RESUMO
We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers: This 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10(-10)M, which is smaller than (7.13±1.20)×10(-10)M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10(-10)M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.
Assuntos
Proteínas de Peixes/imunologia , Peptídeos/imunologia , Receptores Colinérgicos/imunologia , Torpedo/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Sequência de Bases , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Western Blotting , Desenho de Fármacos , Proteínas de Peixes/química , Proteínas de Peixes/genética , Soros Imunes/imunologia , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Miastenia Gravis/sangue , Miastenia Gravis/imunologia , Peptídeos/química , Peptídeos/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Ratos , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Torpedo/genéticaRESUMO
MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , MicroRNAs/imunologia , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Colinérgicos/imunologia , Anticorpos de Cadeia Única/imunologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Animais , Antígenos CD20/imunologia , Linfócitos B/metabolismo , Western Blotting , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Feminino , Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Miastenia Gravis Autoimune Experimental/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Anticorpos de Cadeia Única/genética , Torpedo/imunologia , Torpedo/metabolismoRESUMO
In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Miastenia Gravis Autoimune Experimental/sangue , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Colinérgicos/sangue , Animais , Autoanticorpos/sangue , AMP Cíclico/análogos & derivados , Citometria de Fluxo , Imunização , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos C57BL , Torpedo/imunologiaRESUMO
Myasthenia gravis (MG) and experimental autoimmune MG are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL), composed of the tandemly arranged two single amino acid analogs of two myasthenogenic peptides, p195-212 and p259-271, down-regulated in vitro and in vivo MG-associated T cell responses. In the present study, we investigated the role of CD8(+)CD28(-) regulatory cells in the mechanism of action of the dual APL. We demonstrated that treatment of mice with the dual APL concomitant with immunization with a myasthenogenic peptide resulted in an increased population of CD8(+)CD28(-) cells that express forkhead box P3 (Foxp3). The dual APL inhibited the proliferation of lymph node (LN) cells of the Torpedo acetylcholine receptor-immunized WT C57BL/6 mice, whereas the inhibition was abrogated in CD8(-/-) knockout mice. Moreover, the dual APL did not inhibit the secretion of IFN-gamma by LN cells from CD8(-/-) mice immunized with Torpedo acetylcholine receptor. However, the mRNA expression of IL-10 and TGF-beta by LN cells from CD8(-/-) mice was up-regulated similarly to that of the WT mice. Furthermore, the dual APL elevated the proapoptotic markers caspases 3 and caspase 8, whereas it down-regulated the antiapoptotic marker Bcl-xL in both CD8(-/-) and WT mice. Finally, the dual APL-induced CD4(+)CD25(+)Foxp3(+) cells were up-regulated in CD8(-/-) mice to a similar extent to that observed in the WT mice. Thus, we suggest that CD8(+)CD28(-) regulatory cells play a partial role in the mechanism of action by which the dual APL suppresses experimental autoimmune MG-associated T cell responses.
Assuntos
Antígenos CD28/metabolismo , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Miastenia Gravis/metabolismo , Peptídeos/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/genética , Caspase 3/metabolismo , Caspase 8/metabolismo , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Imunização , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ligantes , Camundongos , Camundongos Knockout , Miastenia Gravis/imunologia , Receptores Nicotínicos/imunologia , Torpedo/imunologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
Immunoblotting can be used for screening a population of antibodies to acetylcholine receptor subunits circulating in the blot of patients with myasthenia. Torpedo Californica acetylcholine receptor served as the antigen. We found that in generalized myasthenia autoantibodies bind to alpha1- or alpha1- and gamma-subunits, while in ophthalmic form they bind only gamma-subunit of acetylcholine receptor. No antibodies to any of the acetylcholine receptor subunits were detected in patients with endocrine ophthalmopathy and in healthy volunteers. This method can be used for differential diagnosis of ophthalmic myasthenia and endocrine ophthalmopathy and for predicting generalization of the pathological process in patients with myasthenia.
Assuntos
Autoanticorpos/sangue , Oftalmopatias/diagnóstico , Immunoblotting/métodos , Miastenia Gravis/diagnóstico , Receptores Nicotínicos/imunologia , Tireoidite Autoimune/complicações , Adolescente , Adulto , Animais , Diagnóstico Diferencial , Oftalmopatias/etiologia , Feminino , Humanos , Masculino , Torpedo/imunologiaRESUMO
Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4+ T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deficient in IL-4 (IL-4-/-) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4+ T cells respond not only to the TAChR and TAChR subunit peptides, but also to several mouse AChR subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR.
