Persistence of CD16+/CD56-/2B4+ natural killer cells: a highly dysfunctional NK subset expanded in ocular myasthenia gravis.
Nguyen Stéphanie,Morel Véronique,Le Garff-Tavernier Magali,Bolgert Francis,Leblond Véronique,Debré Patrice,Vieillard Vincent
Journal of neuroimmunology
We report a case of myasthenia gravis associated with marked expansion of an unusual CD16(+)CD56(-)2B4(+) NK subset. These atypical cells were characterized by poor cytotoxicity against CD48(+) target cells and high proliferation due to 2B4/CD48 interaction. IL18, IFN-gamma and TGF-beta levels were profoundly different in this patient than in healthy donors. Immunosuppressive treatment induced disease remission and decreased the CD16(+)CD56(-)2B4(+)NK cells count. Our data suggest that expansion of this NK subset in myasthenia gravis patients may account for the deleterious NK cell functioning that occurs in this autoimmune disease.
[Identification of correlations between numbers of CD4+ CD25+ Treg cells, levels of sera anti-AChR antibodies and transfer growth factor-beta in patients with myasthenia gravis].
Wang Hua-bing,Zhang Jun-bao,Chui Li-ying
Zhonghua yi xue za zhi
OBJECTIVE:The purpose of the current study is to demonstrate possible involvement of CD4+ CD25+ Treg cells and TGF-beta in immune activation in patients with myasthenia gravis (MG). If so, how does CD4+ CD25+ Treg cells and TGF-beta, collaborate to impact on the production of pathogenic anti-AChR antibodies (Ab)? METHODS:40 MG in-patients with recent onset or deterioration and age and gender-matched 38 healthy subjects were consecutively enrolled. Flow cytometry was employed to detect circulating CD4+ CD25+ Treg cells. Levels of AChR-Abs and TGF-beta1 in serum were detected by radioimmunoassay and enzyme-linked immunoabsorbance assay respectively. RESULTS:Numbers of CD4+ CD25+ Treg cells were significantly decreased in MG patients (3.0% +/- 2.5%) compared with healthy controls(4.6% +/- 3.7% , P = 0.03). Decreased production of CD4+ CD25+ Treg cells was associated with late-onset, longer-duration, positive-MG sera for AChR-Abs, normal or atrophic thymus, and non-thymectomy treatment et al, respectively. Although CD4+ CD25+ Treg cells were not linear-correlated with serum anti-AChR Ab titers, but were conversely correlated with each other in MG patients without thymoma (non-MGT) (r = -0.37, P = 0.02). Likewise, levels of TGF-beta1 in 31 non-MGT patients (112 ng/L +/- 83 ng/L) were decreased compared with those of healthy subjects (215 ng/L +/- 134 ng/L, P = 0.00), and was conversely correlated with titers of anti-AChR Abs (r = -0.37, P = 0.02). The titer of anti-AChR Abs were correlated with Osserman classification and MGFA grade (r = 0.34, P = 0.03). CONCLUSION:Numbers of CD4+ CD25+ Treg cells and levels of TGF-beta1 in MG patients were significantly decreased compared with healthy controls, and may thus contribute to the pathogenesis of MG. Numbers of CD4+ CD25+ Treg cells were conversely correlated with levels of anti-AChR Abs in non-MGT patients.
[Expression of CD45RA and CD45RO thymocyte subset and related cytokine in patients with myasthenia gravis].
Li Qian-ru,Du Ying,Zhao Hang,Gao Feng,Zhang Qing-yong,Zhang Qin-xian
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
AIM:To study the relationship between the abnormal differentiation thymocytes and the pathogenesis of myasthenia gravis (MG). METHODS:The gene expression of some ILs, IFN and their receptors was examined by cDNA array. The percentage of CD45RO(+) and D45RA(+) thymocytes was measured by flow cytometry (FCM). The expression and distribution of CD45RA and CD45RO in the samples of thymus tissues were observed by immunohistochemistry. RESULTS:The levels of IL-1R, IL-4R, IFNgammaR1, IFNgammaR2, IL-6 and IL-8 in the patients with MG were lower than those of normal controls and there were significant differences between them. But the levels of IL-1, IL-2, IL-4, IL-10, IL-7, IFN-alpha, IFN-beta and IFN-gamma showed no significant changes in patients with MG in comparison with normal controls. The percentage of CD56(+) thymocytes in MG patients 0.56+/-0.33 was significantly lower than that in normal controls(1.78+/-0.69).The CD45RO(+) and CD1a(+) in MG patients increased more significantly those in normal controls. The immunohistochemistry and RT-PCR showed the same result. CONCLUSION:Aberrant transformation from CD45R0(+)T to CD45RA(+)T has been found in the development of thymocytes of the patients with MG.
Circulating CD4+CD25+ T regulatory and natural killer T cells in patients with myasthenia gravis: a flow cytometry study.
Battaglia A,Di Schino C,Fattorossi A,Scambia G,Evoli A
Journal of biological regulators and homeostatic agents
The number of circulating CD4+ T cells constitutively expressing CD25 (T regulatory, Treg) and natural killer T (NK T) cells, the two major lymphocyte populations that help to maintain immune homeostasis, was studied in 22 unselected myasthenia gravis (MG) patients, 16 healthy subjects and 24 patients with cancer, the latter as a disease model of a relative immune suppression status. Treg cells were assessed according to their intermediate or high level of expression of CD25, i.e., CD25int and CD25bright, and to the expression of HLA-DR, CD62L, CD45RO and CD152. There were no differences in the number of NK T cells and CD4+CD25bright cells among the three series of individuals. MG patients and healthy subjects had also similar numbers of CD4+CD25int cells. However, the whole CD4+ cell compartment in MG patients was in an activated status, as indicated by the higher level of expression of CD152. By contrast, and consistent with a relative immune suppression status, cancer patients had higher numbers of CD4+CD25int cells and larger proportions of HLA-DR expressing CD4+CD25int and CD4+CD25bright cells. Immunomagnetically purified CD4+CD25+ cells from MG, healthy subjects and cancer patients were anergic and suppressed the proliferative response of other T cells.
