IL-12 is involved in the induction of experimental autoimmune myasthenia gravis, an antibody-mediated disease

IL-12 has been shown to be involved in the pathogenesis of Th1-mediated autoimmune diseases, but its role in antibody-mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL-12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell-dependent, autoantibody-mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL-12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR-specific IFN-gamma production and increased anti-ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL-12-treated mice, and reduced numbers of AChR. By contrast, when IL-12-deficient mice were immunized with ToAChR, ToAChR-specific Th1 cells and anti-ToAChR IgG2a serum antibodies were reduced compared to ToAChR-primed normal B6 mice, and the IL-12-deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL-12 in the induction of an antibody-mediated autoimmune disease, suggest that Th1-dependent complement-fixing IgG2a anti-AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti-AChR levels and clinical disease seen in many earlier studies.     

The Th2 cytokine IL-4 is not required for the progression of antibody-dependent autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG), a disorder of the neuromuscular junction, is mediated by autoantibodies against muscle nicotinic acetylcholine receptor (AChR). The roles of IFN-gamma (Th1) and IL-4 (Th2) cytokines in the initiation and progression of this disease are not fully understood. Recently, we have demonstrated that IFN-gamma is necessary for the initiation of tAChR-induced EAMG in mice. However, the role of IL-4 in the progression of clinical EAMG remained undetermined. In this study we have addressed the contribution of IL-4 in the disease progression in IL-4(-/-) C57BL/6j mice whose IL-4 gene has been disrupted. Following immunization with Torpedo (t) AChR, the IL-4(-/-) mice readily developed signs of muscle weakness and succumbed to clinical EAMG with kinetics similar to the susceptibility of IL-4(+/+) mice. The tAChR-primed lymph node cells from IL-4(-/-) mice vigorously proliferated to tAChR and to its dominant alpha146-162 sequence associated with disease pathogenesis. However, these T cells secreted higher levels of IFN-gamma and IL-2, suggesting the development of a Th1 default pathway in these mice. Nevertheless, the IL-4 mutation had no effect on the recruitment of CD4+ Vbeta6+ T cells specific to the dominant tAChR alpha146-162 sequence in vivo. Immune sera from IL-4(-/-) mice showed a dramatic increase in mouse AChR-specific IgG2a levels followed by a concomitant decrease in IgG1 levels, but these mice did not exhibit an accelerated disease. In conclusion, we have demonstrated for the first time that IL-4 is not required either for the generation of a pathogenic anti-AChR humoral immune response or for progression of clinical EAMG in mice.     

Pathogenic autoimmunity to affinity-purified mouse acetylcholine receptor induced without adjuvant in BALB/c mice

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated disorders in which anti-acetylcholine receptor (anti-AChR) antibodies cause loss of muscle AChR and subsequent weakness. Many species are susceptible to induction of EAMG with purified xenogeneic AChR in adjuvant, but injection of Torpedo AChR without adjuvants can also induce evidence of EAMG. To see whether pathogenic autoimmunity could be induced in mice by isolated mouse AChR we injected BALB/c mice with several doses (1 pmole; about 0.1 microgram) of affinity-purified AChR (from the BC3H1 cell line but thought to be identical with denervated mouse muscle) intraperitoneally, without adjuvant, over a period of 10-22 weeks. Some of the mice became ill and died. High levels of serum anti-mouse AChR, directed mainly towards the main immunogenic region, were found and, in the survivors, correlated with loss of muscle AChR. Thus BALB/c mice can mount an autoimmune response to minute amounts of mouse AChR, without the use of adjuvants, and this response is very similar to that found in MG. This novel finding has implications regarding the etiology of the human disease.     

Pharmacokinetic mechanisms for obtaining high renal coelimination of phencyclidine and a monoclonal antiphencyclidine antigen-binding fragment of immunoglobulin G in the rat

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.     

