Regulatory T cells induced by GM-CSF suppress ongoing experimental myasthenia gravis

We had previously observed that treatment utilizing granulocyte–macrophage colony-stimulating factor (GM-CSF) had profound effects on the induction of experimental autoimmune myasthenia gravis (EAMG), a well-characterized antibody-mediated autoimmune disease. In this study, we show that EAMG induced by repeated immunizations with acetylcholine receptor (AChR) protein in C57BL6 mice is effectively suppressed by GM-CSF treatment administered at a stage of chronic, well-established disease. In addition, this amelioration of clinical disease is accompanied by down-modulation of both autoreactive T cell, and pathogenic autoantibody responses, a mobilization of DCs with a tolerogenic phenotype, and an expansion of regulatory T cells (Tregs) that potently suppress AChR-stimulated T cell proliferation in vitro. These observations suggest that the mobilization of antigen-specific Tregs in vivo using pharmacologic agents, like GM-CSF, can modulate ongoing anti-AChR immune responses capable of suppressing antibody-mediated autoimmunity.     

Interferon-gamma-modified dendritic cells suppress B cell function and ameliorate the development of experimental autoimmune myasthenia gravis

This study was designed to investigate the therapeutic effects of interferon (IFN)-gamma-modulated dendritic cells (DC) in experimental autoimmune myasthenia gravis (EAMG). We induced EAMG in Lewis rats by immunization with Torpedo nicotinic acetylcholine receptor (nAChR) and adjuvant. On day 33 post-immunization (p.i.), splenic DC were prepared, exposed to IFN-gamma alone (IFN-gamma-DC) or to IFN-gamma in combination with 1-methyl-DL-tryptophan (1-MT), the specific inhibitor of indoleamine 2,3-dioxygenase (IDO) (IFN-gamma + 1-MT-DC), and injected subcutaneously into rats with incipient EAMG on day 5 p.i. A control group of EAMG rats received naive DC on day 5 p.i., while another group received 1-MT every other day, intraperitoneally (p.i.), from days 5 to 41 p.i. The severity of clinical signs of EAMG was reduced dramatically in IFN-gamma-DC-treated rats compared to rats receiving naive DC, IFN-gamma + 1-MT-DC or 1-MT alone. The number of plasma cells secreting nAChR antibodies was reduced and the expression of B cell activation factor (BAFF) on splenic and lymph node mononuclear cells (MNC) was down-regulated in rats treated with IFN-gamma-DC. In vitro co-culture of MNC derived from EAMG rats with IFN-gamma-DC produced relatively few cells secreting nAChR antibodies. Addition of 1-MT to the co-culture significantly increased the number of cells secreting nAChR antibodies. We conclude that IFN-gamma-DC reduced the number of plasma cells secreting nAChR antibodies in an IDO-dependent manner and ameliorated the development of EAMG in Lewis rats.     

S1P receptor antagonists fingolimod and siponimod do not improve the outcome of experimental autoimmune myasthenia gravis mice after disease onset

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness and fatigue in the presence of circulating antibodies against components of the neuromuscular junction. Most patients have a good prognosis, but some are refractory to standard‐of‐care immunosuppressive treatment and suffer from recurrent myasthenic crises. Functional sphingosine‐1‐phosphate (S1P) antagonists like fingolimod and siponimod (BAF312) are successfully used for the treatment of multiple sclerosis, and fingolimod was shown to prevent the development of myasthenic symptoms in experimental autoimmune myasthenia gravis (EAMG), the standard model of MG. Here, we investigated whether fingolimod or siponimod improves outcome in EAMG mice when administered after disease onset, modeling the clinical setting in human MG. Both S1P antagonists inhibited lymphocyte egress, resulting in peripheral lymphopenia. After stimulation, there were differences in T‐cell responses, but no change in either antibody titers or total or antigen‐specific plasma cell populations after treatment. Most importantly, disease incidence and severity were not influenced by fingolimod or siponimod therapy. Although fingolimod and siponimod did lead to subtle changes in T‐cell responses, they had no significant effect on antibody titers and disease severity. In conclusion, our data show no evidence of a therapeutic potential for S1P receptor antagonists in MG treatment.     