Regulatory B Cells in Seropositive Myasthenia Gravis versus Healthy Controls.
Karim Md Rezaul,Zhang Hong-Yan,Yuan Jiang,Sun Qiang,Wang Yun-Fu
Frontiers in neurology
OBJECTIVE:To find out if the failure in immunotolerance of myasthenia gravis (MG) is a possible aspect of deduction in Breg cells and to characterize B cell subsets in MG. METHODS:Flow cytometry detection and enzyme-linked immunosorbent assays in peripheral blood films of 10 MG patients and 10 healthy controls (HCs) were performed after isolation of B cells. The CD19CD5CD1d Breg cells percentages were measured to complement a B cell phenotype assay and frequencies of B cell subsets. The clinical outcome measures and immunological variables of patients with MG were compared with HCs. RESULTS:Patients with MG had relatively lowered percentages of CD19CD5CD1d Breg cells as compared to HCs. The production of interleukin (IL)-10 and transforming growth factor (TGF)-β1 was relatively lesser in patients with MG than HCs, which were linked with more severe of MG disease status according to Myasthenia Gravis Foundation of America (MGFA) clinical classification. The reduction of cytokine production was more significant for IL-10 than TGF-β1 when compared to HCs. CONCLUSION:It has been observed that the reduced number of B cells is able to produce IL-10 in MG patients but lesser than compared to HCs. The Bregs reduction mainly was regarded by the severity of disease status, which was highly significant and also by disease duration which was statistically significant as well. The findings of the measurement of B cell phenotype assay and frequencies of B cell subsets between MGs and HCs give us new ideas to develop B cell-mediated therapies of MG such as (1) isolated B cells of MGs could be cultured with steroids, e.g., dexamethasone to see if it induces the CD19CD5CD1d Breg cells, (2) it may observe whether induced CD19CD5CD1d Bregs have higher production of IL-10 and TGF-β1, as both are linked with disease severity, and (3) after completion steps, through further research to observe whether it improves the function of MG disease status.
Clinical implication of peripheral CD4+CD25+ regulatory T cells and Th17 cells in myasthenia gravis patients.
Masuda Masayuki,Matsumoto Moe,Tanaka Sachiko,Nakajima Kanako,Yamada Nao,Ido Nobuhiro,Ohtsuka Takao,Nishida Masashi,Hirano Toshihiko,Utsumi Hiroya
Journal of neuroimmunology
Myasthenia gravis (MG) is an autoimmune disorder generally mediated by antibodies against the acetylcholine receptors (AChR) of the skeletal muscles. CD4 T cells help B cells to produce antibodies through their production of cytokines or chemokines. In this study, we evaluated the frequency of regulatory (Treg) and IL-17 producing CD4 T-cell subsets (Th17) in peripheral blood mononuclear cells (PBMCs) of patients with MG. The transcription factor, forkhead transcription factor (Foxp3), is essential for T-cell regulatory function, and the orphan nuclear receptor, RORgammaT, is important in Th17 cell differentiation. In MG patients, Foxp3 mRNA expression in PBMCs was lower than those in healthy subjects (p=0.007), while there was no significant difference of RORgammaT mRNA expression between MG patients and healthy subjects. Glucocorticoid-induced tumour-necrosis-factor receptor-related protein (GITR) is expressed predominantly on CD4(+)CD25(+) Treg cells. We found that the number of GITR(+)CD4(+)CD25(+) T cells in peripheral lymphocytes in MG patients was lower than that in healthy subjects (P<0.01). In addition, there was a significant positive correlation between the change of the frequency of GITR(+)CD4(+)CD25(+) T cells and the changing rate in quantitative myasthenia gravis scores (%) (p=0.03). Furthermore, there was a significant negative correlation between the change of the percentage of GITR(+)CD4(+)CD25(+) T cells (% lymphocytes) and the changing rate of daily PSL doses (%) (P=0.002). The relative RORgammaT levels in PBMCs negatively correlated with the Th1/Th2 ratio in CD4(+) cells in MG patients (p=0.014). In conclusion, our findings suggest that Th17 cells affect the production of autoantibodies through their influence on the Th1- and Th2-cytokine balance in PBMCs of MG patients. On the other hand, Treg cells are suggested to be involved in the clinical condition or severity of MG disease.
Modulation of B cell regulatory molecules CD22 and CD72 in myasthenia gravis and multiple sclerosis.
