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.     

Decreased frequency of intrathymic regulatory T cells in patients with myasthenia-associated thymoma

Naturally occurring CD4+CD25+ regulatory T cells (Treg) are essential for the control of unwanted autoimmune responses. In this study, we analysed their frequency in peripheral blood and in the thymus/thymomas of patients with myasthenia gravis (MG). We found a marked decrease in the number of CD4+CD25+ thymocytes in MG-associated thymomas, but no differences in the peripheral compartment, suggesting that the thymic development of Treg might be impaired in these patients.     

Changes in peripheral blood lymphocyte subset frequencies in myasthenia gravis patients are related to immunosuppression

Surface antigens on peripheral blood lymphocytes from myasthenia gravis patients were investigated. The expression of DR⁺ and CD8⁺/DR⁺ T lymphocytes was increased and the expression of CD4⁺ T cells reduced. Neither thymectomy, clinical condition nor anti-acetylcholine receptor antibody titre correlated with any of the changes in peripheral blood lymphocyte subsets observed. However, immunosuppressive therapy correlated with the significant reduction in CD4⁺ and CD2⁺/CD4⁺ T cells in these patients.     

Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression in patients with multiple sclerosis

Objectives – To determine whether percentages of CD4⁺CD25^high T cells (a group of regulatory T cells, Treg) differ in patients with multiple sclerosis (MS) in relapse vs remission after glucocorticoid treatment and whether treatment for relapses changes Treg population and the expression of Foxp3, a key Treg‐associated molecule. Materials and methods – Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with MS during relapse, just before and 2 days after starting steroid treatment (i.v. methylprednisolone 1 g/day for 3 days) and then 6 weeks after treatment. CD4⁺CD25^hi cells were analysed by using flow cytometry. Cytokines were measured by using an ELISA and Foxp3, CD3 and CD25 expression by using quantitative real‐time PCR. Results – The percentage of CD4⁺CD25^hi cells, plasma IL‐10 and Foxp3/CD3 ratio increased 48 h after methylprednisolone initiation and returned to baseline values by 6 weeks post‐treatment. Conclusions – Results suggest that glucocorticoids increase Treg cell functional molecules and percentages. This may be a mechanism whereby steroids expedite recovery from MS relapses.     

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.     

重症肌无力患者外周血CD4+T细胞OX40的表达及作用机制研究

目的 分析重症肌无力(MG)患者外周血CD4+T细胞协同刺激分子OX40表达及其对FoxP3+CD4+CD25+调节性T细胞(Treg)的调控作用,初步探讨OX40在MG免疫学发病中的作用机制.方法 以流式细胞技术检测42例MG患者及38名健康对照的外周血OX40+CD4+T细胞、FoxP3+CD4+CD25+Treg表达水平,比较OX40表达在MG患者不同临床疾病状态、Osserman分型、临床绝对评分、胸腺病理类型等情况下的差异,并分析OX40对FoxP3+CD4+CD25+Treg细胞的影响.结果 (1) MG患者外周血OX40+CD4+T细胞占淋巴细胞百分比高于健康对照组(P<0.01).(2)MG患者OX40+CD4+T细胞百分比在发作或加重期高于缓解期(P＜0.05);在临床绝对评分呈中、重度患者OX40+CD4+T细胞百分比高于轻度患者(均P＜0.05);Osserman Ⅱ、Ⅳ型患者OX40+CD4+T细胞百分比高于Ⅰ型患者(均P＜0.05);胸腺增生及胸腺瘤患者OX40+CD4+T细胞百分比高于胸腺正常患者(P＜0.05,P＜0.01).(3)MG患者外周血OX40+CD4+T细胞百分比与FoxP3+CD4+CD25+Treg细胞百分比呈负相关(r=-0.843,P=0.01).结论 协同刺激分子OX40参与MG发病,可能通过抑制FoxP3+CD4+CD25+Treg细胞生成发挥作用.     

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).     

