CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR alpha subunit. Several epitopes were identified. Only the most severely affected patients recognized alpha subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several alpha subunit peptide sequences could be detected when CD(8+)-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any alpha subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48-67, 101-137, 293-337, and 308-437).     

Subcutaneous administration of T-epitope sequences of the acetylcholine receptor prevents experimental myasthenia gravis.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.     

Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4+ epitope sequences of the acetylcholine receptor

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.     

Presentation of endogenous acetylcholine receptor epitope by an MHC class II-transfected human muscle cell line to a specific CD4 T cell clone from a myasthenia gravis patient

Muscle or thymic myoid cells, if induced to express MHC class II in addition to endogenous acetylcholine receptor (AChR), might present epitopes derived from the AChR to specific CD4⁺ T cells. These T cells could in turn initiate or maintain the anti-AChR response that is responsible for AChR loss in myasthenia gravis (MG). We transfected the AChR⁺ TE671 (rhabdyyosarcomyosarcoma) cells with HLA-DR4 and co-cultured them with the DR4-restricted, CD4⁺ T cell clone (PM-A1; raised from a hyperplastic thymus of an MG patient and previously shown to recognise all forms of the AChR that contain the sequence α144–156). Significant T cell activation, demonstrated both by ³H-thymidine incorporation and by lysis of the TE671 cells, was found in the presence of added α144–156 and, more importantly, in the absence of exogenous antigen. These results show that MHC class II-expressing muscle or other AChR-expressing cells could present endogenous AChR to pathogenic T cells. This process may be important in the aetiology of MG.     

The predictive value of the presence of different antibodies and thymus pathology to the clinical outcome in patients with generalized myasthenia gravis

Objectives

To analyze the predictive value of anti-acetylcholine receptor antibodies (anti-AChR Ab) and anti-muscle specific kinase antibodies (anti-MuSK Ab), as well as the thymus pathology to the clinical outcome in patients with generalized myasthenia gravis (MG).

Methods

We analyzed 138 patients with generalized MG, who were thymectomized and assayed for anti-AChR Ab and anti-MuSK Ab.

Results

Anti-AChR Ab were detected in 84% of patients, while anti-MuSK Ab were present in 36% of the AChR Ab negative patients. Severe forms of the disease were more frequent in MuSK Ab positive, compared to the AChR Ab positive and complete seronegative patients. Thymic lymphoid follicular hyperplasia (LFH) was present in 60%, thymoma in 23%, atrophic thymus in 9% and the normal thymus in 8% of patients. LFH was more frequent among women, while thymoma and atrophic thymus were more frequent in men. The younger patients mainly had LFH and normal thymus, while thymoma and atrophic thymus were more frequent in older patients. The mildest clinical presentation was present in patients with normal thymus, while severe forms of the disease were registered in the patients with thymoma. The AChR Ab positive patients had more often LFH and thymoma, while within MuSK Ab positive patients atrophic thymus was most common.

Conclusion

The best disease outcome was observed in patients with normal thymus or LFH with anti-AChR Ab or without both types of antibodies.     

Combined effects of a thymic peptide, thymopoietin and myasthenic patient sera in rat myotube culture.

We investigated in a rat myotube assay the combined effect of 26 myasthenic (MG) patient sera and a thymic peptide, thymopoietin (Tpo) which had previously been shown to bind Torpedo and human AChR and to compete with alpha-bungarotoxin (alpha-Bgt) binding. Cultures were first exposed to Tpo alone for 3 h (0.3, 7.5, 15 nM), then MG sera (5% final dilution) were added for an additional 18 h. Reduction in the amount of 125I-alpha-Bgt binding sites in the presence of various concentrations of Tpo were similar with control sera and in all the patients with low or undetectable anti-AChR Ab (11 cases). In cultures exposed to Tpo and sera with high anti-AChR Ab titre (15 cases), Tpo and anti-AChR Ab have an additive capacity to reduce the number of alpha-Bgt binding sites. The results are compatible with the hypothesis that anti-AChR Ab and Tpo could impair neuromuscular transmission by complementary mechanisms.     