Lu Jiayin,Li Jing,Zhu Tai-qing,Zhang Longbo,Wang Yuzhong,Tian Fa-fa,Yang Huan
B cell activation mediated by cluster of differentiation (CD) molecules plays an important role in B cell-related autoimmune diseases. CD22 and CD72 have been demonstrated to act as B cell inhibitory receptors in many autoimmune diseases. Activated B cells are involved in the pathogenesis of myasthenia gravis (MG) by secretion of anti-acetylcholine receptor (AchR) antibodies. However, the roles of CD22 and CD72 on B cells of MG are unknown. In this study, we detected the expression of CD22 and CD72 on B cells of MG, compared to multiple sclerosis (MS) patient controls and healthy controls by flow cytometry and quantitative real-time polymerase transcription chain reaction. Our data demonstrated that aberrant expression of CD72 exists on B cells of MG and MS patients and expression level of CD72 molecule has a significantly negative correlation with anti-AchR antibody levels in MG, which suggests that CD72 may be involved in the pathogenesis of MG and MS. There were no significant differences between study patients (MG, ocular MG, generalized MG, and MS) and healthy controls.
The increased expression of CD21 on AchR specified B cells in patients with myasthenia gravis.
Yin Weifan,Allman Windy,Ouyang Song,Li Yi,Li Jing,Christadoss Premkumar,Yang Huan
Journal of neuroimmunology
CD21, a major complement receptor expressed on B cells, is associated with autoimmune disorders. In the present study, we investigated the role of CD21 in pathogenesis of myasthenia gravis (MG) in relationship to anti-acetylcholine receptor (AchR) IgG (anti-AchR IgG) secretion. We detected increased surface expression of CD21 on AchR specified B cells as well as decreased surface expression of CD21 on total B cells in peripheral blood of patients with generalized MG (gMG). In addition, the serum concentrations of soluble secreted CD21 (sCD21) were decreased in patients with gMG. We also found that the level of CD21(+) AchR specified B cells correlated positively with serum anti-AchR IgG level, while the serum concentration of soluble CD21 correlated negatively with serum anti-AchR IgG level. Our data suggests that CD21 might facilitate its function on AchR specified B cell activation, resulting in the secretion of anti-AchR IgG.
Comparison of peripheral blood B cell subset ratios and B cell-related cytokine levels between ocular and generalized myasthenia gravis.
Hu Yuxiang,Wang Jun,Rao Jie,Xu Xiaoxuan,Cheng Yi,Yan Li,Wu Yajun,Wu Na,Wu Xiaorong
BACKGROUND:The pathogenesis of myasthenia gravis (MG) depends upon T and B cells, as well as complement and cytokines. The activation of functional subpopulations of T and B cells is the basis of the immune response. However, the activation levels of T and B cell subsets remain unclear in the pathogenesis of MG. Here, we evaluated the proportions of T and B cell subsets and related cytokines in ocular MG (oMG) patients and generalized MG (gMG) patients, and analyzed the potential roles of T and B cell subsets in the pathogenesis of oMG. METHODS:In total, 16 patients with oMG, 31 patients with gMG, and 20 healthy controls (HCs) were included in this study. Peripheral blood mononuclear cells (PBMCs) were separated from venous blood via density centrifugation. The percentages of CD3CD4 T cells, CD3CD8 T cells, CD4CD25CD127 regulatory T cells (Tregs), CD19CD27CD38 memory B cells, CD19CD24CD27 regulatory B cells (Bregs), CD19CD38CD138 plasma cells, and CD19CD40 B cells in PBMCs were detected by flow cytometry. The levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, and interferon (IFN)-γ in serum were detected by enzyme linked immunosorbent assay (ELISA). Differences in T and B cell subsets and related cytokines were compared among the three groups of participants. RESULTS:The proportions of CD19CD27CD38 memory B cells were significantly higher in the oMG and gMG groups than in the HC group (P = 0.004; P < 0.001), whereas the proportion of CD19CD27CD38 memory B cells was significantly lower in the oMG group than in the gMG group (P < 0.001). Moreover, the proportion of CD19CD24CD27 Bregs was significantly higher in the oMG group than in the gMG and HC groups (P = 0.001). The proportion of CD4CD25CD127 Tregs was significantly lower in the gMG group than in the oMG and HC groups (P < 0.001). Finally, the level of serum IL-10 was significantly higher in the oMG group than in the gMG and HC groups (P < 0.05). Compared with the HC group, serum IL-2 levels were significantly increased in the oMG and gMG groups (P = 0.016; P = 0.002). DISCUSSION:The reduced ratios of Tregs and Bregs may contribute to the progression of oMG to gMG, and the increased proportion of memory B cells may be closely related to the progression of oMG. IL-2 and IL-10 are important in the development of oMG.
Inhibition of peripheral blood natural killer cell cytotoxicity in patients with myasthenia gravis treated with plasmapheresis.
Chien P J,Yeh J H,Chiu H C,Hsueh Y M,Chen C T,Chen M C,Shih C M
European journal of neurology
BACKGROUND AND PURPOSE:Myasthenia gravis (MG) is an autoimmune disorder that may involve natural killer (NK) cells. Although NK cells are part of the innate immune system, they also influence adaptive immune responses. Double-filtration plasmapheresis (DFP) is an effective therapy for MG crisis. Thus, we examined the effects of DFP on the cytotoxicity of NK cells. METHODS:A total of 20 patients with MG and 16 healthy controls were recruited for the study. Ficoll-Paque-isolated peripheral blood mononuclear cells (PBMCs) and K562 cells were used as the effector and target cells, respectively. NK cell cytotoxicity was analyzed using flow cytometry immediately before and after DFP and upon course completion. RESULTS:Double-filtration plasmapheresis treatment decreased significantly the NK cell cytotoxicity in patients with MG, especially in good responders, those who were positive for acetylcholine receptor (AChR) antibodies, and those receiving immunosuppressants. CONCLUSIONS:The decrease in NK cell cytotoxicity after DFP and the decline of AChR antibody titer were observed in good responders indicating that this could benefit patients with MG.