Pathogenic CD8+ T cells in multiple sclerosis

Traditionally, autoimmune pathogeneses have been attributed to CD4⁺ T lymphocytes, as in multiple sclerosis (MS), rheumatoid arthritis, type 1 diabetes mellitus, and/or to B lymphocytes, as in myasthenia gravis and systemic lupus erythematosus. That is because their primary genetic associations are mostly with certain human leukocyte antigen class II alleles, whose gene products present antigens to CD4⁺ T cells. Because few autoimmune diseases show stronger associations with major histocompatibility complex class I alleles (ankylosing spondylitis, Behçet's disease, and psoriasis), CD8⁺ T cells, which interact with major histocompatibility complex class I molecules, have been largely ignored in autoimmunity research. However, a variety of findings has recently revived interest in this population, particularly in MS. First, it shows associations with major histocompatibility complex class I alleles. Second, its lesions show a predominance of CD8⁺ T cells. Third, these represent effectors that can directly damage central nervous system target cells. Furthermore, several clinical trials of monoclonal antibodies specifically against CD4⁺ T cells, or the polarizing cytokines on which they depend, have failed to show any therapeutic benefit in MS, unlike broader‐spectrum antibodies that deplete all T cells. Here, we review the evidence that CD8⁺ T cells play a role in MS pathogenesis. Ann Neurol 2009;66:132–141     

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.     

What’s new in chronic inflammatory demyelinating polyradiculoneuropathy in 2007–2008?

Abstract Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)–related research has made progress in the field of pathogenesis, genetics, and treatment. The number of circulating CD4⁺ CD25⁺ T‐regulatory cells was shown to be reduced in CIDP patients. Increased frequency of genotype GA13‐16 of the SH2D2A gene encoding for a T‐cell‐specific adapter protein in CIDP patients may result in a defective control and elimination of autoreactive T cells. IVIg treatment has been shown to increase numbers and function of peripheral CD4⁺ CD25⁺ T‐regulatory cell in a mouse model. These findings shed new light on the understanding of why peripheral tolerance is breached in CIDP patients and why the disease becomes chronic and adds another possible mechanism of action of intravenous immunoglobulin to the already long list. Long‐term effectiveness of IVIg has now been proven. Subcutaneous immunoglobulin could be an alternative for IVIg, but this has to be explored further in well‐designed trials. Autologous stem cell transplantation has been tried in refractory patients, but larger trials are necessary to assess safety and effect of this treatment.     

CD4+CD25+ T Cells are Essential for Behavioral Effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice

Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour.We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6C^hi monocytes were found to be decreased in JB-1 fed mice with intact regulatory T cells, but not in JB-1 fed mice following depletion. Furthermore, adoptive transfer of CD4⁺CD25⁺ cells, from JB-1 treated donor mice, but not from controls, induced antidepressant- and anxiolytic-like effects in recipient mice. Ly6C^hi monocytes were also significantly decreased in mice receiving CD4⁺CD25⁺ cells from JB1 fed donors.This study identifies cells within the CD4⁺CD25⁺ population, most likely regulatory T cells, as both necessary and sufficient in JB-1-induced antidepressant- and anxiolytic-like effects in mice, providing novel mechanistic insight into microbiota-gut-brain communication in addition to highlighting the potential for immunotherapy in psychiatric disorders.     

Repeated bouts of aerobic exercise enhance regulatory T cell responses in a murine asthma model