Tolerance to a dominant T cell epitope in the acetylcholine receptor molecule induces epitope spread and suppresses murine myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. T cells reactive to a dominant peptide alpha 146-162 of acetylcholine receptor (AChR) alpha subunit participate in murine MG pathogenesis. To suppress the autoimmune response to AChR, a high dose of alpha146-162 peptide in IFA was administered parenterally as a tolerogen, after the development of a primary T cell immune response to AChR. This form of AChR T cell peptide tolerance suppressed the in vitro T cell proliferative response to AChR and its dominant alpha146-162 and subdominant alpha182-198 peptides through epitope spread. Administration of alpha146-162 peptide in IFA after the primary immune response to AChR also significantly suppressed the serum anti-AChR Ab of the IgG2b isotype and clinical incidence of MG in C57BL/6 mice. Furthermore, the production of IFN-gamma, IL-2, and IL-10 cytokines by AChR, alpha146-162, and alpha182-198 peptide-reactive cells was suppressed by alpha146-162 peptide tolerance, and the epitope spread observed could be attributed to the reduction in the above cytokine production. Therefore, AChR T cell-dominant peptide tolerance could be adapted in the Ag-specific therapy of MG.     

Polymorphism at the HLA-DQ locus determines susceptibility to experimental autoimmune myasthenia gravis

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-A beta0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-A beta0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.     

Induction of tolerance in experimental autoimmune myasthenia gravis with solubilized MHC class II:acetylcholine receptor peptide complexes

Stimulation of T lymphocytes through the T cell receptor in the absence of costimulatory signal(s) induces a state of unresponsiveness to subsequent antigen presentation. We have employed solubilized complexes consisting of rat class II MHC molecules containing an immunodominant peptide of the acetylcholine receptor (AChR alpha 100-116) to induce unresponsiveness in the autoreactive T lymphocytes mediating an animal model of myasthenia gravis. In vitro incubation of rat T cell lines specific for peptide AChR alpha 100-116 with solubilized complexes of MHC II and AChR alpha 100-116 (MHC II:AChR alpha 100-116) rendered the T cells unresponsive to subsequent stimulation by antigen presenting cells and the peptide. T cell lines with a broader specificity to the entire AChR protein pentamer had an 81% reduction in proliferation to AChR following a preincubation with solubilized MHC II:AChR alpha 100-116. Treatment with the solubilized MHC II:AChR alpha 100-116 induced phosphatidylinositol 4,5-bisphosphate hydrolysis, an early signalling event associated with binding to the TCR. Rats primed with AChR and injected intravenously with MHC II:AChR alpha 100-116 had reduced in vitro T cell proliferation to the AChR alpha 100-116 peptide and to whole AChR. Solubilized MHC II:AChR alpha 100-116 injected i.v. into rats exhibiting serological clinical symptoms of experimental autoimmune myasthenia gravis (EAMG) prevented death in 67% of the treated animals, compared to a 0-20% survival rate in all other control groups. These results demonstrate that solubilized MHC II complexed with an immunodominant autoantigenic peptide is tolerogenic and improves the survival rate of rats with EAMG, suggesting the basis for an antigen-specific therapy in autoimmune diseases such as MG.     

Autoimmune responses against acetylcholine receptor: T and B cell collaboration and manipulation by synthetic peptides

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are induced by antibodies (Abs) against self acetylcholine receptor (AChR). We have mapped the T and B cell epitopes on AChR alpha subunit in human MG and in EAMG-susceptible (C57BL/6, B6) and nonsusceptible mouse strains. A T-cell epitope within residues alpha 146-162 (P14) of Torpedo californica (t)AChR plays an important role in EAMG pathogenesis of the auto Ab-induced disease. P14-specific T cell (P14Th) lines from tAChR-primed B6 mice activated, in vivo and in vitro, tAChR-primed B cells that secreted anti-AChR Abs directed against four other regions on the tAChR alpha-chain, but not against P14 itself. P14Th cells are pathogenic because they help B cells that make Abs against a conserved tAChR region (t alpha 122-150) involved in ACh binding. These Abs cross-react with region alpha 122-150 of mouse (m)AChR, thereby disrupting its normal physiological function. Thus, a T cell epitope not recognized by Abs plays an active role in B cell responses against other epitopes on the protein. We have found that in B6, the MHC region 62-76 of I-A beta(b) is involved in the presentation of P14 to T cells. Anti-peptide Abs, prepared in BALB/c, were found to inhibit in vitro the proliferation of P14-specific T-cells. Furthermore, this MHC peptide elicited Abs in B6 mice and we are investigating whether immunization of B6 with this peptide, before priming with tAChR, would suppress in vivo the T-cell response to the epitope in P14. Thus, these preliminary results would suggest that immunization with the MHC peptide might be employed for control of the autoimmune disease.     