Expansion of circulating counterparts of follicular helper T cells in patients with myasthenia gravis.
Luo Chuanming,Li Yan,Liu Weibin,Feng Huiyu,Wang Haiyan,Huang Xin,Qiu Li,Ouyang Juan
Journal of neuroimmunology
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.
CD4+ CD25(high) regulatory T cell in childhood ocular myasthenia gravis.
Sakuma Hiroshi,Katayama Ayako,Saito Yoshiaki,Komaki Hirofumi,Nakagawa Eiji,Sugai Kenji,Sasaki Masayuki
Brain & development
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.
[Number of memory T cells in myasthenia gravis].
Huang Xin,Li Yan,Liu Weibin,Feng Huiyu,Qiu Li,Wang Haiyan
Zhonghua yi xue za zhi
OBJECTIVE:To determine the altered number of memory T cells in myasthenia gravis (MG) patients and confirmed the existence of immune memory disorder. METHODS: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. RESULTS: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). CONCLUSION:MG patients had immune disorders. And increased number of memory T cells and their activation may be pathogenesis of MG.
Effects of Mitophagy on Regulatory T Cell Function in Patients With Myasthenia Gravis.
Wang Na,Yuan Jiang,Karim Md Rezaul,Zhong Ping,Sun Yan-Peng,Zhang Hong-Yan,Wang Yun-Fu
Frontiers in neurology
This study was conducted to determine whether regulatory T cells (CD4CD25T, Tregs) show abnormal mitophagy as well as the function of Tregs in patients with myasthenia gravis (MG). CD4T cells and CD4CD25Treg cells were obtained from 15 patients with MG (MG group) and 15 controls (N group). Tregs from the MG group were subjected to rapamycin-induced culture for 48 h (Rapa group) and 3-methyladenine-induced culture for 48 h (3-MA group). The levels of mitophagy in Tregs were then observed through electron and confocal microscopy. Expression of the autophagy-related protein LC3-II was detected by western blotting, and mitochondrial function in each group was evaluated by flow cytometry. Inhibition of Treg cell proliferation was detected by flow cytometry. Mitophagy in the MG group was lower than that in the N group; it was higher in the Rapa group compared to that in the MG group and lowered in the 3-MA group than in the MG group. Expression of the autophagy-related protein LC3-II was lower in the MG group than in the N group, higher in the Rapa group than in the MG group, and lower in the 3-MA group than in the MG group. The mitochondrial membrane potential was lower in the MG group compared to that in the N group; it was higher in the Rapa group than in the MG group and lowered in the 3-MA group than in the MG group. Inhibition of Treg proliferation was lower in the MG group than in the N group; it was higher in the Rapa group than in the MG group and lowered in the 3-MA group than in the MG group. The decreased mitochondrial membrane potential and mitophagy in Tregs in the MG group may be related to a decreased inhibition of Treg proliferation. The mitochondrial membrane potential was increased after adding the autophagy agent Rapa to enhance mitophagy, and the proliferation inhibition function of Tregs was also enhanced. The autophagy agent 3-MA down-regulated mitophagy, which decreased the mitochondrial membrane potential and inhibitory effect of Tregs. These results reveal the possible cellular immune mechanism of Treg dysfunction in MG.
[Thymoma T helper type 17 cells and related cytokines in myasthenia gravis].
Wang Zhong-kui,Wang Wei,Chen Yu-ping,Wei Dong-ning
Zhonghua nei ke za zhi
OBJECTIVE:To investigate the immunoregulatory role of Th17 cell and the related cytokines in myasthenia gravis. METHODS:Totally 51 myasthenia gravis (MG) patients were divided into MG with thymomas (TM group) and the MG with normal thymus (NT group), as well as 22 healthy subjects as controls. Th17 cells from peripheral blood mononuclear cells were measured by flow cytometry. Th17 related cytokines were detected by ELISA and real-time quantitative-PCR. RESULTS:The quantity of Th17 cells in MG patients with thymomas (1.53 ± 0.59)% were significantly increased compared with that of healthy control (0.94% ± 0.32%, P < 0.05). There was no significant difference in the number of Th17 cells between healthy controls and NT group. The expression levels of IL-17 mRNA (23.7 ± 4.5) were up-regulated significantly versus those in healthy controls (13.4 ± 3.2, P < 0.01). The levels of mRNA expression of IL-1β, IL-6 and IL-23 were up-regulated significantly in TM group. The mean concentration of IL-17 was up-regulated significantly in TM group (30.4 ± 7.3) ng/L versus healthy controls [(19.2 ± 4.9) ng/L, P < 0.05]. Serum levels of IL-23 and IL-1β were always increased in TM group versus healthy controls. CONCLUSION:The elevated levels of IL-17 and other Th17 related cytokines in thymomas may aggravate the autoimmunity disorder.
Effects of Follicular Helper T Cells and Inflammatory Cytokines on Myasthenia Gravis.