Activation of the adenosine A2A receptor attenuates experimental autoimmune myasthenia gravis severity

The adenosine A2A receptor (A2AR) is the major cellular adenosine receptor commonly associated with immunosuppression. Here, we investigated whether A2AR activation holds the potential for impacting the severity of experimental autoimmune myasthenia gravis (EAMG) induced following immunization of Lewis rats with the acetylcholine receptor (AChR) R97-116 peptide. This report demonstrates reduced A2AR expression by both T cells and B cells residing in spleen and lymph nodes following EAMG induction. A2AR stimulation inhibited anti-AChR antibody production and proliferation of AChR-specific lymphocytes in vitro. Inhibition was blocked with the A2AR antagonists or protein kinase A inhibitor. We also determined that the development of EAMG was accompanied by a T-helper cell imbalance that could be restored following A2AR stimulation that resulted in increased Treg cell levels and a reduction in Th1-, Th2-, and Th17-cell subtypes. An EAMG-preventive treatment regimen was established that consisted of (2-(p-(2-carbonylethyl)phenylethylamino)-5-N-ethylcarboxamidoadenosine) (CGS21680; A2AR agonist) administration 1 day prior to EAMG induction. Administration of CGS21680 29 days post EAMG induction (therapeutic treatment) also ameliorated disease severity. We conclude that A2AR agonists may represent a new class of compounds that can be developed for use in the treatment of myasthenia gravis or other T-cell- and B-cell-mediated autoimmune diseases.     

Suppression of experimental autoimmune myasthenia gravis by autologous T regulatory cells

Adoptive transfer of regulatory T (Treg) cells have been employed effectively for suppression of several animal models for autoimmune diseases. In order to employ Treg cell therapy in patients, it is necessary to generate Treg cells from the patient's own cells (autologous) that would be able to suppress effectively the disease in vivo, upon their reintroduction to the patient. Towards this objective, we report in the present study on a protocol for a successful immune-regulation of experimental autoimmune myasthenia gravis (EAMG) by ex vivo – generated autologous Treg cells. For this protocol bone marrow (BM) cells, are first cultured in the presence of GM-CSF, giving rise to a population of CD11c⁺MHCII⁺CD45RA⁺CD8^– DCs (BMDCs). Splenic CD4⁺ T cells are then co-cultured with the differentiated BM cells and expand to 90% of Foxp3⁺ Treg cells. In vitro assay exhibits a similar dose dependent manner in the suppression of T effector cells proliferation between Treg cells obtained from either healthy or sick donors. In addition, both Treg cells inhibit similarly the secretion of IFN-γ from activated splenocytes. Administration of 1 × 10⁶ ex-vivo generated Treg cells, I.V, to EAMG rats, modulates the disease following a single treatment, given 3 days or 3 weeks after disease induction. Similar disease inhibition was achieved when CD4 cells were taken from either healthy or sick donors. The disease suppression was accompanied by reduced levels of total AChR specific antibodies in the serum. Moreover, due to the polyclonality of the described Treg cell, we have examined whether this treatment approach could be also employed for the treatment of other autoimmune diseases involving Treg cells. Indeed, we demonstrated that the ex-vivo generated autologous Treg cells suppress Adjuvant Arthritis (AA) in rats.This study opens the way for the application of induced autologous Treg cell therapy for myasthenia gravis, as well as for other human autoimmune diseases involving Treg cells.     

Neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG)

Chronic experimental autoimmune myasthenia gravis (EAMG) was induced in rats by immunization with acetylcholine receptor (AChR) purified from the electroplax of Torpedo californica. 35–40 days after immunization, serum anti-AChR antibody titers were about 40 nM. At this stage, electrophysiology was performed on isolated M. omohyoideus muscle-preparations from myasthenic and from normal (control) rats.For the study of the equilibrium interaction between acetylcholine (ACh) and AChR, dose-response curves were obtained by quantitative ionophoretic application of ACh to voltage-clamped end-plates. Analysis of dose-response curves yielded the following parameters: maximum end-plate conductance per unit surfaceg _max (EAMG)=10.3±1.1 nS/m²,g _max (normal)=20.2±1.8 nS/m²; apparent dissociation constant K (EAMG)=96±5 M, K (normal)=58±6 M; Hill-coefficient n_H (EAMG)=2.3±0.1, n_H (normal)=2.3±0.1. Single channel properties were derived from an analysis of ACh-induced end-plate current noise: the mean single channel conductance was (EAMG)=29.1±2.2 pS, (normal)=27.6±1.8 pS and the mean channel life-time (EAMG)=1.39±0.09 ms, (normal)=1.32±0.08 ms (T=22°C).The electrophysiological data are interpreted as follows: (1) At myasthenic end-plates there is a 50–60% reduction of functioning AChR (decrease ofg _max). A total number of about 2×10⁶ (1×10⁶) channels per end-plate was calculated for control (myasthenic) rats. (2) The affinity of AChR for ACh is reduced and/or there is an impediment of the conformational change from the closed- to the open-channel configuration (increase of K). (3) Single channel properties are essentially unaffected.This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, project N and We 667/6     

Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals

Nasal administration of μg doses of acetylcholine receptor (AChR) is effective in preventing the development of B cell-mediated EAMG in the Lewis rat, a model for human MG. In order to investigate whether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally with AChR 2 weeks after immunization with AChR and Freund's complete adjuvant. Ten-fold higher amounts of AChR given nasally (600 μg/rat) were required to ameliorate the manifestations of EAMG compared with the amounts necessary for prevention of EAMG. In lymph node cells from rats receiving 600 μg/rat of AChR, AChR-induced proliferation and interferon-gamma (IFN-γ) secretion were reduced compared with control EAMG rats receiving PBS only. The anti-AChR antibodies in rats treated nasally with 600 μg/rat of AChR had lower affinity, reduced proportion of IgG2b and reduced capacity to induce AChR degradation. Numbers of AChR-reactive IFN-γ and tumour necrosis factor-alpha (TNF-α) mRNA-expressing lymph node cells from rats treated nasally with 600 μg/rat of AChR were suppressed, while IL-4, IL-10 and transforming growth factor-beta (TGF-β) mRNA-expressing cells were not affected. Collectively, these data indicate that nasal administration of AChR in ongoing EAMG induced selective suppression of Th1 functions, i.e. IFN-γ and IgG2b production, but no influence on Th2 cell functions. The impaired Th1 functions may result in the production of less myasthenic anti-AChR antibodies and contribute to the amelioration of EAMG severity in rats treated with AChR 600 μg/rat by the nasal route.     

Requirement of the Fc receptor common gamma-chain for gamma delta T cell-mediated promotion of murine experimental autoimmune encephalomyelitis

Immunoglobulin Fcgamma receptors (FcgammaR) are comprised of a ligand-binding alpha-chain that sometimes associates with a cell signaling common gamma-chain. These receptors comprise an important family of effector molecules that link humoral and cell-mediated adaptive immunity and regulate innate immunity. Recent animal studies suggest that FcgammaR in general, and FcR alpha-chains in particular, are required for full development of experimental autoimmune encephalomyelitis (EAE). We show here that deletion of the gamma-chain renders mice resistant to EAE, whereas deletion of the alpha-chains of FcgammaRI, FcgammaRIIB and FcgammaRIII has no protective effect. Susceptibility to EAE is fully restored in common gamma-chain-/- mice into which wild-type splenocytes are adoptively transferred, but EAE is not restored in common gamma-chain-/- mice given wild-type splenocytes depleted of gammadelta T cells. These data indicate that although the common gamma-chain is required for full development of EAE in mice, this requirement is likely FcgammaR alpha-chain-independent. Expression of the common gamma-chain by gammadelta T cells, probably in conjunction with the T cell receptor/CD3 complex, is likely the key requirement for full development of EAE.     

Highly reduced binding to high and low affinity mouse Fc gamma receptors by L234A/L235A and N297A Fc mutations engineered into mouse IgG2a

The effects of the Fc silencing mutations such as leucine (L) to alanine (A) substitution at the position 234 and 235 (LALA) and the alanine (A) to asparagine (N) substitution at position 297 (N297A) are well investigated for human IgG. However, the effects of the same two silencing Fc mutations in a mouse IgG backbone are not yet well investigated in respect to binding to mouse Fc gamma receptors (FcγRs), complement and subsequent effector functions. By using a mouse IgG2a tool antibody directed against mouse OX40L, we demonstrate a strongly reduced binding of the two Fc mutants to high and low affinity recombinant and cell expressed mouse FcγRs, when compared to the mouse IgG2a with the wild type (wt) backbone. Reduced FcγR binding by the two investigated Fc mutants could further be confirmed on primary mouse macrophages expressing their native FcγRs. In addition, we reveal that the LALA and N297A mutations in the mIgG2a also slightly reduced binding to C1q of human origin. Thus, here we provide experimental evidence that the two investigated Fc mutations in the mouse IgG backbone lead to similar “silencing” properties as previously demonstrated for the human IgG and thus represent a useful method to alter effector functions in tool antibodies to be used in mouse models.     

