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.     

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.     

Cytokines and the pathogenesis of myasthenia gravis

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4⁺ and CD8⁺ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production. IL-6 and IL-10 have similar effects. The Th1 cytokine IFN-γ is important in inducing B-cell maturation and in helping anti-AChR antibody production and, thereby, for induction of clinical signs and symptoms. Results from studies of time kinetics of cytokines imply that IFN-γ is more agile at the onset of EAMG, probably being one of the initiating factors in the induction of the disease, and IL-4 may be mainly responsible for disease progression and persistance. Even though other Th1 cytokines like IL-2, tumor necrosis factor α (TNF-α), and TNF-β as well as the cytolytic compound perforin do not directly play a role in T-cell-mediated help for anti-AChR antibody production, they are actually involved in the development of both EAMG and MG, probably by acting in concert with other cytokines within the cytokine network. In contrast, transforming growth factor β (TGF-β) exerts immunosuppressive effects which include the down-regulation of both Th1 and Th2 cytokines in MG as well as EAMG. Suppressive effects are also exerted by interferon α (IFN-α). Based on elucidation of the role of cytokines in EAMG and MG, treatments that up-modulate TGF-β or IFN-α and/or suppress cytokines that help B-cell proliferation could be useful to improve the clinical outcome. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 543–551, 1997     

CD4CD25 regulatory T cell in childhood ocular myasthenia gravis

Dysfunction of CD4⁺CD25⁺ regulatory T cell (Treg) has been demonstrated to play an important role in the development of autoimmune myasthenia gravis. This T cell subset, which has potent regulatory properties against immune response, has been reported to have a numerical or functional defect in patients with myasthenia gravis. We examined various T cell subsets, including CD4⁺CD25⁺Treg in peripheral blood mononuclear cells using flow cytometry in a pediatric patient suffering from ocular myasthenia gravis. Contrary to previous reports, the percentage of CD4⁺CD25⁺Treg in peripheral blood decreased significantly after successful treatment with prednisolone. This discrepancy could result from diversity within the immunopathogenesis of myasthenia gravis and may underpin a particular subgroup of myasthenia gravis seen in the East-Asian pediatric population.     

Suppression of experimental autoimmune myasthenia gravis by nasal administration of acetylcholine receptor

Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non-specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freund's adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-γ-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR.     

Immunohistological studies of the thymus in myasthenia gravis : Correlation with clinical state and thymocyte culture responses

In frozen sections of thymus from 9 out of 12 myasthenia gravis patients, the medulla contained follicles of B lymphocytes with germinal centres showing the same immunofluorescence staining pattern as is seen normally in reactive lymphoid tissues. Only 1 similar follicle was seen in 9 normal thymus samples. There was a positive association between the extent of germinal centres, plasma anti-acetylcholine receptor (AChR) titre, and spontaneous anti-AChR production by thymocytes in vitro. These thymic changes were not universally found, and are thus probably not central to the initiation of myasthenia.Between the follicles, in 9 cases, there was an apparent increase in interdigitating cells with closely associated ‘inducer’ (OKT₄⁺)T lymphocytes. Thymic antigen presenting cells — either here or in the germinal centres — could be involved in breaking self-tolerance, or in perpetuating the autoimmune response, and it may be their removal that is therapeutic.     

Changes in lymphocyte subsets in myasthenia gravis: correlation with level of antibodies to acetylcholine receptor and age of patient.

We report a detailed analysis of the subsets of lymphocytes in patients with myasthenia gravis (MG). There was a slight, nonsignificant increase in the level of CD5+ B lymphocytes among MG patients as compared with normal controls. The proportion of CD5+ T cells in MG was similar to that in controls. However, whereas age had no effect on the level of these cells in normal individuals, a significant age-related decrease of these cells was present in MG patients. The proportion of double-positive CD4+CD8+ T cells was significantly increased in MG. The level of the CD29+CD4+ (helper-inducer) subset was significantly higher in MG patients than in controls. There was no correlation between the titer of autoantibodies to acetylcholine receptor and the level of either CD29+CD4+ T cells or CD5+ B cells among MG patients. The only T-cell subset that correlated with the autoantibody titer was the CD45RA+CD4+ (suppressor-inducer) subset of CD4+ T cells.     