Wang Lifang,Zhang Yu,Zhu Mingqin,Feng Jiachun,Han Jinming,Zhu Jie,Deng Hui
Current molecular medicine
BACKGROUND:Myasthenia gravis (MG) is an autoimmune disorder mediated by antibodies against the acetylcholine receptors (AChR) of the skeletal muscles. An imbalance in various T helper (Th) cells, including Th1, Th2, Th17, Th22 and follicular helper T (TFH) cells, has been found associated with immunological disturbances. OBJECTIVE:In this study, we aim to investigate the role of the Th cells in peripheral blood of MG patients. MATERIALS AND METHODS:A total of 33 MG patients and 34 age matched controls were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient centrifugation assay. The proportion of TFH cells in PBMC were analyzed using flow-cytometry assay by determining the levels of cellular markers CD4, CXCR5, CD45RO, CD45RA and ICOS and PD-1. The levels of IFN-γ, IL-4, IL-17 and IL-21 in serum were analyzed by Cytometric Bead Array. The serum IL-22 level was analyzed by ELISA. RESULTS:The frequency of TFH cells in PBMCs was higher than those in healthy subjects and correlated to the severity of MG patients. The levels of pro-inflammatory cytokines IFN-γ, IL-17 and IL-21 were elevated in the serum of MG patients, while there were no significant differences regarding the levels of IL-4 and IL-22 between MG patients and control subjects. CONCLUSION:Our findings suggest that Th cells and their cytokines balance of MG patients are involved in the clinical condition or severity of MG disease.
B Cell Immunophenotyping and Transcriptional Profiles of Memory B Cells in Patients with Myasthenia Gravis.
Min Young Gi,Park Canaria,Kwon Young-Nam,Shin Je-Young,Sung Jung-Joon,Hong Yoon-Ho
Myasthenia gravis (MG) is an autoimmune neuromuscular junction disorders mediated by various autoantibodies. Although most patients with MG require chronic immunosuppressive treatment to control disease activity, appropriate surveillance biomarkers that monitor disease activity or potential toxicity of immunosuppressants are yet to be developed. Herein, we investigated quantitative distribution of peripheral blood B cell subsets and transcriptional profiles of memory B cells (CD19+ CD27+) in several subgroups of MG patients classified according to the Myasthenia Gravis Foundation of America (MGFA) Clinical Classification. This study suggests potential immunologic B-cell markers that may guide treatment decision in future clinical settings.
Disturbed B cell subpopulations and increased plasma cells in myasthenia gravis patients.
Kohler Siegfried,Keil Thomas Oskar Philipp,Swierzy Marc,Hoffmann Sarah,Schaffert Hanne,Ismail Mahmoud,Rückert Jens Carsten,Alexander Tobias,Hiepe Falk,Gross Christian,Thiel Andreas,Meisel Andreas
Journal of neuroimmunology
Whether there is a general perturbation of B and plasma cell subsets in myasthenia gravis (MG) has not been investigated so far. Here we performed a detailed flow cytometric analysis of blood and if available thymic tissue in order to detect MG-specific and therapy-induced changes. We observed significant differences in the distribution of B cell subsets in MG patients, yet these were mainly attributable to medical treatment. Furthermore MG is associated with significantly increased frequencies of plasma cells that were especially activated in purely ocular disease manifestation. In contrast to thymoma, B cell subset distribution in hyperplastic thymus could be distinguished from peripheral blood, however both tissues were not significantly enriched with plasma cells. Thus B cell differentiation in general is not defective in MG, but modified by therapy and enhanced frequencies of plasma cells can be detected in MG patients.
Up-regulated expression of Fas antigen in peripheral T cell subsets in patients with myasthenia gravis.
Mai Weihua,Liu Xingwei,Fan Yunping,Liu Hanwei,Hong Hai Yu,Han Rongrong,Zhou Wenying
Clinical and investigative medicine. Medecine clinique et experimentale
PURPOSE:Recent reports have linked various autoimmune diseases to defective Fas-mediated apoptosis or Fas expression. Here we aimed to determine whether Fas-mediated apoptosis is involved in the pathogenesis of myasthenia gravis (MG). METHODS:The expression of Fas antigen in peripheral T cell subsets from 17 Chinese patients with MG and 13 healthy individuals was determined by flow cytometry, and its associations with clinical classification, thymus pathology, the concomitance with hyperthyroidism (HT) and corticosteroid treatment were investigated. RESULTS:Compared with normal controls, a significantly up-regulated expression of Fas antigen was observed in the peripheral CD4+, CD4+CD8- and CD4-CD8- T cell subsets from patients with MG. Fas expression in CD4-CD8+ T cells of MG patients with normal thymus was significantly higher than that of patients with thymoma. Fas expressions in CD4+CD8+ T cells in MG patients with HT was significantly higher than controls and the ones without HT. Enhanced Fas expressions was found in CD4-CD8+ and CD4-CD8- T cells of MG patients with corticosteroid treatment, but no significant difference of Fas expression in peripheral T cells between patients with ocular MG (OMG) and general MG (GMG) was observed. CONCLUSION:Fas antigen may play a role in the pathogenesis of MG. It may be involved in the mechanisms of corticosteroid treatment, and with the occurrence of HT. OMG may represent a systemic disease, similar to that of GMG.
Impaired regulatory B cells in myasthenia gravis.
Sheng Jian Rong,Rezania Kourosh,Soliven Betty
Journal of neuroimmunology
Regulatory B cells (Bregs) attenuate the severity of experimental autoimmune myasthenia gravis (EAMG) in an interleukin-10 (IL-10)-dependent manner. The goal of this study was to investigate the role of human Bregs in MG focusing on CD19(+)CD1d(hi) CD5(+) and CD19(+)CD24(hi)CD38(hi) subsets. We found that MG patients exhibited a decrease in the frequency of both Breg subsets and IL-10 producing B cells within each subset, which correlated with disease severity. In addition, there was impaired suppression of Th1 polarization in MG. These findings, taken together with EAMG data, indicate that Bregs play an important role in regulating the severity of MG.
Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.
