Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.     

Mechanisms of nasal tolerance induction in experimental autoimmune myasthenia gravis: identification of regulatory cells

Autoantigen administration via nasal mucosal tissue can induce systemic tolerance more effectively than oral administration in a number of experimental autoimmune diseases, including Ab-mediated experimental autoimmune myasthenia gravis, a murine model of myasthenia gravis. The mechanisms underlying nasal tolerance induction are not clear. In this study, we show that nasal administration of acetylcholine receptor (AChR) in C57BL/6 mice, before immunizations with AChR in adjuvant, results in delayed onset and reduced muscle weakness compared with control mice. The delayed onset and reduced muscle weakness were associated with decreased AChR-specific lymphocyte proliferation and decreased levels of anti-AChR Abs of the IgG2a and IgG2b isotypes in serum. The clinical and immunological changes in the AChR-pretreated C57BL/6 wild-type (wt) mice were comparable with those observed in AChR-pretreated CD8-/- mice, indicating that CD8+ T cells were not required for the generation of nasal tolerance. AChR-pretreated wt and CD8-/- mice showed augmented TGF-beta and reduced IFN-gamma responses, whereas levels of IL-4 were unaltered. Splenocytes from AChR-pretreated wt and CD8-/- mice, but not from CD4-/- mice, suppressed AChR-specific lymphocyte proliferation. This suppression could be blocked by Abs against TGF-beta. Thus, our results demonstrate that the suppression induced in the present model is independent of CD8+ T cells and suggest the involvement of Ag-specific CD4+ Th3 cells producing TGF-beta.     

IL-1 receptor antagonist-mediated therapeutic effect in murine myasthenia gravis is associated with suppressed serum proinflammatory cytokines, C3, and anti-acetylcholine receptor IgG1

In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.     

Autoimmune rat T lymphocytes monospecific for acetylcholine receptors: purification and fine specificity

We prepared highly purified acetylcholine receptor (AChR)-specific T lymphocytes from rats with experimental autoimmune myasthenia gravis (EAMG). Inbred rats were primed with AChR frm 3 different sources: from the electric organs of Electrophorus electricus and Torpedo californica and from denervated rat muscle. After 20 to 30 days, lymphocytes from regional lymph nodes (primary cells) were challenged with soluble AChR in vitro. The activated blast cells were isolated by density gradient centrifugation and allowed to revert back to small secondary lymphocytes in the absence of antigen. These secondary anti-AChR cells were highly responsive to the type of AChR with which they had been primed. Their reactivity critically depended on help by syngeneic accessory cells. Anti-Electrophorus AChR primary and secondary cells cross-reacted detectably with rat AChR and vice versa, whereas anti-Torpedo AChR primary and secondary cells did not significantly cross-react with Electrophorus or rat AChR. Secondary T cells strongly reactive against rat AChR could be selected in vitro from Electrophorus AChR-primed populations by using rat AChR as selecting stimulant. These cells responded equally well against Electrophorus and rat AChR and thus include autoreactive T cell clones.     

Immunotherapy for myasthenia gravis: a murine model

In vivo therapy with monoclonal antibody (mAb) GK1.5, which recognizes a glycoprotein antigen designated L3T4 on murine helper T lymphocytes, either prevented or suppressed the development of murine lupus, autoimmune encephalomyelitis, and collagen arthritis. The L3T4 antigen in the mouse is analogous to the human Leu-3/T4 antigen expressed on helper T lymphocytes, because they both participate in the T cell response to class II major histocompatibility complex (MHC) antigens. Class II MHC genes and I-A antigens mediate murine experimental autoimmune myasthenia gravis (EAMG) induced by acetylcholine receptor (AChR) autoimmunity. We studied the efficacy of mAb GK1.5 as an immunotherapeutic agent for murine EAMG. Therapy with mAb GK1.5 not only suppressed established autoimmunity to AChR but also prevented loss of muscle AChR in mice with EAMG. Moreover, permanent remission of clinical muscle weakness was induced if mAb GK1.5 therapy was initiated after the onset of clinical disease. Because the function of the Leu-3/T4 determinant on human helper T lymphocytes is analogous to the murine L3T4 determinant, use of antibody to the Leu-3/T4 determinant as an immunotherapeutic agent may provide a way to control the progression of human MG.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.     