V beta-specific immunotoxin selectively kills acetylcholine receptor-reactive T lymphocytes from mice with experimental autoimmune myasthenia gravis

Immunization of C57BL/6 mice with purified acetylcholine receptor (AChR) is known to induce a T cell-dependent antibody response that results in experimental autoimmune myasthenia gravis (EAMG). Since past observations link V beta 6+ T cells with a prominent AChR epitope specificity, a V beta 6-specific immunotoxin (VIT6) was tested in vitro for its ability to selectively kill monoclonal and polyclonal T cells that demonstrate reactivity against AChR. Results described below clearly demonstrate the ability to selectively kill AChR-reactive T cells based on their expression of a particular V beta-associated antigen receptor.     

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell costimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28-/-, CD40L-/-). Compared to wild-type mice, the CD28-/- mice became less susceptible, and CD40L-/- mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28-/- mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28-/- mice. In the CD40L-/- mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28-/- mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.     

Two high-affinity monoclonal IgG2a antibodies with differing thermodynamic stability demonstrate distinct antigen-induced changes in protein A-binding affinity

Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis (EAMG) results in increased degradation of acetylcholine receptor (AChR) and increased synthesis of AChR alpha-subunit mRNA. Passive transfer of anti-Main Immunogenic Region (MIR) mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChR subunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR alpha-subunit mRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats, in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediated degradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats.     

B-cell activation in vitro by helper T cells specific to region alpha 146-162 of Torpedo californica nicotinic acetylcholine receptor

We have previously mapped the T and B cell epitopes on the alpha-subunit of acetylcholine receptor (AChR) in human myasthenia gravis (MG) and in experimental autoimmune MG-susceptible (C57BL/6 (B6)) and nonsusceptible mouse strains. In addition to regions recognized by both T and B cells, the AChR alpha-subunit has regions that are recognized solely by T cells. An exclusive T cell epitope within residues alpha 146-162 of Torpedo californica (t), tAChR, plays an important role in experimental autoimmune MG pathogenesis in B6 mice. To study its function, we established, from tAChR-primed B6 mice, two t alpha 146-162-specific T cell lines (P14Th) which comprised Th2-type cells because they secreted IL-4 but not IL-2. P14Th did not recognize the corresponding region on mouse (m) AChR (m alpha 146-162). They caused in vitro differentiation of tAChR-primed B cells into plasma cells that secreted anti-AChR Abs directed, in decreasing order, against the following tAChR alpha regions: t alpha 122-138 > t alpha 134-150 > t alpha 45-60 > t alpha 170-186 > t alpha 56-71. Little or no Ab response could be detected against peptides t alpha 182-198 or t alpha 146-162 itself. The major enhancement was in the Abs against region t alpha 122-150 (spanning the t alpha 122-138/t alpha 134-150 overlap) that is involved in ACh binding. These Abs cross-reacted completely with m alpha 122-150, the corresponding region on mAChR. Therefore, t alpha 146-162-specific T cells, although unable to recognize m alpha 146-162, are nevertheless pathogenic because they help B cells responding to a tAChR region that is conserved in mAChR and involved in ACh binding. These Abs cross-react with the corresponding effector-binding region of mAChR, thereby disrupting the normal physiologic function of the mouse receptor.     

Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis

Immunization of C57BL/6 mice with AChR provokes symptoms similar to those seen in the disease myasthenia gravis. To elucidate the structural requirements for T cell recognition of AChR and to identify TcR features which might provide targets for immunotherapy, a panel of T cell hybridomas was generated after immunization of mice with the immunodominant peptide of the AChR alpha chain. The TcR genes expressed by these hybridomas were sequenced. TcR-V beta 6 was preferentially employed, but other V beta genes were also observed. A conserved acidic residue was present in all CDR3 regions, regardless of the V beta. The TcR-V alpha repertoire was somewhat skewed with three V alpha families accounting for 82% of the sequences. The utilization of multiple T cell receptor V beta genes may contribute to the inability to inhibit EAMG by elimination of V beta 6+ T cells.     