Rituximab treatment of myasthenia gravis: A systematic review

Rituximab is a chimeric mouse/human anti‐CD20 monoclonal immunoglobulin. We reviewed the efficacy and safety of rituximab in 169 myasthenia gravis (MG) patients from case reports and series. Antibodies to the acetylcholine receptor (AChR) were present in 59% and muscle‐specific tyrosine kinase (MuSK) in 34%. Modified Myasthenia Gravis Foundation of America postintervention scale of minimal manifestations (MM) or better occurred in 44%, and combined pharmacologic and chronic stable remission in 27% overall; MM or better was achieved in 72% of MuSK MG and 30% of AChR MG (P < 0.001). Posttreatment relapses decreased more in MuSK MG (P = 0.05). Response predictors were MuSK MG, less severe disease, and younger age at treatment. Among a responder subset, 26% of AChR and 82% of MuSK MG patients showed decreased posttreatment antibody titers. Rituximab was generally well tolerated. Detectable serum rituximab and depleted CD20⁺ B‐cells were observed up to 20 and 16 weeks, respectively, after 4 weekly infusions. Muscle Nerve 56 : 185–196, 2017     

Double filtration plasmapheresis benefits myasthenia gravis patients through an immunomodulatory action

Double filtration plasmapheresis (DFPP) is used to treat myasthenia gravis (MG). However, the definite mechanism is unclear. This study investigated whether DFPP improves MG through an immunomodulatory action. Thirty-five MG patients were randomly divided into two treatment groups: Group A (DFPP combined with oral methylprednisolone) and Group B (oral methylprednisolone alone). Their antibody levels, clinical scores, cytokine levels, and CD4⁺CD25^highFoxp3⁺ (regulatory T cell [Treg]) levels were then determined. Anti-titin antibody levels were significantly lower in Group A compared with Group B after treatment. The clinical remission rate in Group A was significantly higher than in Group B. The changes in cytokine levels (interleukin [IL]-2, IL-4, IL-10, and interferon-γ) in sera and the peripheral blood mononuclear cell culture supernatants did not significantly differ before and after the treatments in both groups (p < 0.05). The soluble intercellular adhesion molecule-1 (sICAM-1) levels were lower in Group A than in Group B (p < 0.05). MG patients exhibited a lower percentage of Treg cells than normal patients. DFPP combined with methylprednisolone treatment increased the Treg cell percentage more than treatment with methylprednisolone alone (p < 0.05). DFPP treatment more effectively lowers sICAM-1 and increases Treg cell expression, consequently benefiting MG patients.     

Interleukin‐10 producing‐B cells and their association with responsiveness to rituximab in myasthenia gravis

Introduction: A subset of regulatory B cells in humans and mice has been defined functionally by their ability to produce interleukin (IL)‐10. We characterized IL‐10‐producing B (B10) cells in myasthenia gravis (MG) patients and correlated them with disease activity and responsiveness to rituximab therapy. Methods: Frequencies of B10 cells from MG patients and healthy controls were monitored by fluorescence‐activated cell sorting (FACS). Results: MG patients had fewer B10 cells than controls, which was associated with more severe disease status. Moreover, patients who responded well to rituximab therapy exhibited rapid repopulation of B10 cells, whereas in patients who did not respond well to rituximab, B10 cell repopulation was delayed. The kinetics of B10 cells were related to the responsiveness to rituximab in MG. Conclusions: We have characterized a specific subset of B10 cells in MG patients which may serve as a marker for disease activity and responsiveness to immune therapy. Muscle Nerve 49:487–494, 2014     

Neonatal tolerance to an immunodominant T cell reactivity does not confer resistance to EAMG induction in Lewis rats

The overall goal of this study was to determine, during induction of experimental autoimmune myasthenia gravis (EAMG) in Lewis rats, the relative importance of acetylcholine receptor (AChR)-reactive helper T cells associated with one particular immunodominant fine specificity. Thus, experiments presented below were designed to evaluate the immunopathological role played by helper T cells with reactivity against the AChR alpha subunit region associated with amino acid residues 100–116 (i.e., α100–116); in particular, the relationship between T cell reactivity with this specificity and disease induction was assessed. In order to examine the importance of this T cell reactivity, Lewis rat neonates were made T cell tolerant to a synthetic peptide α100–116 and subsequently evaluated for anti-AChR antibody production and resulting neuromuscular dysfunction. Results indicated that although T cell reactivity against the α100–116 peptide could be effectively removed from the Lewis T cell repertoire, tolerized Lewis rats immunized with AChR could undergo an active anti-AChR antibody response that produced symptoms of EAMG. Thus, other AChR T cell reactivities appeared capable of providing adequate help to B cells leading to production of anti-AChR antibodies with pathogenic potential.     