Specific killing of lymphocytes that cause experimental autoimmune myasthenia gravis by ricin toxin-acetylcholine receptor conjugates

Experimental autoimmune myasthenia gravis (EAMG) is an autoimmune disease in which antibodies to acetylcholine receptor (AChR) cause loss of AChR from muscle, thereby impairing neuromuscular transmission. Here we report the use of a hybrid molecule that contains ricin toxin, irreversibly coupled to AChR to specifically suppress the immune response to AChR in vitro. Lymph node cell cultures from rats with EAMG pretreated with ricin toxin-AChR conjugates exhibited suppressed T helper cell proliferation and B cell antibody synthesis in response to the subsequent addition of AChR. Nonspecific toxicity of the conjugates was measured by suppression of the T cell proliferative response to the mitogen concanavalin A and the antigen keyhole limpet hemocyanin (KLH), and B cell antibody production to KLH. We have evaluated different pretreatment conditions and ricin toxin covalently coupled to AChR in different molar ratios to optimize specific immunosuppression. By varying the number of ricin molecules covalently bound to AChR in the immunotoxin, we were able to minimize the nonspecific toxicity while still maintaining specific killing of AChR-reactive lymphocytes. Furthermore, B cells were more susceptible to specific killing than were the T cells. The specific immunosuppression was potentiated by performing the pretreatment with immunotoxin in the presence of chloroquine. Chloroquine raises lysosomal pH and probably delays the degradation of immunotoxin in the cell. It should be noted that ricin toxin was covalently coupled to AChR by using a novel, non-reducible reaction. These in vitro results suggest that it may be feasible to use immunotoxin molecules to specifically suppress this autoimmune response in vivo.     

Absence of suppression in natural and induced tolerance to F antigen

The role of suppression in natural and induced tolerance to F antigen was investigated in two sets of experiments. In the first, CBA mice were submitted to pretreatments which decrease suppression and the antibody response to self- or allo-F type was investigated. The second set of experiments involved the transfer of spleen cells from tolerized or from naturally tolerant mice into normal mice which were then primed with allo-F, as well as the co-transfer of tolerant and primed lymphocytes into normal mice, to test whether tolerant lymphocytes present suppressor cells. The results indicate that the immune response against allo-F antigen is normally kept in a low level by a suppressive mechanism, and that F-specific suppressor T cells are absent from tolerant mice.Abbreviations used in this paper ATx adult thymectomy - BSS buffered salt solution - CFA Freund's complete adjuvant - CY cyclophosphamide - F.1 type-1 F antigen - F.2 type-2 F antigen - PBS phosphate-buffered saline - RIA radioimmunoassay - Th T helper cell - Ts T suppressor cell     

Experimental models of myasthenia gravis: lessons in autoimmunity and progress toward better forms of treatment

The nicotinic acetylcholine receptor (AChR) is a large membrane protein found in muscle cells. It is involved in the transformation of acetylcholine packets into a membrane depolarization, which thereby leads to a muscle twitch. This large, complex molecule is the target of the autoimmune attack in myasthenia gravis, and much has been learned in the past decade about myasthenia by the induction of autoimmunity to AChR in experimental animals. Experimental autoimmune myasthenia gravis (EAMG) has been produced in a variety of animals by immunization with AChR or AChR-like material, or by the passive transfer of anti-AChR antibodies or lymphocytes from afflicted animals into normal animals. EAMG is a remarkably faithful model of human myasthenia and has provided much information about how the immune response to AChR progresses and how weakness and damage to the neuromuscular junction ensure. EAMG has also allowed the development of a number of revolutionary forms of treatment in which only the abnormal response to AChR is restrained, and other necessary immune functions are left intact. These advances in treatment are not far from being tested in human myasthenia gravis. The experience gained in applying these concepts in EAMG and human myasthenia will be helpful in developing similar forms of treatment for other autoimmune diseases.     