Assuntos
Interleucina-4/deficiência , Interleucina-4/genética , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/sangue , Antígeno B7-1/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/biossíntese , Doença Crônica , Epitopos de Linfócito T/imunologia , Predisposição Genética para Doença , Imunização , Injeções Subcutâneas , Interferon gama/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miastenia Gravis Autoimune Experimental/sangue , Fragmentos de Peptídeos/imunologia , Receptores Colinérgicos/administração & dosagem , Receptores Colinérgicos/imunologia , Índice de Gravidade de Doença , Baço/citologia , Baço/imunologia , Baço/metabolismo , Torpedo/imunologiaRESUMO
Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent Ab-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1-type cells and costimulatory factors such as CD40 ligand (CD40L) contribute to disease pathogenesis by producing proinflammatory cytokines and by activating autoreactive B cells. In this study we demonstrate the capacity of CD40L blockade to modulate EAMG, and analyze the mechanism underlying this disease suppression. Anti-CD40L Abs given to rats at the chronic stage of EAMG suppress the clinical progression of the autoimmune process and lead to a decrease in the AChR-specific humoral response and delayed-type hypersensitivity. The cytokine profile of treated rats suggests that the underlying mechanism involves down-regulation of AChR-specific Th1-regulated responses with no significant effect on Th2- and Th3-regulated AChR-specific responses. EAMG suppression is also accompanied by a significant up-regulation of CTLA-4, whereas a series of costimulatory factors remain unchanged. Adoptive transfer of splenocytes from anti-CD40L-treated rats does not protect recipient rats against subsequently induced EAMG. Thus it seems that the suppressed progression of chronic EAMG by anti-CD40L treatment does not induce a switch from Th1 to Th2/Th3 regulation of the AChR-specific immune response and does not induce generation of regulatory cells. The ability of anti-CD40L treatment to suppress ongoing chronic EAMG suggests that blockade of CD40L may serve as a potential approach for the immunotherapy of MG and other Ab-mediated autoimmune diseases.
Assuntos
Antígenos de Diferenciação/biossíntese , Ligante de CD40/imunologia , Regulação para Baixo/imunologia , Soros Imunes/administração & dosagem , Imunoconjugados , Imunossupressores/administração & dosagem , Miastenia Gravis Autoimune Experimental/imunologia , Células Th1/imunologia , Regulação para Cima/imunologia , Abatacepte , Animais , Especificidade de Anticorpos , Antígenos CD , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígeno CTLA-4 , Diferenciação Celular/imunologia , Células Cultivadas , Doença Crônica , Citocinas/biossíntese , Feminino , Humanos , Hipersensibilidade Tardia/imunologia , Imunoglobulina G/biossíntese , Isotipos de Imunoglobulinas/biossíntese , Injeções Intraperitoneais , Injeções Subcutâneas , Ativação Linfocitária/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Miastenia Gravis Autoimune Experimental/prevenção & controle , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/administração & dosagem , Receptores Colinérgicos/imunologia , Células Th1/metabolismo , Células Th1/patologia , Células Th2/imunologia , Células Th2/metabolismo , Torpedo/imunologiaRESUMO
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.
