Cells In Myasthenia Gravis Research Articles

In vitro modulation of T cells in myasthenia gravis by low-dose IL-2.

Follicular helper (Tfh), peripheral helper (Tph), and regulatory (Treg) T cells are involved in myasthenia gravis (MG) pathogenesis, an autoimmune disorder arising from autoantibodies targeting neuromuscular junction proteins. This study explores the impact of low-dose IL-2 on Tfh, Tph, and Treg cells in vitro in MG. Acetylcholine-receptor antibody-positive MG (AChR-MG), muscle-specific kinase antibody-positive MG (MuSK-MG) patients, and healthy controls (HC) were studied. Blood cells were cultured with/without IL-2 and compared by the ratios of IL-2 stimulated/unstimulated cultures. In both AChR-MG and MuSK-MG patients, CD25+FoxP3+Tregs were lower, while CXCR5+PD-1+ or ICOS+Tfh and CXCR5-PD-1+ or ICOS+Tph cells were higher compared with HC. Among the MG group, the FoxP3+ Treg cells in AChR-MG patients were even lower compared with MuSK-MG patients. In vitro IL-2 stimulation increased Tregs in all groups while decreasing PD-1+/ICOS+Tfh and PD-1+/ICOS+Tph populations. The fold-increase ratio of Tregs and the fold-decrease ratio of PD-1+ or ICOS+Tfh and ICOS+Tph cells in AChR-MG and MuSK-MG patients were greater than in HCs. Low-dose IL-2 treatment may balance Tfh, Tph, and Treg cells in MG patients, offering a potential opportunity for disease modulation.

Exhausted signature and regulatory network of NK cells in myasthenia gravis.

NK cells are dysfunctional in myasthenia gravis (MG), but the mechanism is unclear. This study aims to measure associations and underlying mechanisms between the NK cells and the development of MG. Twenty healthy controls (HCs) and 53 MG patients who did not receive glucocorticoids and immunosuppressants were collected. According to the Myasthenia Gravis Foundation of America (MGFA) classification, MG patients were categorized into MGFA I group (n = 18) and MGFA II-IV group (n = 35). Flow cytometry, cell sorting, ELISA, mRNA-sequencing, RT-qPCR, western blot, and cell culture experiments were performed to evaluate the regulatory mechanism of exhausted NK cells. Peripheral NK cells in MGFA II-IV patients exhibit exhausted phenotypes than HCs, marked by the dramatic loss of total NK cells, CD56dimCD16- NK cells, elevated PD1 expression, reduced NKG2D expression, impaired cytotoxic activity (perforin, granzyme B, CD107a) and cytokine secretion (IFN-γ). Plasma IL-6 and IL-21 are elevated in MG patients and mainly derived from the aberrant expansion of monocytes and Tfh cells, respectively. IL-6/IL-21 cooperatively induced NK-cell exhausted signature via upregulating SOCS2 and inhibiting the phosphorylation of STAT5. SOCS2 siRNA and IL-2 supplement attenuated the IL-6/IL-21-mediated alteration of NK-cell phenotypes and function. Inhibition of IL-6/IL-21/SOCS2/STAT5 pathway and recovery of NK-cell ability to inhibit autoimmunity may be a new direction in the treatment of MG.

MicroRNA expression profiles of peripheral blood and mononuclear cells in myasthenia gravis: A systematic review

BackgroundStudies have described the role of microRNAs (miRNAs) in thymic function, along with directly observing the altered expression of miRNAs in thymuses of myasthenia gravis (MG) patients; so, miRNAs became a core component in the pathophysiology of MG. However, because the miRNA analysis results are contradictory, the identification of MG-related miRNAs is daunting. ObjectiveWe did a systematic review of studies analyzing the miRNA expression profile of peripheral blood and mononuclear cells for patients with MG. MethodsWe ran a database search in PubMed, Scopus, and Web of Science on August 17, 2021. Original articles that analyzed miRNA profiles in peripheral blood (serum, plasma, and whole blood) and peripheral blood mononuclear cells (PBMCs) for patients with MG in comparison with a non-MG or healthy control (HC) group were eligible. The quality of studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2). Results26 studies were included. The quality of studies was fair (median score, 5). Among 226 different miRNAs that were deregulated in at least one study (range, 1–87), ten miRNAs were significantly deregulated in three or more studies. Five miRNAs (50%) showed the same deregulation: miR-106b-3p and miR-21-5p were consistently upregulated, and miR-20b, miR-15b, and miR-16 were consistently downregulated. Also, there were five miRNAs that were mostly upregulated, miR-150-5p, miR-146a, miR-30e-5p, and miR-338-3p, or downregulated, miR-324-3p, across studies. ConclusionThese miRNAs contribute to different pathways, importantly neural apoptosis and autophagy, inflammation, T regulatory cell development, and T helper cell balance. Prior to being used for diagnostic and therapeutic purposes, it is required to pursue molecular mechanisms these consistently and mostly dysregulated miRNAs specifically use in the context of MG.