A sensitive rosetting assay for detection of acetylcholine receptor antibodies using BC3H-1 cells: positive results in 'antibody-negative' myasthenia gravis

Antibodies to acetylcholine receptor (AChR) were measured in a group of patients with myasthenia gravis (MG), some of whom had previously been classified as 'antibody negative' using the standard anti-AChR radioimmunoassay (RIA). AChR antibodies were measured using the rosetting assay, a new detection method which utilizes protein A-coated red blood cells and live BC3H-1 cells, a murine cell line which expresses muscle nicotinic AChR. The results of the rosetting assay were compared with those obtained in the anti-AChR RIA. 76% of all myasthenic sera tested showed rosetting at titers higher than any of the control sera (from patients with non-myasthenic neurologic disease and normal individuals). Of the myasthenic patients previously classified as 'antibody negative' in the RIA using human AChR, 71% demonstrated positive rosetting. There was no correlation between the anti-AChR antibody titer obtained in the rosetting assay and that obtained in the RIA using either human or denervated rat AChR. The results suggest that the rosetting assay may measure a subpopulation of antibodies that differs from those detected in the RIA.     

Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes

Nasal administration of synthetic CD4(+) epitopes of the acetylcholine receptor (AChR) prevents experimental myasthenia gravis (EMG) in C57Bl/6 mice, but not in IL4-deficient C57Bl/6 (IL4(-/-)) mice. Here we verify that nasal tolerance requires IL4, by showing that CD4(+) cells from C57Bl/6 mice treated nasally with a pool of AChR CD4(+) epitopes protected IL4(-/-) mice from EMG and caused a reduced production of anti-AChR antibody. CD4(+) cells from C57Bl/6 mice treated with unrelated peptides or sham-treated did not induce protection. CD4(+) cells from C57Bl/6 mice treated with just one AChR peptide protected IL4(-/-) mice from EMG without affecting antibody synthesis.     

Circulating CD4+CD8+ cells in myasthenia gravis: supplementary immunological parameter for long-term prognosis

Summary Twenty patients with myasthenia gravis (MG) were studied prospectively for up to 5 years after thymectomy, in order to clarify the relationships between disease severity, anti-acetylcholine receptor antibody (anti-AChR) titres, proportions of circulating CD4⁺CD8⁺ cells (CD4⁺CD8⁺ cell level) and major lymphocyte subsets. The CD4⁺CD8⁺ cell levels were closely related to the clinical change within 1 year after surgery in 8 patients who showed a preoperative elevation in the cell levels. This group of patients consisted of six thymomatous and two non-thymomatous patients; the latter were both negative for anti-AChR. The anti-AChR titres generally changed in parallel with the clinical state in 9 of the 16 patients who were followed up for more than a year after thymectomy, and the CD4⁺CD8⁺ cell levels were useful in predicting the clinical course in 6 of the above 9 patients and 3 other patients, including antibody-negative cases. The present study suggests that the CD4⁺CD8⁺ cell levels may serve as an indicator for long-term prognosis of MG.     

CD4+CD25+ regulatory T cells and CD1-restricted NKT cells do not mediate facial motoneuron survival after axotomy

CD4+ T cells rescue facial motoneurons (FMN) from axotomy-induced cell death. The objective of this study is to determine if the CD4+ T regulatory subsets, CD4+CD25+ T or CD1d-restricted NKT cells are critical for FMN survival after facial nerve axotomy. Surviving FMN within facial motor nuclei from axotomized and control sides 4 weeks after axotomy were counted to determine percent FMN survival. Data generated by applying this paradigm to recombination activating gene-2-deficient mice reconstituted with CD4+ T cells depleted of CD4+CD25+ T cells and to CD1-/- mice, deficient in CD1d-restricted NKT cells, suggest that neither regulatory CD4+ T subset is critical for FMN survival.     

Intrathecal immunoactivation in the patients with myasthenia gravis--autoantibodies in the cerebrospinal fluid

We examined the cerebrospinal fluid (CSF) of 17 myasthenia gravis (MG) patients for certain immunological parameters in order to assess signs of intrathecal immunoactivation. We have found oligoclonal IgG bands in the CSF of 6 of 17 by the agarose gel electrophoresis and enlarged lymphoid cells in CSF of 8 of 17 patients of MG. We measured anti-nicotinic acetylcholine receptor (AChR) antibody and anti-striational muscle antibody (AMA) in serum and CSF from MG patients and compared the serum: CSF ratio with total IgG. No evidence of intrathecal anti-AChR antibody synthesis was demonstrated while AMA was partly (50%) synthesized in myasthenic CSF. This difference suggested that there is either different immunological regulation in synthesis between anti-AChR antibody and AMA in the CSF or the different behavior after transfer to CSF.     