Depletion of CD8+ T cells suppresses the development of experimental autoimmune myasthenia gravis in Lewis rats

To understand the role of CD8⁺ T cells in experimental autoimmune myasthenia gravis (EAMG), CD8⁺ T cells were depleted by injecting a monoclonal anti-rat CD8 antibody (OX8) into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). CD8-depleted EAMG rats showed strikingly less muscle weakness and lower anti-AChR IgG antibody levels compared to Hy2-15-injected control EAMG rats. Moreover, the numbers of AChR-specific IgG antibody-secreting cells, AChR-reactive interferony-γ-secreting T helper type 1-like cells and lymphocyte proliferation to AChR were lower in the CD8-depleted group than in control EAMG rats. These differences were significant among mononuclear cells from inguinal and popliteal lymph nodes, mesenteric lymph nodes and spleen, but not from thymus when examined 3, 5 and 7 weeks post-immunization. We suggest that CD8⁺ T cells are involved in the induction and persistance of EAMG by directly or indirectly affecting AChR-reactive T cells and anti-AChR IgG antibody-secreting cells.     

Cellular mRNA expression of interferon-gamma (IFN-γ), IL-4 and IL-10 relates to resistance to experimental autoimmune myasthenia gravis (EAMG) in young Lewis rats

Age-related alterations in the immune system, including changes in lymphocyte subset composition, result in changes of cytokine patterns and might thereby influence the incidence and severity of autoimmune diseases. To investigate the age-related resistance to EAMG, an animal model for human MG, young (4-week-old) and adult (8–10-week-old) female Lewis rats were immunized with Torpedo acetylcholine receptor (AChR) and Freund's complete adjuvant (FCA). Adult Lewis rats showed severe weight loss and progressive muscular weakness after immunization, while young rats developed minor clinical signs of EAMG after a prolonged interval post-immunization. By comparison with adult rats, the young had lower AChR-specific T and B cells responses, and less muscle AChR loss. In situ hybridization performed on mononuclear cells (MNC) from lymph nodes revealed that young rats had lower levels of AChR-specific IFN-γ, IL-4 and IL-10 mRNA-expressing cells compared with adult rats. Since IFN-γ, IL-4 and IL-10 promote the development of EAMG, the low expression of these cytokines might contribute to EAMG resistance in young Lewis rats.     

雷帕霉素通过下调Th17细胞/调节性T细胞比值减轻实验性自身免疫性重症肌无力大鼠症状

目的 初步研究雷帕霉素(RAPA)对实验性自身免疫性重症肌无力(EAMG)大鼠的治疗作用,并研究相关免疫机制.方法 应用鼠源性乙酰胆碱受体α亚基97～116肽段(R97-116)免疫Lewis大鼠,建立EAMG动物模型,随机分为完全Freund佐剂对照组(CFA组)、EAMG模型对照组、RAPA处理组[1 mg/(kg...     

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determinedin vitro by a classical rosette assay in 12 postmen-opausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, -mannosyl [IgG(Con A)] or -galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P<0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs. In addition, when both groups of women were compared, all three Fc-dependent functions measuredin vitro using IgG(PNA)-SRBCs were significantly lower (P<0.01 at least) in women without E than in women on therapy. In another series of experiments, we also found that the rosetting and phagocytosing capacities of MCs were dramatically and transiently reduced in three of three young women during the menstrual period, only when the IgG(PNA)-SRBCs were used as targets. Taken together, our data show that MC phagocytosis of SRBCs coated with IgG antibody exhibiting terminal, and/or accessible, -galactosyl residues in their Fc domain is selectively impaired by a physiological E deficiency and is restored when this deficiency is artificially or spontaneously corrected. They therefore suggest that these hormones are capable of affecting the PNA-like activity of IgG(Fc) receptors of human MCs.     