Yi J S,Guidon A,Sparks S,Osborne R,Juel V C,Massey J M,Sanders D B,Weinhold K J,Guptill J T
Journal of autoimmunity
Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.
miR-20b Inhibits T Cell Proliferation and Activation via NFAT Signaling Pathway in Thymoma-Associated Myasthenia Gravis.
Xin Yanzhong,Cai Hongfei,Lu Tianyu,Zhang Yan,Yang Yue,Cui Youbin
BioMed research international
. We examined the role of miR-20b in development of thymoma-associated myasthenia gravis, especially in T cell proliferation and activation. . Using qRT-PCR, we assessed expression levels of miR-20b and its target genes in cultured cells and patient samples and examined the proliferation of cultured cells, using MTT cell proliferation assays and flow cytometry based cell cycle analysis. Activation of T cells was determined by both flow cytometry and qRT-PCR of activation-specific marker genes. . Expression of miR-20b was downregulated in samples of thymoma tissues and serum from patients with thymoma-associated myasthenia gravis. In addition, T cell proliferation and activation were inhibited by ectopic overexpression of miR-20b, which led to increased T cell proliferation and activation. NFAT5 and CAMTA1 were identified as targets of miR-20b. Expression levels of NFAT5 and CAMTA1 were inhibited by miR-20b expression in cultured cells, and the expression levels of miR-20b and NFAT5/CAMTA1 were inversely correlated in patients with thymoma-associated myasthenia gravis. . miR-20b acts as a tumor suppressor in the development of thymoma and thymoma-associated myasthenia gravis. The tumor suppressive function of miR-20b in thymoma could be due to its inhibition of NFAT signaling by repression of NFAT5 and CAMTA1 expression.
Altered naive CD4 T cell homeostasis in myasthenia gravis and thymoma patients.
Kohler Siegfried,Keil Thomas,Alexander Tobias,Thiel Andreas,Swierzy Marc,Ismail Mahmoud,Rückert Jens Carsten,Meisel Andreas
Journal of neuroimmunology
In Myasthenia Gravis (MG) thymic pathologies are often present and thymectomy is used as treatment. By flow cytometry we elucidated alterations of naïve CD4 T cell homeostasis in MG patients and patients with thymoma. MG patients showed increased absolute numbers of CD31naïve CD4 T cells. Thymoma patients displayed a significantly higher fraction of peripheral blood CD31naive T cells. We show an altered naive CD4 T cell homeostasis in MG patients that might predispose to autoimmunity. Aberrant generation of T cells in thymoma can be detected by an increased frequency of CD31naive CD4 T cells in the periphery.
CD19+ Tim-1+ B cells are decreased and negatively correlated with disease severity in Myasthenia Gravis patients.
Zhang Yong,Zhang Xiuying,Xia Yan,Jia Xiao,Li Hao,Zhang Yanyan,Shao Zhen,Xin Ning,Guo Mingfeng,Chen Jing,Zheng ShuangShuang,Wang YuZhong,Fu Linlin,Xiao Chenghua,Geng Deqin,Liu Yonghai,Cui Guiyun,Dong Ruiguo,Huang Xiaoyu,Yu Tingyan
T cell immunoglobulin mucin domain-1(Tim-1) was recently identified to be critical and essential for optimal regulatory B cells function in maintaining immune tolerance. We aimed to measure the expression levels of Tim-1 on B cells from patients with Myasthenia Gravis (MG) and to investigate whether the expression of Tim-1 is associated with pathogenesis of MG. A total of 34 patients with MG (18 generalized MG (GMG) and 16 ocular MG (OMG) and 24 healthy donors were recruited in this study. The quantitative myasthenia gravis score (QMGS) was used to evaluate the clinical severity. Real-time PCR and flow cytometry were used to measure the levels of Tim-1 expressed on peripheral B cells. Peripheral CD138+ plasma cells were assayed by flow cytometry. Serum Th17-related cytokines (IL-6, IL-1β and IL-17) and anti-AChR antibody (Ab) titers were tested by enzyme-linked immunosorbent assay (ELISA). Our data demonstrated that the mRNA and protein expression levels of B cell Tim-1 in both the GMG and OMG groups were significantly lower than those in healthy controls, with lower expression in GMG than in OMG. Tim-1 expression on B cells from OMG/GMG was negatively correlated with clinical severity, plasma cells frequency, serum Th17-related cytokines and anti-AChR Ab levels. Our results indicated that aberrant expression of Tim-1 exists on B cells and may contribute to the Th17 polarization and antibody-secreting plasma cells differentiation in MG patients.
Reduction in peripheral regulatory T cell population in childhood ocular type myasthenia gravis.
Nishimura Takafumi,Inaba Yuji,Nakazawa Yozo,Omata Taku,Akasaka Manami,Shirai Ikuko,Ichikawa Motoki
Brain & development
OBJECTIVE:Myasthenia gravis (MG) is a T-cell dependent and antibody mediated autoimmune disease. Recent studies of adult patients and animal models have shown that regulatory T cells (Tregs) play an important role in the pathogenesis of MG, but little is known about MG in children. This study evaluated the role of peripheral blood Tregs in childhood ocular MG and assessed if Tregs could be an index for estimating immunological status. PATIENTS AND METHODS:Clinical data and peripheral lymphocytes were obtained from 13 children with serum AChR antibody-positive ocular type MG and 18 age-matched controls. Committed cells from MG patients were divided into two clinical stages: active (n=12) and remission (n=11). Tregs and Th17 cells were analyzed by flow cytometric analysis based on CD4(+)CD25(+) intracellular Foxp3(+) and CD4(+) intracellular IL-17A(+) fractions, respectively. RESULTS:The percentage of Tregs among peripheral blood CD4(+) T cells in active stage, remission stage, and control groups was 3.3±1.3%, 4.8±1.7%, and 5.0±0.6%, respectively. The Treg population was significantly lower in the active stage than in the remission stage and controls. Furthermore, Treg percentage was significantly lower during relapse of myasthenia symptoms. We witnessed no remarkable associations between the percentage of Tregs and immune suppressant dosages. CONCLUSIONS:A significant reduction in the peripheral Treg population is considered to contribute to the pathophysiology of ocular type childhood MG and may be a marker of immunological state in these patients.