Increased Frequency of Myeloid-Derived Suppressor Cells in Myasthenia Gravis After Immunotherapy.

Myeloid-derived suppressor cells (MDSCs) are a population of myeloid progenitor cells with immunoregulatory functions and their role in myasthenia gravis (MG) was unknown. In this study, we investigated the phenotypic and functional alterations of MDSCs in MG before and after immunotherapy. The frequency of MDSCs significantly increased and negatively correlated to that of Th1 or Th17 cells after immunotherapy. MDSCs from untreated patients with MG showed an impaired suppression of IFN-γ production in T-cells and improved immunosuppressive function was identified after immunotherapy. The MFI of Arg-1 in MDSCs also increased after immunotherapy. These findings suggested the functional difference in MDSCs before and after immunotherapy, and MDSCs might play a role in disease remission.

Differential Expression of miRNA in the Peripheral Blood Mononuclear Cells in Myasthenia Gravis with Muscle-Specific Receptor Tyrosine Kinase Antibodies.

To determine if differential profile of miRNAs in peripheral blood mononuclear cells (PBMCs) could be identified in muscle-specific receptor tyrosine kinase antibody positive myasthenia gravis (MuSK-MG) and linked to disease stage, a case-control method was used to compare the difference in miRNA expression profiles of PBMCs using next generation sequencing (NGS) in MuSK-MG patients and healthy controls (HCs). Six significant miRNAs from the discovery set were then validated using RT-qPCR in 11 MuSK-MG patients and 10 HCs. A unique miRNA prediction algorithm was used to predict the target genes of differentially expressed miRNAs and a network of miRNA gene pathways. Compared with HCs, 101 differentially expressed miRNAs were screened in MuSK-MG, of which 5 miRNAs were upregulated, and 96 miRNAs were downregulated. The top six differentially expressed molecules were selected for verification; four of them (miR-340-5p, miR-106b-5p, miR-27a-3p, and miR-15a-3p) were significantly different. The network analysis of miRNA gene pathways revealed that differentially expressed miRNAs were involved in a complex set of biological processes. Clinically, the four miRNAs that were validated are not correlated to MuSK antibody titers and quantitative myasthenia gravis score. Four miRNAs that were validated in this study have specificity to distinguish MuSK-MG from HCs.

Immunoregulatory Cells in Myasthenia Gravis.

Myasthenia gravis (MG) is a T cell-dependent, B-cell mediated autoimmune disease caused by antibodies against the nicotinic acetylcholine receptor or other components of the post-synaptic muscle endplate at the neuromuscular junction. These specific antibodies serve as excellent biomarkers for diagnosis, but do not adequately substitute for clinical evaluations to predict disease severity or treatment response. Several immunoregulatory cell populations are implicated in the pathogenesis of MG. The immunophenotype of these populations has been well-characterized in human peripheral blood. CD4+FoxP3+ regulatory T cells (Tregs) are functionally defective in MG, but there is a lack of consensus on whether they show numerical perturbations. Myeloid-derived suppressor cells (MDSCs) have also been explored in the context of MG. Adoptive transfer of CD4+FoxP3+ Tregs or MDSCs suppresses ongoing experimental autoimmune MG (EAMG), a rodent model of MG, suggesting a protective role of both populations in this disease. An imbalance between follicular Tregs and follicular T helper cells is found in untreated MG patients, correlating with disease manifestations. There is an inverse correlation between the frequency of circulating IL-10–producing B cells and clinical status in MG patients. Taken together, both functional and numerical defects in various populations of immunoregulatory cells in EAMG and human MG have been demonstrated, but how they relate to pathogenesis and whether these cells can serve as biomarkers of disease activity in humans deserve further exploration.