Acetylcholine receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis

Muscle-specific tyrosine kinase (MuSK) antibodies are found in some patients with "seronegative" myasthenia gravis (MG), but how they cause myasthenic symptoms is not clear. We visualized acetylcholine receptors (AChRs) and complement component 3 (C3) in muscle biopsies from 10 Japanese MG patients with MuSK antibodies, compared with 42 with AChR antibodies. The AChR density was not significantly decreased in MuSK antibody (Ab)-positive end-plates compared with AChR antibody-positive end-plates, and C3 was detected in only two of eight MuSK Ab-positive patients. MuSK antibodies do not appear to cause substantial AChR loss, complement deposition, or morphological damage. Effects on MuSK function need to be explored.     

乙酰胆碱受体诱导实验性自身免疫性重症肌无力模型的建立

目的采用纯化乙酰胆碱受体(acetylcholine receptors,AChR)免疫大鼠和小鼠以建立实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)动物模型。方法以亲和层析法从电鳐电器官提取和纯化AChR,并用其免疫接种Lewis大鼠和C57BL/6小鼠,观察接种动物的临床症状、电生理变化以及AChR抗体产生的情况。结果动物模型临床肌无力症状、翻转悬挂时间(小鼠,P<0.05)、重复神经电刺激(repetitive nerve stimulation,RNS)动作电位衰减率、抗体吸光度(P<0.05)及新斯的明试验均为阳性或有统计学意义。结论纯化电器官AChR作为免疫原,成功诱导产生EAMG动物模型。Lewis大鼠和C57BL/6小鼠均对AChR免疫易感而产生EAMG表现,7~8周龄的C57BL/6更易诱导出类似人类MG表现的EAMG。     

MG患者外周血Th17细胞与AChR抗体产生的关系研究

目的探讨重症肌无力(MG)患者外周血中Th17细胞及相关细胞因子白细胞介素17(IL-17)在MG发病中的作用。方法收集40例MG患者和10名健康人(对照组)外周血标本,采用流式细胞术检测外周血单个核细胞(PBMCs)中Th17细胞比例,反转录酶-聚合酶链锁反应(RT-PCR)检测PBMCs中维甲酸受体相关孤儿受体γt(RORγt)mRNA水平,ELISA检测血清中IL-17水平,放射免疫沉淀法检测血清中抗乙酰胆碱受体抗体(AChR-Ab)滴度;分离PBMCs中CD4~+T细胞和CD19~+B细胞与金黄色葡萄球菌肠毒素B(SEB)进行共培养,培养系统中加入人IL-17和(或)IL-21中和抗体,放射免疫测定法检测培养液中AChR-Ab滴度。采用MG评分(quantitative MG scoring system,QMGs)对MG的严重程度进行评估,并对MG患者的Th17细胞比例、RORγt mRNA和IL-17水平与病情QMGs的相关性,以及MG患者抗AChR-Ab滴度与PBMCs中Th17细胞比例的相关性进行分析。结果 MG患者PBMCs中Th17细胞比例[1.11%(0.90%,1.34%)]高于健康对照组Th17细胞比例[0.26%(0.08%,0.36%)](z=5.494,P0.001),且与疾病严重程度呈正相关(r=0.4394,P=0.0046);血清中IL-17水平和PBMCs中RORγt mRNA相对表达[分别71.46(53.91,104.76)pg/mL、2.63(1.94,3.12)]均较健康对照组[分别18.82(12.73,29.80)pg/mL、1.13(0.98,1.28)]显著增高(均P0.001);MG患者血清中抗AChR-Ab滴度[2.34(1.19,3.60)nmol/L]较健康对照组[-0.08(-0.24,-0.03)nmol/L]显著增高(z=4.662,P0.001),且与Th17细胞比例呈正相关(r=0.7066,P=0.0001)。MG患者外周血T、B细胞与SEB共培养后抗AChR-Ab水平高于未加入SEB时及健康对照(均P0.01);加入抗人IL-21或IL-17中和抗体后,两者AChR-Ab滴度与未加入抗体时AChR-Ab滴度比较均降低(均P0.05),且均仍高于MG患者未加入SEB时及健康对照(P0.01);在培养上清中同时加入抗人IL-21和IL-17中和抗体时AChR-Ab滴度明显低于加入单种抗体时,而与未加入SEB时及健康对照差异无统计学意义(均P0.05)。结论 MG患者外周血中Th17细胞可能通过IL-17促进AChR-Ab产生,参与疾病的病理过程。     