CCR9 AND CCR7 are overexpressed in CD4 CD8 thymocytes of myasthenia gravis patients.
Li Qianru,Liu Pingping,Xuan Xiaoyan,Zhang Junfeng,Zhang Yun,Zhu Zhengkun,Gao Feng,Zhang Qingyong,Du Ying
Muscle & nerve
INTRODUCTION:Chemokine CC motif receptors 9 and 7 (CCR9 and CCR7) play a major role in the migration of T-cell precursors to the thymus to initiate T thymopoiesis. However, their role in development of T-cells in myasthenia gravis (MG) patients has not been fully elucidated. METHODS:Expression and distribution of CCR9 and CCR7 cells were detected by flow cytometry and immunofluorescence. Real-time polymerase chain reaction was used to check the adhesion molecules on CD4 CD8 double-negative (DN) thymocytes. RESULTS:CCR9 and CCR7 expression by DN thymocytes increased in the MG thymus; the levels of CCR9, CCR7, interleukin-7R mRNA increased, and CXCR4 levels decreased compared with levels in the non-MG thymus. More CCR7 and CCR9 double-positive (DP) thymocytes were gathered near the subcapsular region in MG thymus. CONCLUSIONS:Enhanced expression of CCR9 and CCR7 may complicate the differentiation of DP thymocytes from the DN stage in MG thymus. Muscle Nerve, 2016 Muscle Nerve 55: 84-90, 2017.
MuSK autoantibodies in myasthenia gravis detected by cell based assay--A multinational study.
Tsonis A I,Zisimopoulou P,Lazaridis K,Tzartos J,Matsigkou E,Zouvelou V,Mantegazza R,Antozzi C,Andreetta F,Evoli A,Deymeer F,Saruhan-Direskeneli G,Durmus H,Brenner T,Vaknin A,Berrih-Aknin S,Behin A,Sharshar T,De Baets M,Losen M,Martinez-Martinez P,Kleopa K A,Zamba-Papanicolaou E,Kyriakides T,Kostera-Pruszczyk A,Szczudlik P,Szyluk B,Lavrnic D,Basta I,Peric S,Tallaksen C,Maniaol A,Casasnovas Pons C,Pitha J,Jakubíkova M,Hanisch F,Tzartos S J
Journal of neuroimmunology
Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.
Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools.
Makino Tomohiro,Nakamura Ryuichi,Terakawa Maki,Muneoka Satoshi,Nagahira Kazuhiro,Nagane Yuriko,Yamate Jyoji,Motomura Masakatsu,Utsugisawa Kimiaki
The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.
High frequencies of circulating Tfh-Th17 cells in myasthenia gravis patients.
Yang Yongxiang,Zhang Min,Ye Yuqin,Ma Shan,Fan Lingling,Li Zhuyi
Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology
Recent studies show that the frequencies of circulating follicullar helper T (cTfh) cells are significantly higher in myasthenia gravis (MG) patients compared with healthy controls (HC). And, they are positively correlated with levels of serum anti-acetylcholine receptor antibody (anti-AchR Ab). It is unclear whether cTfh cell subset frequencies are altered and what role they play in MG patients. In order to clarify this, we examined the frequencies of cTfh cell counterparts, their subsets, and circulating plasmablasts in MG patients by flow cytometry. We determined the concentrations of serum anti-AChR Ab by enzyme-linked immunosorbent assay (ELISA). We assayed the function of cTfh cell subsets by flow cytometry and real-time polymerase chain reaction (RT-PCR). We found higher frequencies of cTfh cell counterparts, cTfh-Th17 cells, and plasmablasts in MG patients compared with HC. The frequencies of cTfh cell counterparts and cTfh-Th17 cells were positively correlated with the frequencies of plasmablasts and the concentrations of anti-AChR Ab in MG patients. Functional assays showed that activated cTfh-Th17 cells highly expressed key molecular features of Tfh cells including ICOS, PD-1, and IL-21. Results indicate that, just like cTfh cell counterparts, cTfh-Th17 cells may play a role in the immunopathogenesis and the production of anti-AChR Ab of MG.
Effect of low-dose rituximab treatment on T- and B-cell lymphocyte imbalance in refractory myasthenia gravis.