Melatonin exerts immunoregulatory effects by balancing peripheral effector and regulatory T helper cells in myasthenia gravis

Myasthenia gravis (MG) is a prototypic organ-specific autoimmune disorder that, in most cases, is mainly mediated by antibodies against the acetylcholine receptor. Evidence implicates CD4+ T helper (Th) cells in the development of MG, whereas regulatory T cells (Tregs) are associated with disease resolution. Melatonin has important immunoregulatory effects in many T cell-mediated autoimmune diseases. However, there are few studies on the role of melatonin in MG. In the present study, we investigated serum melatonin levels and melatonin receptor expression in MG patients and healthy controls (HCs). We also evaluated the impact of melatonin administration on peripheral CD4+ Th cells and related cytokine production. Serum melatonin levels were lower in MG patients than in HCs, and MT1 expression was lower in PBMCs from MG patients than in those from HCs. Administration of melatonin significantly decreased Th1 and Th17 cell responses and proinflammatory cytokine production. Further investigation in vitro revealed that melatonin administration increased FoxP3 and IL-10 expression in CD4+ T cells from MG patients and enhanced the suppressive function of Tregs. These findings indicate that melatonin exerts immunoregulatory activity in MG by balancing effector and regulatory Th cell populations as well as by suppressing proinflammatory cytokine production.

Defects of CTLA-4 Are Associated with Regulatory T Cells in Myasthenia Gravis Implicated by Intravenous Immunoglobulin Therapy.

Myasthenia gravis (MG) is a CD4+ T cell-dependent autoimmune disease resulting from aberrant immune response mediated by circulating autoantibodies at the neuromuscular junction. Intravenous immunoglobulin (IVIg) is an expensive and commonly used immunotherapeutic approach to treat patients with MG. The mechanisms of actions involved in IVIg treatment, however, remain to be investigated. In an effort to examine the roles of various subsets of CD4+ T cells in the periphery blood of MG and uncover the mechanisms that contribute to the therapeutical effects of IVIg, we first demonstrated that a subset of CD4+ T cells, CTLA-4-expressing regulatory T (Treg) cells, were underrepresented and functionally defective in MG patients. The dynamic profiling during the IVIg therapy course further revealed an inverse relationship between the frequency of CTLA-4+ Treg and the quantitative MG (QMG) score that represents disease severity. Our mechanistic studies indicated that IVIg expands CTLA-4-Treg cells via modulating antigen-presenting dendritic cells (DCs). To determine the molecular defects of CTLA-4 in abnormities of Treg in MG patients, we demonstrated hypermethylation at -658 and -793 CpGs of CTLA-4 promoter in MG Tregs. Interestingly, IVIg therapy significantly reduced the methylation level at these two sites in MG patients. Overall, our study may suggest a role of CTLA-4 in functionally defected Treg cells in MG and its actions involved in IVIg therapy.

Inhibition of ROCK activity regulates the balance of Th1, Th17 and Treg cells in myasthenia gravis

Aberrant ROCK activation has been found in patients with several autoimmune diseases, but the role of ROCK in myasthenia gravis (MG) has not yet been clearly investigated. Here, we demonstrated that ROCK activity was significantly higher in peripheral blood mononuclear cells (PBMCs) from MG patients. ROCK inhibitor Fasudil down-regulated the proportions of Th1 and Th17 cells in PBMCs of MG patients in vitro. Intraperitoneal injection of Fasudil ameliorated the severity of experimental autoimmune myasthenia gravis (EAMG) rats and restored the balance of Th1/Th2/Th17/Treg subsets. Furthermore, Fasudil inhibited the proliferation of antigen-specific Th1 and Th17 cells, and inhibited CD4 + T cells differentiated into Th1 and Th17 through decreasing phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through increasing phosphorylated Stat5. We conclude that dysregulated ROCK activity may be involved in the pathogenic immune response of MG and inhibition of ROCK activity might serve as a novel treatment strategy for MG.

Regulatory B cells in myasthenia gravis are differentially affected by therapies.