Human muscle acetylcholine receptor reactive T and B lymphocytes in the peripheral blood of patients with myasthenia growth

The prevalence of T and B cells reactive with the acetylcholine receptor (AChR) of human skeletal muscle was studied in 33 patients with myasthenia gravis (MG), 18 patients with other neurological diseases (OND) or autoimmune disorders (AD) and 27 age- and sex-matched healthy controls. T cell stimulation was estimated by enumerating cells secreting interferon (IFN)-γ and interleukin (IL)-2 in response to the AChR, whereas B cell reactivity was estimated by enumerating cells secreting IgG antibodies binding to the AChR. AChR-reactive T cells were increased in the peripheral blood of patients with MG as compared to patients with OND, AD and healthy individuals. Of the patients with MG, 29/33 (87.7%) had numbers of IFN-γ secreting cells higher than the mean ± 2 SD of the mean of controls as compared to 4/18 (22.2%) of patients with OND or AD and 2/27 (7.4%) of the controls. The mean value of the numbers of AChR-reactive T cells in the patients with MG was 19.6/10⁵ PBMC, corresponding to 1/5100 PBMC. Comparable results were obtained also for IL-2-secreting cells. Anti-AChR IgG antibody-secreting cells were detected in the blood of 30/33 (91%) of the patients with MG, 3/18 (16.7%) of the patients with OND or AD and 2/25 (8%) of the controls. The mean value of the antibody-secreting cells in MG was 11.7 cells/10⁶ PBMC corresponding to 1/70400 PBMC in the patients with MG, compared to a mean value of antibody-secreting cells in the patients with OND or AD of 0.33 and controls of 0.16 cells/10⁶ PBMC.     

Normalization of elevated CD4-/CD8- (double-negative) T cells after thymectomy parallels clinical remission in myasthenia gravis associated with thymic hyperplasia but not thymoma

T-cell-dependent B-cell help is likely to be of major importance in the pathogenesis of myasthenia gravis, but mechanisms provoking a pathological anti-acetylcholine receptor (AChR) response are poorly understood. We report on the dysregulation of recently identified CD4-/CD8- (double-negative) T cells (DN T cells), which have been shown to participate in immunoregulation and antibody augmentation. Compared with healthy controls, significantly increased frequencies of DN T cells were found in the blood of myasthenia gravis patients with lymphofollicular hyperplasia. After thymectomy, however, normalization in the number of these cells was seen in parallel with clinical improvement and reduction in anti-AChR antibody titers. The effect of thymectomy was observed irrespective of adjuvant treatment and held true for up to 4 years of follow-up. In marked contrast, frequencies similar to control values were found in myasthenia gravis patients with thymoma, with thymectomy having no further reducing effect. These data indicate that CD4-/CD8- T cells not only participate in the pathogenesis of myasthenia gravis but also correlate with disease activity and histological findings.     

Paraneoplastic diseases associated with thymoma

Abstract Background Thymoma is frequently associated with paraneoplastic diseases (PDs), most commonly with myasthenia gravis (MG). This association is thought to depend on thymoma's capacity to produce and export T lymphocytes. Objective (1) To determine the frequency and characteristics of thymoma-associated PDs other than MG; (2) to evaluate T cell maturation in thymomas with and without PDs. Methods We studied 260 patients with thymoma (associated with MG in 228). The occurrence of PDs was monitored together with the tumor outcome. Phenotypic characterization of thymocyte subsets in 14 thymoma samples (7 with and 7 without MG) was performed by FACS. Results A total of 47 PDs was diagnosed in 41/260 patients (15.8 %). Neurological PDs included neuromyotonia, limbic encephalitis, polymyositis, subacute hearing loss, psychosis and sleep disorders. A broad spectrum of nonneurological PDs were observed, among these, hematological and cutaneous diseases prevailed. Like MG, these disorders occurred either in the presence of the thymoma or at different times after thymomectomy; their onset often heralded a tumor recurrence. In thymomas from MG subjects, we found an increased proportion of fully mature CD4 single positive (SP) thymocytes and a reduced frequency of CD4SPCD25⁺ cells; the latter finding may reflect a deficient generation of T regulatory cells, a reduced intratumorous activation of T cells, or both. Conclusions We confirm the strong association of thymoma with PDs. These disorders often occurred in MG patients and their course in relation to thymoma was similar to that of MG. In accordance with previous observations, we found some alterations in the intratumorous production of mature CD4⁺ T cells that could be involved in the pathogenesis of paraneoplastic autoimmunity.     

Analysis of CD27 surface expression on T cell subsets in MS patients and control individuals

Within the peripheral blood, CD4⁺CD27⁻ T cells only reside within the CD45RAT ⁻(memory or primed) T cell subset. Cells with this phenotype have characteristics of specialized effector T cells according to their cytokine secretion profiles and the expression of tissue-specific adhesion molecules. This subset was previously found to be increased in certain diseases that are associated with immune activation. Therefore we analyzed CD27 expression of peripheral blood and CSF T cells in MS patients. Within the CD4⁺ T cell subset no differences were seen between MS patients and controls in proportions of CD45RA⁻CD27⁻ cells. However, when the CD3⁺ T cell compartment was analyzed, CD27⁻ cells were also found within the CD45RA⁺ subset. These cells, most likely CD8⁺, are significantly reduced in PBL and CSF of MS patients as compared with OND patients. In MS and OND groups the level of CD27⁻ cells in peripheral blood correlated significantly with that in CSF, indicating a balanced migration of CD27⁻ cells between the two compartments. In OIND patients, however, this equilibrium was lost. The correlation of the level of CD27⁺ cells with the amount of intrathecally produced IgG in MS patients may suggest that CD27⁺ cells are responsible for B cell help in this disease.     