Assuntos
Interferon gama/deficiência , Interferon gama/genética , Interleucina-12/deficiência , Interleucina-12/genética , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Animais , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Sítios de Ligação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Predisposição Genética para Doença , Imunidade Inata , Imunoglobulina G/biossíntese , Imunoglobulina G/classificação , Imunoglobulina G/metabolismo , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miastenia Gravis/metabolismo , Junção Neuromuscular/imunologia , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/administração & dosagem , Receptores Colinérgicos/imunologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Torpedo/imunologiaRESUMO
Characterizing AChR-specific T lymphocyte clones is an important step towards the ability to induce antigen-specific tolerance in myasthenia gravis (MG). However, the limited supply of relatively inefficient autologous antigen presenting cells (APCs) makes establishing AChR-specific T lymphocyte lines difficult. In this study we targeted AChR to autologous surface IgM+ (sIgM+) APCs using heterobifunctional antibodies (bi-Ab) consisting of anti-sIgM linked to anti-AChR antibodies. FACScan analysis and whole cell-based radioimmunoassay (RIA) showed binding of bi-Ab/AChR conjugates to sIgM+ APCs. Using antigen targeting, AChR-presentation to a well-characterized AChR-specific T cell clone, and to T cell lines raised de novo from MG thymocytes, was improved. Thus, antigen targeting using bi-Ab improved the efficiency of presentation of the scarce autoantigen AChR, suggesting that this method might allow the use of relatively impure antigen preparations and normally inefficient non-antigen-specific APCs, including those which can be immortalized, to accelerate the characterization of the AChR epitopes recognized by pathogenic T helper lymphocytes.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Biespecíficos/imunologia , Apresentação de Antígeno , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunoglobulina M/imunologia , Isoanticorpos/imunologia , Miastenia Gravis/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores Colinérgicos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Doenças Autoimunes/patologia , Doenças Autoimunes/cirurgia , Autoimunidade , Subpopulações de Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Linhagem Celular Transformada/imunologia , Células Clonais , Herpesvirus Humano 4 , Humanos , Hibridomas/imunologia , Miastenia Gravis/patologia , Miastenia Gravis/cirurgia , Ratos , Subpopulações de Linfócitos T/patologia , Timectomia , Timoma/imunologia , Timoma/patologia , Timoma/cirurgia , Neoplasias do Timo/imunologia , Neoplasias do Timo/patologia , Neoplasias do Timo/cirurgia , Torpedo/imunologiaRESUMO
Experimental autoimmune myasthenia gravis (EAMG), a disorder of the neuromuscular junction, is mediated by autoantibodies against muscle nicotinic acetylcholine receptor (AChR). The roles of IFN-gamma (Th1) and IL-4 (Th2) cytokines in the initiation and progression of this disease are not fully understood. Recently, we have demonstrated that IFN-gamma is necessary for the initiation of tAChR-induced EAMG in mice. However, the role of IL-4 in the progression of clinical EAMG remained undetermined. In this study we have addressed the contribution of IL-4 in the disease progression in IL-4(-/-) C57BL/6j mice whose IL-4 gene has been disrupted. Following immunization with Torpedo (t) AChR, the IL-4(-/-) mice readily developed signs of muscle weakness and succumbed to clinical EAMG with kinetics similar to the susceptibility of IL-4(+/+) mice. The tAChR-primed lymph node cells from IL-4(-/-) mice vigorously proliferated to tAChR and to its dominant alpha146-162 sequence associated with disease pathogenesis. However, these T cells secreted higher levels of IFN-gamma and IL-2, suggesting the development of a Th1 default pathway in these mice. Nevertheless, the IL-4 mutation had no effect on the recruitment of CD4+ Vbeta6+ T cells specific to the dominant tAChR alpha146-162 sequence in vivo. Immune sera from IL-4(-/-) mice showed a dramatic increase in mouse AChR-specific IgG2a levels followed by a concomitant decrease in IgG1 levels, but these mice did not exhibit an accelerated disease. In conclusion, we have demonstrated for the first time that IL-4 is not required either for the generation of a pathogenic anti-AChR humoral immune response or for progression of clinical EAMG in mice.