Specific immunotherapy of experimental myasthenia gravis in vitro: the "guided missile" strategy

We describe a strategy for specific immunotherapy of myasthenia gravis (MG) based on genetic engineering of antigen presenting cells (APCs) to present the autoantigen acetylcholine receptor (AChR) and express the "warhead" Fas ligand (FasL). For transduction of APCs we prepared recombinant attenuated vaccinia virus vectors carrying the following three gene constructs: (i) AChR fused to LAMP1 to present AChR and target AChR-specific T cells; (ii) FasL to eliminate the targeted T cells; and (iii) truncated FADD to protect APCs from self-destruction by FasL. The engineered APCs effectively expressed the genes of interest and killed AChR-specific T cells in culture by the Fas/FasL pathway. T cells specific for an unrelated antigen were spared. Our in vitro demonstration that engineered APCs target and kill antigen-specific T cells represents a promising novel strategy for specific immunotherapy of MG and other autoimmune diseases.     

Fas/Fas ligand pathway, apoptosis, and clonal anergy involved in systemic acetylcholine receptor T cell epitope tolerance

The cellular mechanisms of high dose systemic acetylcholine receptor (AChR) T cell epitope, alpha 146--162 peptide-induced tolerance in experimental myasthenia gravis were examined. CD4 cells are the prime target for alpha 146--162 peptide-induced tolerance. The expression of CD69, Fas, and B7.2 molecules on AChR-immune lymphocytes was enhanced within 4--12 h after tolerance induction. A high dose of alpha 146--162 peptide in IFA failed to suppress T cell proliferation and/or clinical myasthenia gravis in lpr and gld mice deficient in Fas and Fas ligand, respectively. A high dose of alpha 146--162 peptide in IFA in AChR-immunized mice induced apoptosis of BV6 cells. Further, reconstitution of IL-2 in vitro-recovered alpha 146--162 peptide tolerized T cell proliferation, IFN-gamma, and IL-10 production. The findings implicate the possible role of Fas-/Fas ligand-mediated apoptosis and the resulting clonal anergy as the mechanisms of high dose AChR alpha 146--162 peptide-induced tolerance on CD4 cells.     

Resistance to experimental autoimmune myasthenia gravis in genetically inbred rats. Association with decreased amounts of in situ acetylcholine receptor-antibody complexes

Genetically related susceptibility for experimental autoimmune myasthenia gravis was investigated in nine inbred strains of rats immunized with heterologous acetylcholine (AChR) from Torpedo californica. Wistar Munich and Fischer strain animals consistently developed severe, fatal disease associated with impaired neuromuscular transmission and increased sensitivity to low doses of curare. A lower incidence of disease was induced in Wistar Kyoto, ACI, Brown Norway, Buffalo, and Lewis strain animals. In contrast, Wistar Furth and Copenhagen strain animals were resistant to experimental autoimmune myasthenia gravis, electrophysiologic responses were normal, and animals were insensitive to curare. All strains of animals manifested equivalent amounts of serum antibody to AChR and total muscle AChR was reduced to the same extent in both resistant and susceptible animals. In contrast, the amount of antibody-bound AChR was greater in susceptible Wistar Munich animals than the amount observed in resistant Wistar Furth animals. These data suggest that impaired neurotransmission is correlated with the extent of antibody binding to the AChR. The discordance in the amount of antibody bound to the AChR of resistant and susceptible animals may result from heritable differences in antibody properties. Cross-breeding experiments with Wistar Munich and Wistar Furth animals show that resistance for development of experimental autoimmune myasthenia gravis is recessive and indicate that disease susceptibility is linked to one or two genetic loci.     