Change and predictive ability of circulating immunoregulatory lymphocytes in long-term outcomes of acute ischemic stroke

Lymphocytes play an important role in the immune response after stroke. However, our knowledge of the circulating lymphocytes in ischemic stroke is limited. Herein, we collected the blood samples of clinical ischemic stroke patients to detect the change of lymphocytes from admission to 3 months after ischemic stroke by flow cytometry. A total of 87 healthy controls and 210 patients were enrolled, and the percentages of circulating T cells, CD4⁺ T cells, CD8⁺ T cells, double negative T cells (DNTs), CD4⁺ regulatory T cells (Tregs), CD8⁺ Tregs, B cells and regulatory B cells (Bregs) were measured. Among patients, B cells, Bregs and CD8⁺ Tregs increased significantly, while CD4⁺ Tregs dropped and soon reversed after ischemic stroke. CD4⁺ Tregs, CD8⁺ Tregs, and DNTs also showed high correlations with the infarct volume and neurological scores of patients. Moreover, these lymphocytes enhanced the predictive ability of long-term prognosis of neurological scores when added to basic clinical information. The percentage of CD4⁺ Tregs within lymphocytes showed high correlations with both acute and long-term neurological outcomes, which exhibited a great independent predictive ability. These findings suggest that CD4⁺ Tregs can be a biomarker to predict stroke outcomes and improve existing therapeutic strategies of immunoregulatory lymphocytes.     

Antibodies against saline-soluble components of skeletal muscle in myasthenia gravis

Summary Antibodies against phosphate-buffered-saline extracts (SE) of non-acetylcholine receptor (AChR) skeletal muscle antigens were found in patients with myasthenia gravis (MG). The antigenicity of SE was distributed in three fractions with molecular masses of over 200 kDa, 90–150 kDa and 7–14 kDa on gel filtration. These fractions shared common antigenicities. Further analysis of 90–150 kDa fractions on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed five major bands, ranging from 105 kDa to 275 kDa. The antibodies against SE were detected in 52% (58/112) of the MG patients; incidence and titres were higher in the thymoma group (n=21; 90% and 0.872 respectively) than in the non-thymoma group (n=91; 43% and 0.200, P<0.001). In patients without a thymoma, these antibodies were frequently observed in late-onset disease and the severe generalized form (P<0.01). In 4 of 7 ocular MG patients without anti-AChR antibodies, low but appreciable levels of anti-SE antibodies were found. In 73% (11/15) of generalized MG patients treated with prednisolone and thymectomy, anti-SE antibody titres changed in association with those of anti-AChR antibodies and with the clinical course. Both antibody titres increased synchronously in patients who developed crises.     

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。     

Atorvastatin-modified dendritic cells in vitro ameliorate experimental autoimmune myasthenia gravis by up-regulated Treg cells and shifted Th1/Th17 to Th2 cytokines

Conventional therapies for autoimmune diseases produce nonspecific immune suppression, which are usually continued lifelong to maintain disease control, and associated with a variety of adverse effects. In this study, we found that spleen-derived dendritic cells (DCs) from the ongoing experimental autoimmune myasthenia gravis (EAMG) rats can be induced into tolerogenic DCs by atorvastatin in vitro. Administration of these tolerogenic DCs to EAMG rats on days 5 and 13 post immunization (p.i.) resulted in improved clinical symptoms, which were associated with increased numbers of CD4⁺CD25⁺ T regulatory (Treg) cells and Foxp3 expression, decreased lymphocyte proliferation among lymph node mononuclear cells (MNC), shifted cytokine profile from Th1/Th17 to Th2 type cytokines, decreased level of anti-R97–116 peptide (region 97–116 of the rat acetylcholine receptor α subunit) IgG antibody in serum. These tolerogenic DCs can migrate to spleen, thymus, popliteal and inguinal lymph nodes after they were injected into the EAMG rats intraperitoneally. Furthermore, these tolerogenic DCs played their immunomodulatory effects in vivo mainly by decreased expression of CD86 and MHC class II on endogenous DCs. All these data provided us a new strategy to treat EAMG and even human myasthenia gravis (MG).