Assuntos
Autoanticorpos/fisiologia , Interleucina-4/fisiologia , Miastenia Gravis/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Sequência de Aminoácidos , Animais , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Citocinas/biossíntese , Progressão da Doença , Deleção de Genes , Imunização , Epitopos Imunodominantes/imunologia , Imunoglobulina G/biossíntese , Interleucina-4/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Miastenia Gravis/etiologia , Miastenia Gravis/genética , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores Nicotínicos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Torpedo/imunologiaRESUMO
To study the role in myasthenia gravis (MG) of peptides resulting from acetylcholine receptor (AChR) degradation, we examined the ability of AChR peptides to induce T cell responses that are capable of cross-reacting with intact AChR. The studies were carried out in an experimental autoimmune MG (EAMG)-susceptible mouse strain [C57BL/6 (B6)] as well as in two non-susceptible strains [B6.C-H-2bm12 (bm12) and C3H/He]. A set of overlapping peptides encompassing the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica (t) AChR were used, individually or in equimolar mixtures, as immunogens. In B6, immunization with peptides alpha45-60, alpha111-126, alpha146-162 and alpha182-198 gave T cells that responded in vitro to the correlate immunizing peptide. Only the T cells against the latter three peptides cross-reacted with tAChR. Peptide alpha146-162 exhibited the highest in vitro reaction with the immunizing peptide and cross-reaction with tAChR. T cells obtained by immunization of B6 with an equimolar mixture of the peptides responded in vitro to peptides alpha111-126, alpha146-162 and alpha182-198 and cross-reacted very strongly with tAChR. In bm12 and C3H/He, a number of peptides evoked, when used individually as immunogens, strong or moderate T cell responses that recognized in vitro the correlate immunizing peptide but cross-reacted poorly with tAChR. Immunization with the mixture of the peptides gave T cells that recognized several peptides in each strain butdid not cross-react with alpha146-162 or tAChR. The results indicate that the ability to recognize alpha146-162 or AChR by T cells against peptides resulting from receptor degradation can account for the susceptibility to, and aggravation of, MG in B6.
Assuntos
Autoimunidade , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Autoanticorpos/imunologia , Imunização , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores Colinérgicos/genética , Torpedo/imunologiaRESUMO
Myasthenia gravis is an autoimmune disorder characterized by muscle weakness, due to an antibody-mediated deficit of acetylcholine receptors (AChRs) at neuromuscular junctions. We analyzed the factors that determine the severity of experimental myasthenia gravis (EAMG) induced by immunization with Torpedo AChR, in two congenic strains of mice--B6 mice, which are highly susceptible to EAMG; and bm12 mice, which are relatively resistant, and differ only in a change of three amino acids in MHC Class II. We prepared large numbers of AChR-specific T cell hybridomas from each strain and characterized their epitope specificities and T cell receptor (TCR) gene usage: Half the B6 hybridomas responded to a single AChR peptide (alpha 146-162), and their TCR genes encoded restricted V alpha and V beta chains and CDR3 motifs. bm12 hybridomas had different epitope specificities and different, less restricted TCR genes. APCs were able to present AChR or AChR-derived peptides virtually exclusively to hybridomas of their own strain. Levels of antibodies to Torpedo and autoantibodies to mouse AChR were higher in B6 mice, and were biased toward the IgG2b isotype. We conclude that the "better fit" of MHC II, peptide, and TCR in the B6 mice enhanced cognate interactions of APCs with T cells, and T cells with B cells, resulting in a more abundant and pathogenic AChR antibody response, and thus more severe EAMG.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Animais , Especificidade de Anticorpos , Apresentação de Antígeno , DNA Complementar/genética , Feminino , Rearranjo Gênico do Linfócito T , Hibridomas/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Especificidade da Espécie , Torpedo/imunologiaRESUMO
We have previously mapped the T and B cell epitopes on the alpha-subunit of acetylcholine receptor (AChR) in human myasthenia gravis (MG) and in experimental autoimmune MG-susceptible (C57BL/6 (B6)) and nonsusceptible mouse strains. In addition to regions recognized by both T and B cells, the AChR alpha-subunit has regions that are recognized solely by T cells. An exclusive T cell epitope within residues alpha 146-162 of Torpedo californica (t), tAChR, plays an important role in experimental autoimmune MG pathogenesis in B6 mice. To study its function, we established, from tAChR-primed B6 mice, two t alpha 146-162-specific T cell lines (P14Th) which comprised Th2-type cells because they secreted IL-4 but not IL-2. P14Th did not recognize the corresponding region on mouse (m) AChR (m alpha 146-162). They caused in vitro differentiation of tAChR-primed B cells into plasma cells that secreted anti-AChR Abs directed, in decreasing order, against the following tAChR alpha regions: t alpha 122-138 > t alpha 134-150 > t alpha 45-60 > t alpha 170-186 > t alpha 56-71. Little or no Ab response could be detected against peptides t alpha 182-198 or t alpha 146-162 itself. The major enhancement was in the Abs against region t alpha 122-150 (spanning the t alpha 122-138/t alpha 134-150 overlap) that is involved in ACh binding. These Abs cross-reacted completely with m alpha 122-150, the corresponding region on mAChR. Therefore, t alpha 146-162-specific T cells, although unable to recognize m alpha 146-162, are nevertheless pathogenic because they help B cells responding to a tAChR region that is conserved in mAChR and involved in ACh binding. These Abs cross-react with the corresponding effector-binding region of mAChR, thereby disrupting the normal physiologic function of the mouse receptor.