We have reported previously that moderate intensity aerobic exercise training attenuates airway inflammation in a murine asthma model. Recent studies implicate regulatory T (Treg) cells in decreasing asthma-related airway inflammation; as such, the current study examined the effect of exercise on Treg cell function in a murine asthma model. Mice were sensitized with ovalbumin (OVA) prior to the start of exercise training at a moderate intensity 3×/week for 4 weeks; exercise was performed as treadmill running (13.5 m/min, 0% grade). Mice were OVA challenged repeatedly throughout the exercise protocol. At protocol completion, mice were analyzed for changes in the number and suppressive function of CD4⁺CD25⁺Foxp3⁺ cells isolated from lungs, mediastinal lymph nodes, and spleens. Results show that exercise increased significantly the number of Foxp3⁺ cells within the lungs and mediastinal lymph nodes, but not the spleens, of OVA-treated mice as compared with sedentary controls. Exercise also enhanced the suppression function of CD4⁺CD25⁺Foxp3⁺ Treg cells derived from OVA-treated mice as compared with sedentary controls. Specifically, Treg cells from exercised, OVA-treated mice more effectively suppressed CD4⁺CD25^? cell proliferation and Th2 cytokine production in vitro. Enhanced suppression was associated with increased protein levels of TGF-β and lesser amounts of IL-10 and IL-17; however, blocking TGF-β had no effect on suppressive functions. These data demonstrate that exercise-mediated increases in Treg cell function may play a role in the attenuation of airway inflammation. Further, these results indicate that moderate intensity aerobic exercise training may alter the Treg cell function within the asthmatic airway.     

GM-CSF-induced regulatory T cells selectively inhibit anti-acetylcholine receptor-specific immune responses in experimental myasthenia gravis

We and others have demonstrated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to suppress autoimmunity by increasing the number of CD4⁺CD25⁺ regulatory T cells (Tregs). In the current study, we have explored the critical role of induced antigen specific Tregs in the therapeutic effects of GM-CSF in murine experimental autoimmune myasthenia gravis (EAMG). Specifically, we show that Tregs from GM-CSF treated EAMG mice (GM-CSF/AChR-induced-Tregs) adoptively transferred into animals with EAMG suppressed clinical disease more potently than equal numbers of Tregs from either GM-CSF untreated EAMG mice or healthy mice treated with GM-CSF. In addition, GM-CSF/AChR-induced-Tregs selectively suppressed antigen specific T cell proliferation induced by AChR relative to that induced by an irrelevant self antigen, (thyroglobulin) and failed to significantly alter T cell proliferation in response to an exogenous antigen (ovalbumin). These results are consistent with the hypothesized mechanism of action of GM-CSF involving the mobilization of tolerogenic dendritic cell precursors which, upon antigen (AChR) capture, suppress the anti-AChR immune response through the induction/expansion of AChR-specific Tregs.     

Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression

Accumulating evidence indicates an immunosuppressive role for CD4(+)CD25(+) regulatory T cells (Tregs) in autoimmune diseases. Although an impaired Treg function in patients with relapsing-remitting multiple sclerosis (RR-MS) has been reported recently, no information is available so far about Treg function in the progressive stage of the disease. In the present study, the phenotypic and functional characteristics of CD4(+)CD25(+) T cells isolated from the peripheral blood of patients with RR-MS and secondary progressive multiple sclerosis (SP-MS) were investigated. No significant quantitative or phenotypic abnormalities in CD4(+)CD25(+) T cells from RR- and SP-MS patients were detected. However, whereas a reduced suppressor function of CD4(+)CD25(+) T cells toward proliferation and interferon-gamma production of CD4(+)CD25(-) responder T cells was found in RR-MS patients, SP-MS patients showed a normal Treg function. The suppressive capacity of MS-derived CD4(+)CD25(+) T cells was correlated with disease duration but not with age, indicating that Treg function is more affected in the early phase of the disease process. Consistently with the suppressive capacity, CD4(+)CD25(+) T cells from SP-MS patients showed normal levels of FOXP3 mRNA in contrast to RR-MS patients that had a reduced FOXP3 expression. These data are the first to demonstrate differences in function and FOXP3 expression of CD4(+)CD25(+) T cells from patients with RR- and SP-MS.     