Jing Sisi,Lu Jun,Song Jie,Luo Sushan,Zhou Lei,Quan Chao,Xi Jianying,Zhao Chongbo
Journal of neuroimmunology
We aimed to explore the effects of low-dose rituximab (RTX) on circulating T- and B-cell lymphocytes and the improvement of clinical symptoms in refractory myasthenia gravis (MG) patients. Fifteen patients with refractory MG were treated with a low dose of 600 mg RTX and were evaluated by serial-clinical scales, flow cytometry of peripheral blood T and B cells, and antibody titer before and after six months of RTX treatment. The quantitative MG score (QMGS), manual muscle testing (MMT), MG-related activities of daily living (MG-ADL) and MG-specific quality-of-life (QOL) were significantly improved and the average steroid-dosage reduction was 40% (p = .001) in refractory MG patients at six months after RTX infusion. Compared to eighteen non-refractory MG patients and eighteen Healthy controls, our study showed that the frequencies of circulating regulatory B cells (Bregs) and regulatory T cells (Tregs) were significantly lower and the expression of B-cell activating factor receptors (BAFF-Rs) was greater in refractory MG patients without RTX treatment. Importantly, 600-mg RTX was sufficient to deplete B cells and maintain low B-cell counts for up to six months after infusion. Additionally, a low dose of RTX further increased the frequencies of Tregs. Hence, there is an immune imbalance in circulating T- and B-cell lymphocytes in refractory MG patients compared to non-refractory MG patients. We conclude that remarkable T- and B-cell lymphocyte imbalance exists in refractory MG. Low-dose RTX can improve myasthenic symptom and deplete B cells and increase Tregs%.
Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis.
Marino Mariapaola,Scuderi Flavia,Samengo Daniela,Saltelli Giorgia,Maiuri Maria Teresa,Shen Chengyong,Mei Lin,Sabatelli Mario,Pani Giovambattista,Antonini Giovanni,Evoli Amelia,Bartoccioni Emanuela
BACKGROUND:Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG. METHODS:The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system. RESULTS:We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals. CONCLUSION:Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.
Altered expression of transcription factors IRF4 and IRF8 in peripheral blood B cells is associated with clinical severity and circulating plasma cells frequency in patients with myasthenia gravis.
Zhang Yong,Jia Xiao,Xia Yan,Li Hao,Chen Fei,Zhu Jie,Zhang Xiuying,Zhang Yanyan,Wang YuZhong,Xu Yanan,Pan Meng,Huang Xiaoyu,Yu Tingyan,Fu Linlin,Xiao Chenghua,Geng Deqin
Previous studies have shown that interferon regulatory factor-4 (IRF4) and IRF8 play critical but distinct roles in the differentiation of B cells into plasma cells (PCs). In the present study, we aimed to measure the expression levels of IRF4 and IRF8 in B cells from patients with myasthenia gravis (MG) and to investigate whether the expression of IRF4 and IRF8 associates with pathogenesis of MG. A total of 35 anti-acetylcholine receptor (AChR) antibody (Ab)-positive patients with MG [20 generalized MG (GMG) and 15 ocular MG (OMG) and 25 healthy donors were recruited in this study. The quantitative myasthenia gravis score (QMGS) was used to evaluate the clinical severity. Real-time PCR and Western blot were used to measure the levels of IRF4 and IRF8 expressed in peripheral blood B cells. Peripheral blood CD138 PCs were assayed by flow cytometry. Our data demonstrated that the mRNA/protein levels of IRF4 and IRF8 were significantly higher and lower, respectively, in patients with OMG/GMG groups compared with healthy controls. In addition, IRF4 expression was significantly higher and IRF8 expression was significantly lower in GMG group than in OMG group. Pearson's correlation analysis revealed that IRF8 expression was negatively correlated with clinical severity, PCs frequency and anti-AChR Ab levels, while IRF4 expression and IRF4/IRF8 ratio was positively correlated with these parameters in two MG subgroups. Finally, glucocorticoid treatment can relieve the imbalance of IRF4/IRF8 in peripheral blood B cells, and this restoration is accompanied by reduced PCs frequency and clinical symptoms. These evidences suggest that IRF4 and IRF8 are important in the counter-balancing mechanisms controlling differentiation of PCs in MG. The disruption of the balanced IRF4/IFR8 ratio in B cells may play important roles in the pathogenesis of MG and offer a promising therapeutic target for the development of novel immunotherapy for MG patients.
Clinical efficacy of tacrolimus for treating myasthenia gravis and its influence on lymphocyte subsets.
Bao J,Gao S,Weng Y,Zhu J,Ye H,Zhang X
BACKGROUND:This study aimed to determine the clinical efficacy and effects of tacrolimus in treating myasthenia gravis (MG). METHODS:A total of 45 outpatients and inpatients were divided into a tacrolimus group (n=15) and non-tacrolimus group (n=30): those in the former group were treated with 3mg/day of tacrolimus for 24 weeks, while those in the latter (control) group took other immunosuppressants (prednisone, azathioprine combined with prednisone). Each group was evaluated at weeks 4, 8, 12, 16, 20 and 24 by Myasthenia Gravis Foundation of America Quantitative Myasthenia Gravis (MGFA-QMG) Test, activities of daily living (ADL) profiles, and manual muscle (MMT) and fatigue tests. Dynamic changes in CD4CD25 cells were tested by flow cytometry. Inflammatory cytokines were also evaluated in the tacrolimus group. RESULTS:Efficacy index scores decreased significantly compared with baseline at every test week in both groups (P<0.01), although improvements were more evident with than without tacrolimus treatment (F=9.312, P<0.01 vs. F=24.551, P<0.01 and F=10.710, P<0.01). At week 24, peripheral blood CD4CD25 T cells with tacrolimus decreased significantly (P<0.01), but increased significantly without tacrolimus (P<0.01). During treatment, CD19BAFF-R B cells in peripheral blood decreased in both groups (P<0.05). Interferon (IFN)-γ concentrations in peripheral blood also diminished significantly with tacrolimus (P<0.01). CONCLUSION:A relatively low dose of tacrolimus can affect multiple immune-system targets and, thus, can treat MG effectively in terms of both clinical symptoms and immunological responses.