Reactivity of T cells from seronegative patients with myasthenia gravis to T cell epitopes of the human acetylcholine receptor

Seronegative (SN) patients with myasthenia gravis (MG) have clinical and electrophysiologic features similar to those of seropositive (SP) patients, and they respond to the same therapeutic measures. However, because SN patients lack detectable (by standard radioimmunoassays) serum antibodies to acetylcholine receptor (AChR), which are considered to have a crucial role in MG, the pathophysiologic basis for the disease is not clear. We therefore compared the ability of peripheral blood lymphocytes (PBL) of SN patients (11) and SP patients (39) to respond to myasthenogenic T cell epitopes of human AChR. We tested two aspects that relate to T-cell immunity: 1) T cell responses to myasthenogenic peptides by proliferation and IL-2 production, and 2) the ability of antigen-presenting cells to bind these T-cell epitopes. T cells of SN patients did not differ from those of SP patients in their ability to respond and to bind the two human AChR-derived myasthenogenic peptides. This supports the belief that most SN patients indeed suffer from an autoimmune disease directed against the AChR. The presence of T-cell immunity in the absence of antibodies may emphasize the importance of AChR-specific T cells in MG.     

Myasthenia gravis: recognition of a human autoantigen at the molecular level

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.     

Diverse Fab specific for acetylcholine receptor epitopes from a myasthenia gravis thymus combinatorial library

The muscle weakness in myasthenia gravis (MG) is caused by heterogeneous high-affinity IgG autoantibodies to the nicotinic acetylcholine receptor (AChR), a complex ion channel glycoprotein. These antibodies are clearly responsible for reducing AChR numbers at the neuromuscular junction in myasthenia; however, the origins, diversity, specificity and pathogenicity of individual antibodies have not yet been established. We have cloned and characterized four different AChR-specific Fab from an MG patient's thymus by screening an IgG1/kappa gene combinatorial lambda phage library with soluble human AChR labeled with [125I] alpha-bungarotoxin. Unlike most previously cloned human antibodies, all four Fab immunoprecipitated soluble human muscle AChR. Two Fab strongly inhibited binding of mAb to the main immunogenic region on the alpha subunits and one Fab bound to an epitope on the fetal-specific gamma subunit. In sensitivity and fine specificity, these Fab resembled the anti-AChR antibodies found in many MG patients, including the donor. The closest germline counterparts for their heavy chains were in VH families 1, 3 and 4; however, there were many differences consistent with an antigen-driven response of diverse B cell clones. The combinatorial approach holds promise for further analysis of human autoantibodies.     

VH gene family utilization of anti-acetylcholine receptor antibodies in experimental autoimmune myasthenia gravis

The immunoglobulin heavy chain (VH) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using VH gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by VH genes from at least six different families. The Vgam3.8 family was overrepresented in alpha-bungarotoxin blocking mAbs. Expression of cross-reactive idiotypes by anti-AChR mAbs was irrespective of the VH gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant VH gene family usage of anti-AChR mAbs.     

Prevention and treatment of experimental autoimmune myasthenia gravis with anti-rabbit thymocyte serum and gammaglobulin in rabbits

Twenty-two rabbits received Torpedo californica acetylcholine receptor (AChR) subcutaneously. Six rabbits were treated with 0.5 ml/kg/day of anti-rabbit thymocyte serum (ARTS), and four were treated with 5.0 mg/kg/day of anti-rabbit thymocyte gammaglobulin (ARTG) subcutaneously beginning concomitantly 1, 2 and 3 weeks after the initial AChR immunization. All 12 control animals died of experimental autoimmune myasthenia gravis (EAMG) within 52 days. None of the 10 treated rabbits developed clinical EAMG. ARTS- and ARTG-treated animals had significantly lower anti-AChR antibody titers than control animals at 3 weeks (pre-AChR booster, p < 0.01). At 6 weeks (post-AChR booster), only ARTS-treated animals had significantly lower titers than controls (p < 0.01). ARTS-treated animals developed sterile abscesses at injection sites, which were minimal in the ARTG-treated group. ARTS and ARTG prevent EAMG.     

A pathogenetic role for the thymoma in myasthenia gravis. Autosensitization of IL-4- producing T cell clones recognizing extracellular acetylcholine receptor epitopes presented by minority class II isotypes

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.