Peripheral leukocyte profile in people with temporal lobe epilepsy reflects the associated proinflammatory state

IntroductionMarkers of low-grade peripheral inflammation have been reported amongst people with epilepsy. The mechanisms underlying this phenomenon are unknown. We attempted to characterize peripheral immune cells and their activation status in people with temporal lobe epilepsy (TLE) and healthy controls.Methods and resultsTwenty people with TLE and 19 controls were recruited, and peripheral blood lymphocyte and monocyte subsets evaluated ex vivo by multi-color flow cytometry. People with TLE had higher expression of HLA-DR, CD69, CTLA-4, CD25, IL-23R, IFN-γ, TNF and IL-17 in CD4⁺ lymphocytes than controls. Granzyme A, CTLA-4, IL-23R and IL-17 expression was also elevated in CD8⁺ T cells from people with TLE. Frequency of HLA-DR in CD19⁺ B cells and regulatory T cells CD4⁺CD25⁺Foxp3⁺ producing IL-10 was higher in TLE when compared with controls. A negative correlation between CD4⁺ expressing co-stimulatory molecules (CD69, CD25 and CTLA-4) with age at onset of seizures was found. The frequency of CD4⁺CD25⁺Foxp3⁺ cells was also positively correlated with age at onset of seizures.ConclusionImmune cells of people with TLE show an activation profile, mainly in effector T cells, in line with the low-grade peripheral inflammation.     

重症肌无力外周血调节性T细胞线粒体自噬异常的研究

目的探讨线粒体自噬对重症肌无力(myasthenia Gravis,MG)患者外周血调节性T细胞(CD4~+CD25~+regulatory T cell,Treg)功能的影响及关系。方法选择首次确诊、尚未接受治疗的15例MG患者,以15名健康人作为对照,分别采集外周血,以磁珠分选法获得Treg细胞,经流式细胞仪鉴定纯度后,分别通过透射电镜检测Treg细胞线粒体自噬情况、Western Blot法检测微管相关蛋白轻链3-Ⅱ(microtubule associated protein light chain 3-Ⅱ,LC3-Ⅱ)的表达水平、JC-10荧光探针检测线粒体膜电位(以红色荧光细胞数/绿色荧光细胞数比值表示)变化、羧基荧光素琥珀酰亚胺酯(CFDA-SE,CFSE)检测Treg对正常CD4~+T细胞增殖抑制能力(荧光强度越强,增殖抑制能力越强)。结果与对照组比较,MG组电镜下线粒体自噬明显减少[(25.60±7.81)个、(19.20±5.49)个,t=-2.596,P0.05],且变形线粒体较多;MG组LC3-Ⅱ表达明显减少(0.450±0.137、0.334±0.124,t=-2.413,P0.05);MG组Treg细胞线粒体膜电位下降(2.153±0.537,1.726±0.494,t=2.126,P0.05);MG组CFSE检测平均荧光强度减弱(34.82±10.64、26.49±5.94,t=2.646,P0.05)。结论 MG患者有Treg细胞的线粒体自噬功能降低,这可能与其增殖抑制功能降低相关。     

Influence of human myasthenia gravis thymus on the differentiation of human cord blood stem cells in SCID mice

The normal thymus contributes to T lymphocytes differentiation and induction of tolerance to self-antigens. Myasthenia gravis (MG) is characterized by abnormal thymic hyperplasia. To assess the potential influence of MG-thymus on the differentiation of T lymphocytes differentiation, we used the MG-thymus transplanted severe combined immunodeficiency (SCID) mice model to evaluate the human cord blood stem cells differentiation. Thymus fragments from MG patient and human cord blood stem cells were transplanted into SCID mice successively. SCID mice were observed to develop sustained human T lymphocytes and a functional anti-tumor immune. The levels of various T cell subsets in SCID mice with MG-thymus were different from that of control group. Among that, the frequency of CD4⁺CD25⁺ T cells was significant lower in SCID mice with MG-thymus. The deficiency of CD4⁺CD25⁺ T cells seens to contribute to the pathogenesis of MG.     

Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis

The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3⁺Tim3⁺, CD4⁺Tim3⁺, and CD4⁺CD25⁺Tim3⁺ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4⁺ and CD4⁺CD25⁺ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4⁺CD25(high) T cell subset. The proportions of CD4⁺Tim3⁺ and CD4⁺CD25⁺Tim3⁺ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3⁺Tim3⁺ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS.