab™), which has reached the clinic for treatment of autoimmune disease. We characterized molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action. Epitope mapping using the crystal structure of CD6 to design mutants identified two distinct binding sites on different faces of domain 1, one containing residue R77, crucial for MT605 and T12.1 binding and the other, E63, which is crucial for itolizumab and MEM98. Analysis of binding kinetics revealed that itolizumab has a lower affinity compared with other CD6 domain 1 mAbs. We compared potential agonistic (triggering) and antagonistic (blocking) properties of CD6 mAbs in assays where the mechanism of action was well defined. CD6 domain 1 and 3 mAbs were equally effective in triggering interleukin-2 production by a cell line expressing a chimeric antigen receptor containing the extracellular region of CD6. CD6 domain 1 mAbs hindered binding of multivalent immobilized CD166 but were inferior compared with blocking by soluble CD166 or a CD6 domain 3 mAb. Characterization of CD6 mAbs provides an insight into how their functional effects in vivo may be interpreted and their therapeutic use optimized.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Epitopos/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos CD/química , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/química , Antígenos de Diferenciação de Linfócitos T/genética , Linhagem Celular Tumoral , Humanos , Camundongos , Modelos Moleculares , Mutagênese , Mutação , Conformação Proteica , Domínios Proteicos/imunologia , Domínios e Motivos de Interação entre Proteínas , Ratos , Transdução de Sinais/efeitos dos fármacos
3.
Immunology ; 146(3): 392-400, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26221972

RESUMO

Signalling lymphocyte activation molecule (SLAM) family members regulate activation and inhibition in the innate and adaptive immune systems. Genome-wide association studies identified their genetic locus (1q23) as highly polymorphic and associated with susceptibility to systemic lupus erythematosus (SLE). Here we show that the Val602 variant of the non-synonymous single nucleotide polymorphism (SNP) rs509749 in the SLAM family member CD229 (Ly9, SLAMF3) has a two-fold lower affinity compared with the SLE-associated Met602 variant for the small adaptor protein SAP. Comparison of the two variants in T-cell lines revealed the Val602 variant to be significantly more highly expressed than CD229 Met602 . Activation was diminished in cells expressing CD229 Val602 compared with CD229 Met602 as measured by up-regulation of CD69. There was no correlation between homozygosity at rs509749 and activation in peripheral blood mononuclear cells from healthy donors. These findings identify potential mechanisms by which a single SNP can perturb fine-tuning in the immune system with significant functional consequences.


Assuntos
Antígenos CD/química , Antígenos CD/genética , Polimorfismo de Nucleotídeo Único , Linfócitos T/imunologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Complexo CD3/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Lectinas Tipo C/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária , Fosfotirosina/química , Ligação Proteica , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/metabolismo , Domínios de Homologia de src
4.
Blood ; 119(25): 5955-62, 2012 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-22452981

RESUMO

Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and platelet factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4(K50E) forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.


Assuntos
Anticoagulantes/isolamento & purificação , Anticoagulantes/uso terapêutico , Descoberta de Drogas/métodos , Fator Plaquetário 4/antagonistas & inibidores , Trombocitopenia/tratamento farmacológico , Animais , Anticoagulantes/química , Células Cultivadas , Galinhas , Biologia Computacional , Drosophila , Avaliação Pré-Clínica de Medicamentos , Heparina/efeitos adversos , Heparina/uso terapêutico , Humanos , Modelos Biológicos , Modelos Moleculares , Racionalização , Projetos de Pesquisa , Trombocitopenia/induzido quimicamente
5.
JCI Insight ; 9(16)2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39024572

RESUMO

HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyzed ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8+ T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4ß7, and CCR6. In addition, we found an expansion of YeiH-specific CD8+ T cells in axSpA and B27AAU blood compared with that from individuals acting as healthy controls, whereas influenza-specific CD8+ T cells were equivalent across groups. Finally, we demonstrated the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8+ T cells may occur in enteric organs.


Assuntos
Espondiloartrite Axial , Antígeno HLA-B27 , Receptores CCR6 , Uveíte Anterior , Humanos , Uveíte Anterior/imunologia , Antígeno HLA-B27/genética , Antígeno HLA-B27/imunologia , Receptores CCR6/genética , Receptores CCR6/metabolismo , Receptores CCR6/imunologia , Espondiloartrite Axial/imunologia , Feminino , Masculino , Adulto , Linfócitos T CD8-Positivos/imunologia , Integrinas/metabolismo , Integrinas/imunologia , Pessoa de Meia-Idade
6.
J Biol Chem ; 286(20): 18013-25, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21454581