Control of the autoimmune response by type 2 nitric oxide synthase

Immune defense against pathogens often requires NO, synthesized by type 2 NO synthase (NOS2). To discern whether this axis could participate in an autoimmune response, we immunized NOS2-deficient mice with the autoantigen acetylcholine receptor, inducing muscle weakness characteristic of myasthenia gravis, a T cell-dependent Ab-mediated autoimmune disease. We found that the acetylcholine receptor-immunized NOS2-deficient mice developed an exacerbated form of myasthenia gravis, and demonstrated that NOS2 expression limits autoreactive T cell determinant spreading and diversification of the autoantibody repertoire, a process driven by macrophages. Thus, NOS2/NO is important for silencing autoreactive T cells and may restrict bystander autoimmune reactions following the innate immune response.     

The nature of the interaction between suppressor and helper T cells in the response to LDHB

Purified Lyt-1+2+ T cells were depleted of alloreactive cells by BUdR and light treatment, and then were primed in vitro against LDHB presented on allogeneic APC. Such cells could be restimulated by LDHB on the same allogeneic APC, but not by LDHB on APC syngeneic with the T cells. The restimulated T cells suppressed the proliferative response of Lyt-1+2- T cells primed and restimulated by the same antigen. The suppression, which was antigen specific, occurred after a 6-hr co-culture of the suppressor (Tse) and proliferating helper (Th) cells. The successful interaction (as measured by suppression) between allogeneic Th and Tse cells was found to be determined by the restriction specificity but not the MHC haplotype of Th cells, and the MHC haplotype but not the restriction specificity of Tse cells. Thus, suppression occurred only when the Tse cells carried genes controlling the MHC molecules that served as restriction elements for antigen recognition by the Th cells. No evidence could be obtained for the participation of APC in the Tse-Th interaction. The data suggest the interaction is based on the recognition by the Th cell of the antigen presented in the context of MHC molecules controlled by the Tse cell.     

Myasthenia Gravis: Analysis of Serum Autoantibody Reactivities to 1827 Potential Human Autoantigens by Protein Macroarrays

Background

Myasthenia gravis is a disorder of neuromuscular transmission associated with autoantibodies against the nicotinic acetylcholine receptor. We have previously developed a customized protein macroarray comprising 1827 potential human autoantigens, which permitted to discriminate sera of patients with different cancers from sera of healthy controls, but has not yet been evaluated in antibody-mediated autoimmune diseases.

Objective

To determine whether autoantibody signatures obtained by protein macroarray separate sera of patients with myasthenia gravis from healthy controls.

Methods

Sera of patients with acetylcholine receptor antibody-positive myasthenia gravis (n = 25) and healthy controls (n = 32) were analyzed by protein macroarrays comprising 1827 peptide clones.

Results

Autoantibody signatures did not separate patients with myasthenia gravis from controls with sufficient sensitivity, specificity, and accuracy. Intensity values of one antigen (poly A binding protein cytoplasmic 1, p = 0.0045) were higher in patients with myasthenia gravis, but the relevance of this and two further antigens, 40S ribosomal protein S13 (20.8% vs. 0%, p = 0.011) and proteasome subunit alpha type 1 (25% vs. 3.1%, p = 0.035), which were detected more frequently by myasthenia gravis than by control sera, currently remains uncertain.

Conclusion

Seroreactivity profiles of patients with myasthenia gravis detected by a customized protein macroarray did not allow discrimination from healthy controls, compatible with the notion that the autoantibody response in myasthenia gravis is highly focussed against the acetylcholine receptor.     