We analyzed the number and functionality of regulatory B (Breg) cells in well‐defined myasthenia gravis patients. We first showed a decreased number of circulating CD19+ CD24++ CD38++ Breg cells and an altered functionality of Breg cells in untreated myasthenia gravis patients. Next, we demonstrated that the proportion of circulating Breg cells was restored in myasthenia gravis patients after thymectomy, probably as Breg cells could be sequestered in the myasthenia gravis thymus. In contrast, corticosteroid treatments did not restore and decreased even more the proportion of Breg cells in myasthenia gravis patients. These results clearly demonstrated that two distinct immunomodulatory therapies affect differentially Breg cells.

Imbalance between T follicular helper and T follicular regulatory cells in myasthenia gravis

According to the signals they receive from the local environment, T cells can differentiate into one out of several subtypes, including Th1, Th2, Th3, Th9, Th17, T follicular helper (Tfh) and Treg cells (1). Each of these cell subsets possesses a particular function. The term “Tfh cell” first described a subset of CD4+ T cells present in human lymphoid tissues, namely tonsils, and functions primarily to provide help to B cells (2). Interestingly and similarly to B cells, Tfh cells express the homing receptor, CXCR5, whose ligand is the CXCL13, the main B cell chemoattractant chemokine. Within germinal centers (GC), Tfh cells mediate the selection and survival of B cells that differentiate into plasma cells producing high-affinity antibodies against foreign antigens, and memory B cells (3). Recently, a small subset of T follicular regulatory (Tfr) cells was described, that are Treg cells able to suppress specifically the Tfh cell function (4). Thus it is likely that a balance between Tfr and Tfh cells dictates the fate of the B cell immune response.

Caveolin-3 is aberrantly expressed in skeletal muscle cells in myasthenia gravis

Caveolin-3 is a muscle-specific membrane protein that localizes to the sarcolemma and T-tubule system. Caveolin-3 is needed for muscle repair and skeletal muscle development. The objective of this study was to compare caveolin-3 expression in myasthenia gravis (MG) and control muscles. Caveolin-3 was abnormally expressed in the MG muscle membrane, with partial loss of expression and overexpression in 5/15 and 10/15 patients, respectively. Caveolin-3 mRNA and protein levels were higher in MG than in control muscles, suggesting that partial deficiency of caveolin-3 is occasionally detected in MG muscle and that caveolin-3 overexpression may be required after MG muscle damage.

Number of memory T cells in myasthenia gravis

To determine the altered number of memory T cells in myasthenia gravis (MG) patients and confirmed the existence of immune memory disorder. A total of 27 MG cases (12 females, 15 males) undergoing expanded thymectomy at our hospital between 2009-2012. They were divided into 3 group of eutherapeutic (clinical relative score ≥ 50%), invalid (clinical relative score ≤ 25%) and improved (25% < clinical relative score <50%). Control group was composed of 17 cases of healthy subjects without immune system related disease. Flow cytometry was employed to detect the numbers of CD4⁺, CD8⁺, CD4⁺ CD45RO⁺, CD8⁺ CD45RO⁺, CD4⁺ CD45RO⁺ CCR7⁺, CD8⁺ CD45RO⁺ CCR7⁺, CD4⁺ CD45RO⁺ CD44(high) and CD8⁺ CD45RO⁺ CD44(high) T cells in PBMCs of 27 MG patients and 17 normal controls. As compared with healthy controls, the abnormal rates of CD4⁺ and CD8⁺ T cells were significantly higher in patients (P < 0.05), the number of CD4⁺ CD45RO⁺ CCR7⁺ T cells was significantly higher [(9.9 ± 5.5)% vs (6.6 ± 3.0)%, P = 0.012], the number of CD4⁺ CD45RO⁺ CD44(high) T cells was significantly higher [(6.8 ± 2.4)% vs (5.0 ± 3.0)%, P = 0.04] and the number of CD8⁺ CD45RO⁺ CCR7⁺ T cells was also significantly higher (P < 0.001). MG patients had immune disorders. And increased number of memory T cells and their activation may be pathogenesis of MG.

Survivin as a potential mediator to support autoreactive cell survival in myasthenia gravis: a human and animal model study.

The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.

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

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. Frequencies of B10 cells from MG patients and healthy controls were monitored by fluorescence-activated cell sorting (FACS). 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. 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.