Neuroimmunology of myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease in which the immune system attacks the nicotinic acetylcholine receptors (AChRs) of the neuromuscular junction. Anti-AChR antibodies are present in 85% of patients and bind to distinct epitopes on the surface of the AChR alpha subunits, as defined by competition with monoclonal anti-AChR antibodies. There are at least three types of the disease, defined by thymic histology, age of onset, and HLA associations, and anti-AChR antibodies show some differences in fine specificity between those with thymic hyperplasia and those with thymic tumors. Peripheral blood lymphocytes from MG patients contain T lymphocytes specifically sensitized to AChR. These are stimulated by purified Torpedo AChR and some human alpha subunit synthetic peptides. The T and B cell epitopes on the primary sequence of the alpha subunit are currently being mapped using recombinant human AChR subunit fragments.     

FoxP3+ CD4+ CD25+调节性T细胞在重症肌无力发病机制中的作用

目的 在细胞与分子水平检验重症肌无力(myasthenia gravis,MG)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+Tregs)的表达缺陷,探讨CD4+CD25+Tregs亚群异常与MG发病间的关系.方法 流式细胞技术检测21例MG患者(11例经胸腺切除)与20名健康对照者(healthy controls,HCs)外周血CD4+CD25+Tregs及FoxP3+CD4+CD25+Tregs含量,实时荧光定量聚合酶链反应(RT-FQ-PCR)分析MG患者与HCs外周血CD4+CD25+Tregs中FoxP3 mRNA的表达.结果 MG患者外周血CD4+CD25+ Tregs占CD4+T细胞含量与HCs比较无统计学差异(P＞0.05).MG患者外周血FoxP3+CD4+CD25+ Tregs含量及FoxP3 mRNA表达量与HCs比较均显著性降低(P＜0.05);胸腺切除的MG患者与未经胸腺切除的MG患者外周血FoxP3+CD4+CD25+ Tregs含量及FoxP3mRNA表达量无统计学差异(P＞0.05).结论 MG患者外周血CD4+CD25+ Tregs数量正常,但其表面分子FoxP3的表达下调,这种CD4+CD25+ Tregs亚群的异常发现有助于深入阐明MG的免疫发病机制.     

Surface phenotypes of lymphoid cells altered in the human myasthenic thymus

We investigated surface phenotypes of peripheral blood lymphocytes and thymic lymphoid cells from patients with myasthenia gravis (MG) by fluorocytometry, using monoclonal antibodies to human lymphoid cells. There were no significant differences in peripheral blood lymphocyte subsets in myasthenic patients, with or without a thymus. In the MG hyperplastic thymuses, the percentage of OKIa1+ cells or CCB1+ cells was significantly increased compared with controls. Although there were no significant differences in the percentage of T-cell lineage (CD3+, CD4+, CD8+, or CD1+ cells) between MG hyperplastic thymuses and the controls, the surface densities of T-cell lineage antigens (CD3, CD4, and CD8) were significantly increased on lymphoid cells in MG hyperplastic thymuses, compared with those in control thymuses. There were no significant differences in the percentage or fluorocytograph of lymphoid cells between the MG thymomas and the controls.     