Relation of HLA-DRB1 to IgG4 autoantibody and cytokine production in muscle-specific tyrosine kinase myasthenia gravis (MuSK-MG)

A small subset of myasthenia gravis (MG) patients develop autoantibodies against muscle-specific kinase (MuSK), which are predominantly of the immunoglobulin (Ig)G4 isotype. MuSK-MG is strongly associated with HLA-DRB1*14, HLA-DRB1*16 and HLA-DQB1*05. In this study, the possible effects of these HLA associations on MuSK IgG autoantibody or cytokine production were investigated. Samples from 80 MG patients with MuSK antibodies were studied. The disease-associated HLA types were screened in the DNA samples. The IgG1, IgG2, IgG3 and IgG4 titres of the MuSK antibodies and the levels of interleukin (IL)-4, IL-6, IL-17A and IL-10 were measured in the sera. Comparisons were made among the groups with or without HLA-DRB1*14, HLA-DRB1*16 or HLA-DQB1*05. The IgG4 titres of the MuSK antibodies were higher than those of the IgG1, IgG2 and IgG3 isotypes among the whole group of patients. DRB1*14 (+) DRB1*16 (–) patients had higher levels of IgG4 antibodies than those of DRB1*14 (–) DRB1*16 (+) patients. DRB1*14 (+) DRB1*16 (+) patients also had higher levels of IgG4 antibodies than those of DRB1*14 (–) DRB1*16 (+) and DRB1*14 (–) DRB1*16 (–) patients. Higher IL-10 and lower IL-17A levels were measured in DRB1*14 (+) DRB1*16 (–) patients than in DRB1*14 (–) DRB1*16 (–) patients. The higher IgG4 titres of MuSK autoantibodies in patients carrying HLA-DRB1*14 than those in the other patients suggest a role for HLA in the production of the antibodies. The differences in IL-10 and IL-17A support the role of DRB1 in the etiopathogenesis of this autoimmune response.     

'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.     

Age- and sex-related resistance to chronic experimental autoimmune myasthenia gravis (EAMG) in Brown Norway rats

The influence of age and sex on the induction of chronic EAMG was analysed. Aged male rats, immunized with Torpedo acetylcholine receptor (tAChR), showed no clinical signs of disease or AChR loss. Immunization of young male Brown Norway (BN) rats resulted in both clinical signs of disease and 65% AChR loss. In contrast, both young and aged female BN rats showed comparable AChR loss (58% and 50%, respectively), although aged female rats did not develop clinical signs of disease. Differences in antibody titres, isotype distribution, fine specificity or complement activation could not account for the observed resistance. These results suggest that resistance against EAMG in aged rats is due to resistance of the AChR against antibody-mediated degradation, or to mechanisms able to compensate for AChR loss.     

In vitro neutralization of HSV-2: inhibition by binding of normal IgG and purified Fc to virion Fc receptor (FcR)

We designed experiments to assess the effects of binding of the HSV-2 Fc receptor (FcR) to purified rabbit nonimmune IgG and purified Fc. Purified Fc (75 micrograms) or nonimmune IgG (100 micrograms) when bound to HSV-2 did not reduce infectivity but did protect the virions against thermal inactivation at 37 degrees C. However, preincubation of these two reagents with HSV-2 virions significantly protected against neutralization by specific anti-HSV-2, F(ab')2 purified rabbit antiserum. The blockage of neutralization and protection against thermal inactivation afforded by FcR-Fc interaction on HSV-2 virions provide a tenable mechanism to explain viral persistence in an immune host.     

Activation of the receptor for advanced glycation end products (RAGE) exacerbates experimental autoimmune myasthenia gravis symptoms

RAGE belongs to immunoglobulin superfamily and serves as a ligand for various immunoregulatory molecules including S100B that has been demonstrated important to T cell mediated autoimmune diseases. In this context, we hypothesized that RAGE could also impact B cell mediated, T cell-dependent autoimmune diseases. This was tested using myasthenia gravis (MG) animal model, EAMG. We show that expression of both RAGE and S100B are increased during EAMG and the interaction between RAGE and S100B affected the Th1/Th2/Th17/Treg cell equilibrium, up-regulate AChR-specific T cell proliferation. Furthermore, addition of S100B in vitro stimulated splenocyte activity linked to COX-2 up-regulation. NS-398, a selective COX-2 inhibitor, effectively diminished S100B mediated activity of AChR-specific antibody secreting splenocytes. These findings suggested that a reciprocal relationship between RAGE and S100B promoted the development of EAMG, highlighting the importance of understanding the mechanisms of EAMG disease as a means of developing new therapies for the treatment of MG.