RESUMO

The myeloid inhibitory receptor LILRB4 (also called ILT3, LIR-5, CD85k), a member of the leukocyte immunoglobulin-like receptors (LILRs/LIRs), is an important mediator of immune tolerance. Up-regulated on tolerogenic dendritic cells, it has been shown to modulate immune responses via induction of T cell anergy and differentiation of CD8(+) T suppressor cells and may play a role in establishing immune tolerance in cancer. Consequently, characterizing the molecular mechanisms involved in LILRB4 function and in particular its structure and ligands is a key aim but has remained elusive to date. Here we describe the production, crystallization, and structure of the LILRB4 ectodomain to 1.7 Å using an expression strategy involving engineering of an additional disulfide bond in the D2 domain to enhance protein stability. LILRB4 comprises two immunoglobulin domains similar in structure to other LILRs; however, the D2 domain, which is most closely related to the D4 domains of other family members, contains 3(10) helices not previously observed. At the D1-D2 interface, reduced interdomain contacts resulted in an obtuse interdomain angle of ∼107°. Comparison with MHC class I binding Group 1 LILRs suggests LILRB4 is both conformationally and electrostatically unsuited to MHC ligation, consistent with LILRB4 status as a Group 2 LILR likely to bind novel non-MHC class I ligands. Finally, examination of the LILRB4 surface highlighted distinctive surface patches on the D1 domain and D1D2 hinge region, which may be involved in ligand binding. These findings will facilitate our attempts to precisely define the role of LILRB4 in the regulation of immune tolerance.


Assuntos
Antígenos CD/química , Tolerância Imunológica/fisiologia , Receptores Imunológicos/química , Antígenos CD/genética , Antígenos CD/imunologia , Cristalografia por Raios X , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia
7.
Structure ; 23(8): 1426-1436, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26146185

RESUMO

CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.


Assuntos
Antígenos CD/química , Antígenos de Diferenciação de Linfócitos T/química , Moléculas de Adesão Celular Neuronais/química , Proteínas Fetais/química , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Motivos de Aminoácidos , Animais , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Sítios de Ligação , Células CHO , Moléculas de Adesão Celular Neuronais/genética , Clonagem Molecular , Cricetulus , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fetais/genética , Expressão Gênica , Glicosilação , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Eletricidade Estática
8.
PLoS One ; 9(3): e92184, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24642916

RESUMO

SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 µM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 µM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.


Assuntos
Antígenos CD/metabolismo , Células Matadoras Naturais/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Antígenos CD/genética , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Motivo de Inibição do Imunorreceptor Baseado em Tirosina , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Dados de Sequência Molecular , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Protein Expr Purif ; 47(2): 490-7, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16406677

RESUMO

The leukocyte immunoglobulin-like receptors (LIRs, also known as ILTs, CD85, and LILRs) comprise a family of related immunoregulatory receptors encoded within the leukocyte receptor cluster (LRC) on human chromosome 19. LIRs are transmembrane proteins containing either two or four extracellular immunoglobulin domains, and most family members are expressed predominantly on myeloid cell lineages. Although the inhibitory receptors LIR-1 and LIR-2 are known to bind to a broad range of class I MHC molecules and are thought to play important roles in immune regulation, the majority of LIRs are currently of unknown structure and their ligands remain unidentified. In this study, we describe recombinant production and characterisation of the extracellular portion of LIR-5 (ILT3), a poorly understood inhibitory receptor that transduces tolerising signals to dendritic cells. The two extracellular immunoglobulin domains of LIR-5 were expressed in Escherichia coli to a high level and were found to accumulate in inclusion bodies. Inclusion bodies were purified, solubilised, and receptor then renatured by dilution refolding, with acceptable yields. Size exclusion chromatography and SDS-PAGE analyses confirmed the extracellular portion behaved as a monomer in solution, and purified protein was antibody-reactive. LIR-5 is representative of a subset of LIR receptors that on the basis of structural and sequence comparisons with LIR-1 seem unlikely to bind class I MHC molecules. Successful prokaryotic generation of correctly folded LIR-5 in high levels has implications for production of other LRC receptors and should greatly facilitate attempts to define the structure and ligands of this important regulator of dendritic cell function.


Assuntos
Escherichia coli , Dobramento de Proteína , Receptores Imunológicos/biossíntese , Receptores Imunológicos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Antígenos CD/química , Antígenos CD/metabolismo , Cromatografia em Gel , Células Dendríticas/fisiologia , Eletroforese em Gel de Poliacrilamida , Genes MHC Classe I/fisiologia , Humanos , Tolerância Imunológica/fisiologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ligantes , Família Multigênica/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteínas Recombinantes/química , Transdução de Sinais/fisiologia , Homologia Estrutural de Proteína , Ressonância de Plasmônio de Superfície
10.
J Infect Dis ; 191(11): 1842-53, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15871117

RESUMO

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8+ T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection.


Assuntos
Antígenos CD/metabolismo , Infecções por Citomegalovirus/imunologia , Regulação da Expressão Gênica/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Antígenos HLA/metabolismo , Humanos , Influenza Humana/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Recidiva , Especificidade da Espécie
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