Synthesis of anti-acetylcholine receptor antibodies by CD5- B cells from peripheral blood of myasthenia gravis patients

An increased frequency of CD5⁺ B cells (or, according to a new nomenclature, B 1 cells) has been detected in the peripheral blood of a proportion of patients with myasthenia gravis (MG), as in some other autoimmune diseases. To elucidate the pathogenic significance of this B-cell subset in myasthenia gravis, mononuclear cells from the peripheral blood of six MG patients were separated into T and B lymphocytes by a magnetic cell separation procedure employing superparamagnetic microbeads (MACS). Subsequently, the B-cell fraction was depleted of CD5⁺ B cells in a second separation. The resulting purified CD5^– B-cell fraction was cultured alone or with the addition of autologous T cells. Anti-acetylcholine receptor (AChR) synthesis by CD5^– B cells in cultures with T cells was significantly increased by pokeweed mitogen (176 ±130 fmol/ml per week/2 × 10⁵ B cells) compared with unfractionated cells (75 ± 101) or CD5^– B cells alone (19 ± 4). These results demonstrate that in MG anti-AChR are synthesized, at least in part, by CD5^– B cells which are dependent on T cells. Although this does not exclude the existence of AChR-specific CD5⁺ B cells, it provides evidence against a pivotal role of this B-cell subset in anti-AChR synthesis.     

Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia

In myasthenia gravis (MG), neuromuscular transmission is disrupted due to the production of autoantibodies against acetylcholine receptors (AChR). In previous work, we showed that decay accelerating factor (DAF or CD55), an intrinsic cell surface complement regulator that disables C3/C5 amplification convertases, protects against receptor loss and muscle weakness. In this study, we examined whether, and if so, to what extent CD59, a downstream intrinsic cell surface regulator that prevents assembly of membrane attack complexes (MACs), contributes to this protection. Twenty-four hours after anti-AChR injection, we found that CD59a-/- mice did not significantly differ from WTs, all Daf1-/- CD59a-/- mice either died or required euthanasia. At 48h, Daf1-/- were significantly weaker than CD59a-/- and WT mice, and for these mice immunohistochemistry revealed marked C9 deposition at postsynaptic junctions, radioimmunoassays showed reductions in AChR levels, and electron microscopy demonstrated massive junctional damage. These data indicate that DAF serves as the initial shield that protects the neuromuscular junction whereas CD59 is a further barrier. They argue that complement inhibitor, particularly if targeted to the receptor, could then have therapeutic value in human MG.     

Anticorps dans la myasthénie

In autoimmune myasthenia gravis, 75 to 80% of patients have antiacetylcholine receptor antibodies (anti-AChR abs) quantified by immunoprecipitation. Anti-AChR abs are polyclonal, directed against all AChR subunits, with a major fraction against the main immunogenic region, (alpha-subunit, aminoacids 67-76); they cause AChR loss by three mechanisms: blocking of acetylcholine binding; accelerated degradation or AChR due to bridging of two adjacent AChR molecules (antigenic modulation); lysis of postsynaptic membrane induced by complement. Neither anti-AChR ab level nor antigenic repertoire are correlated with disease severity. Studies performed on rat and human myotubes have shown that capacity of myasthenic patients's sera or immunoglobulins to induce AChR loss was correlated with anti-AChR ab titer but not with severity. Highest anti-AChR ab titers are found in young women with hyperplastic thymus, lowest in older patients and atrophic thymus. Ten to fifteen percent of babies born to myasthenic mothers suffer from a transitory neonatal myasthenic syndrome due to passive transfer of maternal anti-AChR abs. There is no correlation between clinical condition of the baby (presence and severity of neonatal myasthenia) and severity of maternal myasthenia. The risk of neonatal myasthenia is high when maternal ab titer is elevated (≥ 100 nM). Very rare and severe cases of foetal neonatal myasthenia gravis with arthrogryposis, hypomobility are due to presence in maternal serum of anti-AChR ab directed against foetal (gamma) AChR. In generalized myasthenia without anti-AChR ab, antibodies directed against MuSK, a postsynaptic molecule involved in AChR aggregation, are detected in around 40% of patients. Features of MuSK+ myasthenia are the following: strong female preponderance, severity (respiratory and bulbar) requiring immunosuppressants, facial and tongue atrophy, poor response to anticholinesterase inhibitors, atrophic thymus and poor response to thymectomy. Low-affinity anti-AChR abs have been recently reported in myasthenia gravis without anti-AChR and anti-MuSK ABS.