Cooperation of invariant NKT cells and CD4+CD25+ T regulatory cells in the prevention of autoimmune myasthenia

CD1d-restricted NKT cells and CD4+CD25+ regulatory T (Treg) cells are thymus-derived subsets of regulatory T cells that have an important role in the maintenance of self-tolerance. Whether NKT cells and Treg cells cooperate functionally in the regulation of autoimmunity is not known. We have explored this possibility in experimental autoimmune myasthenia gravis (EAMG), an animal model of human myasthenia gravis, induced by immunization of C57BL/6 mice with the autoantigen acetylcholine receptor. We have demonstrated that activation of NKT cells by a synthetic glycolipid agonist of NKT cells, alpha-galactosylceramide (alpha-GalCer), inhibits the development of EAMG. alpha-GalCer administration in EAMG mice increased the size of the Treg cell compartment, and augmented the expression of foxp3 and the potency of CD4+CD25+ cells to inhibit proliferation of autoreactive T cells. Furthermore, alpha-GalCer promoted NKT cells to transcribe the IL-2 gene and produce IL-2 protein. Depletion of CD25+ cells or neutralization of IL-2 reduced the therapeutic effect of alpha-GalCer in this model. Thus, alpha-GalCer-activated NKT cells can induce expansion of CD4+CD25+ Treg cells, which in turn mediate the therapeutic effects of alpha-GalCer in EAMG. Induced cooperation of NKT cells and Treg cells may serve as a superior strategy to treat autoimmune disease.     

Specific vaccines against autoimmune diseases

Copolymer 1 (Cop 1, Copaxone) is a synthetic amino acid copolymer effective in suppression of experimental allergic encephalomyelitis (EAE). The suppressive effect of Cop 1 in EAE is not restricted to a certain species, disease type or encephalitogen used for EAE induction. In phase II and III clinical trials, Cop 1 was found to slow the progression of disability and reduce the relapse rate in exacerbating-remitting multiple sclerosis (MS) patients. In vivo and in vitro studies suggest that the mechanism for Cop 1 activity in EAE and MS involves, as an initial step, the binding of Cop 1 to MHC class II molecules. This binding results in competition with myelin antigens for T-cell activation, both at the MHC and T-cell receptor levels and in induction of specific suppressor cells of the Th2 type. As an antigen-specific intervention, Cop 1 has the advantage of reduced probability for long-term damage to the immune system, and is thus a safe and effective novel therapeutic approach to MS. It also serves to illustrate the new concept of a drug/vaccine specific for a single autoimmune disease. Indeed, we have used a similar approach for myasthenia gravis. Myasthenia gravis (MG) and its experimental animal model, experimental autoimmune MG (EAMG), are immune disorders characterized by circulating antibodies and lymphocyte autoreactivity to nicotinic acetylcholine receptor (AChR). We utilized peptides representing different sequences of the human acetylcholine receptor alpha-subunit to study the role of T cells in the initiation, development and immunomodulation of myasthenia gravis. Here we summarize our studies over the last decade on T cells specific to 'myasthenogenic' epitopes of the alpha-subunit of the human acetylcholine receptor and their relevance for myasthenia gravis.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

Enterically induced immunologic tolerance. I. Induction of suppressor T lymphoyctes by intragastric administration of soluble proteins.

Specific immune unresponsiveness was induced in inbred mice (BDF1) by the administration of soluble ovalbumin (OVA) by gastric intubation. Anti-hapten (DNP) responses likewise were specifically diminished when animals were fed autologous carrier (OVA or keyhole limpet hemocyanin). Adoptive transfer of spleen cells demonstrated that the tolerant state could be maintained in irradiated recipient mice, and specific anergy could be transferred to normal recipient animals. Adoptive suppression was mediated by T lymphocytes, as demonstrated by nylon wool fractionation and susceptibility of the cells to anti-Thy 1.2 and complement. Transferred B cells had neither suppressive nor augmentative effects. Enteric administration of OVA also specifically diminished antigen-induced DNA synthesis of primed lymph node T cells, although suppressor cells were not identified in the lymph nodes per se.