Expansion of circulating counterparts of follicular helper T cells in patients with myasthenia gravis

Growing evidence has demonstrated that dysfunction of follicular helper T (TFH) cells results in an abnormal positive selection of autoreactive B cells, which contributes to the development of autoimmune diseases. This study reveals that the frequency of circulating counterparts of TFH cells in myasthenia gravis (MG) patients is significantly higher compared to healthy controls. Interestingly, the frequencies of circulating TFH cells were positively correlated with the levels of serum anti-AChR Ab in MG patients. Our data suggest the presence of overactivation and expansion of circulating counterparts of TFH cells in MG patients, which may contribute to the immunopathogenesis of MG.

Functional defect in regulatory T cells in myasthenia gravis

Forkhead box P3 (FOXP3) is a transcription factor necessary for the function of regulatory T cells (T(reg) cells). T(reg) cells maintain immune homeostasis and self-tolerance and play an important role in the prevention of autoimmune disease. Here, we discuss the role of T(reg) cells in the pathogenesis of myasthenia gravis (MG) and review evidence indicating that a significant defect in T(reg) cell in vitro suppressive function exists in MG patients, without an alteration in circulating frequency. This functional defect is associated with a reduced expression of key functional molecules, such as FOXP3 on isolated T(reg) cells, and appears to be more pronounced in immunosuppression-naive MG patients. In vitro administration of granulocyte macrophage-colony-stimulating factor (GM-CSF) enhanced the suppressive function of T(reg) cells and upregulated FOXP3 expression. These findings indicate a clinically relevant T(reg) cell-intrinsic defect in immune regulation in MG that may reveal a novel therapeutic target.

Impaired regulatory function in circulating CD4+CD25highCD127low/− T cells in patients with myasthenia gravis

Previous studies have reported alterations in numbers or function of regulatory T (Treg) cells in myasthenia gravis (MG) patients, but published results have been inconsistent, likely due to the isolation of heterogenous "Treg" populations. In this study, we used surface CD4, CD25(high), and CD127(low/-) expression to isolate a relatively pure population of Tregs, and established that there was no alteration in the relative numbers of Tregs within the peripheral T cell pool in MG patients. In vitro proliferation assays, however, demonstrated that Treg-mediated suppression of responder T (Tresp) cells was impaired in MG patients and was associated with a reduced expression of FOXP3 in isolated Tregs. Suppression of both polyclonal and AChR-activated Tresp cells from MG patients could be restored using Tregs isolated from healthy controls, indicating that the defect in immune regulation in MG is primarily localized to isolated Treg cells, and revealing a potential novel therapeutic target.

Changes of Treg-Associated Molecules on CD4+CD25+Treg Cells in Myasthenia Gravis and Effects of Immunosuppressants

Myasthenia gravis (MG) is a CD4(+) T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4(+)CD25(+)Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them. We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs. The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4(+)CD25(+)FOXP3(+)Helios(+)T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4(+)CD25(+)FOXP3(+)Helios(+)T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG. The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.

Circulating and thymic CD4+ CD25+ T regulatory cells in myasthenia gravis: effect of immunosuppressive treatment

Accumulating evidence indicates an immunosuppressive role of the thymus-derived CD4+ T-cell population constitutively expressing high level of CD25, T regulatory (Treg) cells, in autoimmune diseases. Here we show that the number of Treg cells in the blood is significantly lower in untreated myasthenia gravis patients than in age-matched healthy subjects, whereas it is normal or elevated in patients on immunosuppressive therapy (prednisone frequently associated with azathioprine). Therapeutic thymectomy (Tx) for either the thymoma or non-neoplastic thymic alterations that are often associated with myasthenia gravis provided unique material for studying intrathymic Treg cells and correlating them with their peripheral counterparts. We observed that Tx prevents the increase of Treg cells in the circulation that follows immunosuppressive therapy (particularly evident if the thymus is not neoplastic), indicating that the thymus contributes to Treg-cell normalization. However, thymic Treg cells are not modulated by immunosuppressive therapy and even in thymectomized patients Treg-cell numbers in the blood eventually recover. The present findings suggest that a deficiency in Treg cells favours the development of myasthenia gravis and that their normalization is an important clinical benefit of immunosuppressive therapy. Treg normalization appears to be largely thymus independent and possibly reflects the reported capacity of corticosteroids to promote Treg-cell development.