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Epitopos/imunologia , Cooperação Linfocítica , Miastenia Gravis/imunologia , Fragmentos de Peptídeos/imunologia , Receptores Nicotínicos/imunologia , Células Th2/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Reações Cruzadas , Meios de Cultivo Condicionados , Feminino , Imunização , Interleucina-2/análise , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmócitos/imunologia , Alinhamento de Sequência , Especificidade da Espécie , Células Th2/metabolismo , Torpedo/imunologiaRESUMO
C57BL/6 (B6) mice develop a neuromuscular disease, experimental autoimmune myasthenia gravis (EAMG), after two or more immunizations with Torpedo californica acetylcholine receptor (AChR). To determine whether EAMG is related to recognition of particular region(s) on the main extracellular domain of the alpha chain (residues alpha 1-210) in prolonged immunization, we have examined the differences in the antibody and T cell recognition profiles of B6 and SJL (a strain that does not develop EAMG) mice after different periods and a number of immunizations with Torpedo AChR. In a given strain, antibodies and T cells recognized immunodominant regions, which may coincide or may be uniquely B cell or T cell determinants. Both B6 and SJL exhibited similar antibody recognition profiles after the second and through the fourth immunizations with AChR. Major differences between the two strains were found in their T cell recognition of regions in the second part (residues 100-210) of the main extracellular domain of the alpha chain. T cells of SJL recognized consistently only one region (111-126) within this part of the alpha chain, whereas in B6, T cell recognition of three peptides (111-126, 146-162 and 182-198) and next neighbor regions to them persisted throughout the period. Of these three peptides, 146-162 was an immunodominant peptide unique to B6, as the other two peptides (111-126 and 182-198) were also recognized by either T cells or antibodies in SJL. To study the role of the T cells recognizing region 146-162 in EAMG, a T cell line was generated against this region and the cells transferred into B6 mice followed by one Torpedo AChR injection. Enhancement of antibody production toward alpha chain peptides was observed as an influence of T cell transfer compared to profiles at 1 week. In addition, one out of three mice examined showed signs of EAMG. These results suggest the importance of T cells recognizing residues 146-162 in EAMG. It is concluded that the presence of persistent T cell responses to the second half (residues (100-210) of the main extracellular domain of the alpha chain is associated with the development of EAMG in B6 mice, while absence of these responses in SJL mice may enable them to escape the disease. The preservation of the immunodominance of peptide 146-162 in the T cell recognition of B6 is probably most important for the pathogenesis of EAMG in this strain.
Assuntos
Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Feminino , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Dados de Sequência Molecular , Linfócitos T/imunologia , Linfócitos T/transplante , Fatores de Tempo , Torpedo/imunologiaRESUMO
Residues 146-162 of the Torpedo californica acetylcholine receptor alpha-subunit contain the immunodominant T cell epitope for experimental autoimmune myasthenia gravis-susceptible C57BL/6 mice. To develop potential therapeutic peptides, a detailed analysis of the epitope was undertaken. Truncated and substituted synthetic peptides were tested as stimulators of T cell clones and immune lymph node cells. Critical residues spanned positions 151-159. Y151 and V156 were critical for MHC binding. The results indicated a general motif for binding to I-Ab to be an aromatic or hydrophobic residue (preferentially Y or F) at position i followed by an uncharged residue at position (i + 5). Lysine was not tolerated at position (i + 8). Residues D152, K155, S157, and I158 were important T cell contact residues. A peptide corresponding to the murine 146-162 sequence, which differs from the Torpedo sequence at 5 residues, bound to I-Ab but was nonimmunogenic, consistent with the assigned TCR and I-Ab contact residues. These results suggest that tolerance is responsible for the lack of T cell cross-reactivity with the murine acetylcholine receptor. Substituted peptides were tested for the inhibition of T cell clone responses and for TCR antagonism. Although peptides substituted at residues 157 and 158 inhibited most clones, no single peptide could completely inhibit all clones or primed lymph node cells. These findings indicate that antagonist peptides may be useful in inhibiting T cell responses to complex Ag displaying a single immunodominant epitope. Multiple antagonists used in combination may be required for maximum inhibition of the response.
Assuntos
Receptores Colinérgicos/imunologia , Torpedo/imunologia , Sequência de Aminoácidos , Animais , Autoimunidade , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Ligação Competitiva , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/metabolismo , Imunização , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/metabolismo , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miastenia Gravis/etiologia , Miastenia Gravis/terapia , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Homologia de Sequência de Aminoácidos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Torpedo/genéticaRESUMO
To study the organization of the polypeptide chain of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha-subunit, we examined the ability of the native membrane-bound AChR of Torpedo californica (T-AChR) to bind a panel of antibodies against overlapping synthetic peptides which collectively encompassed this entire domain. Antibodies against the alpha-chain peptides alpha 1-16, alpha 89-104 and alpha 158-174 were able to bind to membrane-bound T-AChR. Other anti-peptide antibodies showed little or no binding to T-AChR in the membrane. It is concluded that regions alpha 1-16, alpha 89-104 and alpha 158-174 are highly exposed on the surface of the alpha subunit of membrane-bound AChR.
Assuntos
Anticorpos/imunologia , Fragmentos de Peptídeos/imunologia , Conformação Proteica , Receptores Nicotínicos/química , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Epitopos/imunologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Receptores Nicotínicos/imunologia , Torpedo/imunologiaAssuntos
Animais Recém-Nascidos/imunologia , Doenças Autoimunes/terapia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Epitopos Imunodominantes/imunologia , Terapia de Imunossupressão , Miastenia Gravis/prevenção & controle , Receptores Nicotínicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Sequência de Aminoácidos , Animais , Autoanticorpos/sangue , Doenças Autoimunes/imunologia , Suscetibilidade a Doenças/imunologia , Feminino , Conversão Gênica , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunização , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miastenia Gravis/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Método Simples-Cego , Especificidade da Espécie , Torpedo/imunologiaRESUMO
The alpha-subunit of the nicotinic acetylcholine receptor is the major target of the autoimmune response in myasthenia gravis. We investigated the proliferative response of T cells from patients with myasthenia gravis and healthy volunteers to recombinant polypeptides of the human acetylcholine receptor including the full-length alpha-subunit (alpha 1-437). T cells from 20 (71%) of 28 patients and 7 (37%) of 19 healthy volunteers responded in primary cultures. Subsequently, specific T-cell lines were established: CD4+, CD8-, UCHL1+, and major histocompatibility complex (MHC) class II-restricted. Using a set of fragments of the alpha-subunit, major antigenic sites could be localized on the extracellular, N-terminal part of the molecule as well as close to the C-terminus. The T-cell response was heterogeneous, both among different individuals and among T-cell lines from a single donor. These T cells did not cross-react with Torpedo acetylcholine receptor, which was previously used as a substitute for human muscle acetylcholine receptor, suggesting that the T cells had a bias for unique human sequences. A single antigenic fragment could be presented in the context of different MHC class II molecules, and different fragments could be presented in the context of the same MHC molecule. This supports earlier observations of considerable heterogeneity in dealing with acetylcholine receptor as an autoantigen on the level of both T cells and antigen-presenting cells. The data also demonstrate that acetylcholine receptor-specific T cells are present in the normal immune repertoire, and emphasize the role of immune regulation for maintaining a state of tolerance.
Assuntos
Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Miastenia Gravis/imunologia , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Animais , Autoantígenos/genética , Doenças Autoimunes/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Reações Cruzadas , DNA/genética , Epitopos/imunologia , Antígenos HLA-DR/imunologia , Tolerância Imunológica , Ativação Linfocitária , Fragmentos de Peptídeos/imunologia , Receptores Colinérgicos/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Torpedo/imunologiaRESUMO
Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder characterized by reduced acetylcholine release at the neuromuscular junction. We report a model of the disease developed by active immunization of rats with purely cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Electromyographic studies of neuromuscular transmission in these rats showed a weak initial response followed by a pronounced incremental response to paired supramaximal stimuli (8 msec apart). There was no such response in control rats. There was no evidence of a postsynaptic transmission deficit in the synaptosomes immunized rats. We conclude that immunizing rats with Torpedo cholinergic nerve terminals causes a specific presynaptic dysfunction and may serve as a model for the study of LEMS.