Myasthenia gravis: past, present, and future.

Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure of neuromuscular transmission, which results from the binding of autoantibodies to proteins involved in signaling at the neuromuscular junction (NMJ). These proteins include the nicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved in AChR clustering. Much is known about the mechanisms that maintain self tolerance and modulate anti-AChR Ab synthesis, AChR clustering, and AChR function as well as those that cause neuromuscular transmission failure upon Ab binding. This insight has led to the development of improved diagnostic methods and to the design of specific immunosuppressive or immunomodulatory treatments.

Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains.

Development of antibodies (Ab) that either block the function of coagulation factor VIII (FVIII) (inhibitors) or clear it from circulation, seriously complicate the treatment of haemophilia A patients with FVIII products. Autoantibodies which develop in subjects without congenital FVIII defects, cause acquired haemophilia, a rare but life-threatening coagulopathy. Identification of the FVIII epitopes to which inhibitor Abs bind will help understanding the mechanisms of inhibitor activity, and perhaps development of new therapies. Here, we examined the FVIII peptide sequence regions recognised by anti-FVIII Ab in the plasma of six congenital and one acquired haemophilia patients with high inhibitor titers (24.4-2000 BU/ml). We used indirect ELISA and overlapping synthetic peptides, 20 residues long, spanning the sequence of the A and C FVIII domains. None of the plasma samples reacted with A1, A3 or C1 domain peptides. Six plasmas reacted with A2 and/or C2 peptides. Peptides spanning residues A2-521-690 and C2-2251-2332 were recognised most frequently and strongly. They include residues that contribute to the binding sites for activated factor IX and phosphatidyl serine/von Willebrand factor. These results suggest that anti-FVIII Abs share a pattern of antigen specificity in our panel of patients, and that exposed regions of the FVIII molecule that form functionally important binding sites elicit an intense Ab response.

CD4+ T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

Although human and experimental acquired myasthenia gravis (MG) are prototypic antibody (Ab)-mediated autoimmune diseases, synthesis of the pathogenic anti-acetylcholine receptor (AChR) Abs, which are high affinity IgG, requires intervention of CD4(+) T helper cells and their cytokines. Moreover, cytokine signaling is crucial for development, modulation, and downregulation of immune responses, and therefore influences the initiation and evolution of the anti-AChR response in acquired MG. Cytokines are involved in the growth and differentiation of CD4(+) T cells, and are secreted by activated CD4(+) T cells as effectors of their functions: differentiated CD4(+) T cells are classified into subtypes based on the cytokines they synthesize and secrete. Because cytokines are synthesized by and act on a variety of cells and because they may influence the synthesis and/or the activity of other cytokines, the effects of their signaling, in both normal and autoimmune responses, are complex and sometimes even contrasting, depending on the circumstances when the cytokine action took place. In this chapter, we will review studies on the effects on the development of acquired MG symptoms of several cytokines secreted by activated CD4(+) T cells or influencing the activation of particular CD4(+) T cell subsets.

Markedly enhanced susceptibility to experimental autoimmune myasthenia gravis in the absence of decay-accelerating factor protection.

Myasthenia gravis (MG) is an autoimmune neuromuscular transmission disorder characterized by loss of acetylcholine receptors (AChR's) due primarily to the production of anti-AChR autoantibodies. In this study we investigated whether the presence of decay-accelerating factor (DAF or CD55), an intrinsic complement regulator, protects against the development of disease. Experimental autoimmune MG was induced in Daf1(-/-) mice (devoid of neuromuscular DAF protein) and their Daf1(+/+) littermates by injection of rat anti-AChR mAb McAb-3. After twenty-four hours, grip strength assessment revealed that Daf1(-/-) mice exhibited hold times of less than 30 seconds, compared with more than 8 minutes for the Daf1(+/+) controls. The weakness was reversed by edrophonium, consistent with a myasthenic disorder. Immunohistochemistry revealed greatly augmented C3b deposition localized at postsynaptic junctions, and radioimmunoassays showed more profound reductions in AChR levels. Electron microscopy demonstrated markedly greater junctional damage in the Daf1(-/-) mice compared with the Daf1(+/+) littermates. Control studies showed equivalent levels of other cell surface regulators, i.e., Crry and CD59. The results demonstrate that mice that lack DAF are markedly more susceptible to anti-AChR-induced MG, which simulates the primary mechanism in the human disease, and strongly suggest that in disease flares complement inhibitors might have therapeutic value.

Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients.

Haemophilia A patients treated with human factor VIII (fVIII) may develop antibody (Ab) inhibitors to fVIII. FVIII-specific CD4(+) T cells are common in haemophilia A patients, but also in healthy subjects who do not have a sustained anti-fVIII Ab response. Here, we examined the fVIII-induced IFN gamma-, IL-4- and TGF-beta1-producing CD4(+) T blasts by culturing peripheral blood mononuclear cells (PBMC) from controls and patients with recombinant fVIII. FVIII exposure significantly increased IFN gamma- and IL-4-, but not TGF-beta1-producing CD4(+) T blasts in patients with inhibitors. Patients without inhibitors had fVIII-induced IFN gamma- and TGF-beta1-, but not IL-4-producing CD4(+) T blasts. Controls did not have IL-4-producing CD4(+) T blasts. However, controls whose PMBC proliferated in response to fVIII had fVIII-induced CD4(+) T blasts that produced IFN-gamma, the number of which correlated with the intensity of the proliferative response to fVIII of their PMBC, whereas controls whose PMBC did not proliferate to fVIII had predominantly fVIII-induced CD4(+) T blasts that produced TGF-beta1. The presence in controls and patients without inhibitors of fVIII-induced IFN-gamma-producing CD4(+) T cells, but not IL-4-producing CD4(+) T cells, which are abundant in inhibitor patients, suggests a role of Th1 cells in initiating the immune response to fVIII, and of Th2 cells in the development of strong inhibitor production. The polarized high ratios of Th3/Th1 and Th3/Th2 in controls and patients without inhibitors suggest that a preponderance ofTh3 cells in the response to fVIII may help to maintain tolerance to fVIII.

CD4+ T and B cells cooperate in the immunoregulation of Experimental Autoimmune Myasthenia Gravis.

C57Bl6 mice (B6 mice) immunized with Torpedo acetylcholine receptor (TAChR) in Freund's adjuvants (FA) develop Experimental Autoimmune Myasthenia Gravis (EAMG). In mouse EAMG Th2 cytokines may be protective. Aluminum hydroxide (Alum) was used to immunize B6 mice to the TAChR and prime CD4+ T and B cells secreting Th2 cytokines. Mice immunized with TAChR/Alum developed anti-AChR CD4+ T cells response, but minimal antibody levels and symptoms. TAChR/Alum treatments prior immunization with TAChR/FA protected mice from EAMG. Cell transfer experiments demonstrated that B and CD4+ T cells mediated the protective effect by causing intense reduction of complement-fixing anti-TAChR IgG subclasses.

CD4(+) T cells from healthy subjects and colon cancer patients recognize a carcinoembryonic antigen-specific immunodominant epitope.

The carcinoembryonic antigen (CEA) is an attractive target for immunotherapeutic purposes because of its expression profile, its role in tumor progression, and its immunogenicity. However, CEA belongs to the CD66 immunoglobulin super-gene family that comprises highly homologous molecules expressed on leukocytes, making CEA a potential autoantigen expressed on hematopoietic cells. We used a MHC class II epitope prediction algorithm (TEPITOPE) to select 11 sequence segments of CEA that could form promiscuous CD4(+) T-cell epitopes and used synthetic peptides corresponding to the predicted sequences to propagate in vitro CD4(+) T cells from healthy donors and colon cancer patients. CD4(+) T cells from all subjects strongly recognized the sequence segment (LWWVNNQSLPVSP), repeated at residues 177-189 and 355-367. Importantly, we demonstrated that this highly immunodominant region contains a naturally processed epitope(s). Cross-recognition experiments with peptide analogues present on the CD66 homologous proteins showed that CEA(177-189/355-367)-specific CD4(+) T cells did not recognize the analogues, demonstrating that recognition of the immunodominant epitope is CEA specific. These data suggest that the repertoire of CEA(177-189/355-367)-specific CD4(+) T cells might have been shaped by a selective process to exclude CD4(+) T cells specific for CD66 homologues expressed on leukocyte, while preserving the CEA-specific repertoire. The features of strong immunogenicity and immunodominance in the absence of potential induction of autoimmunity make the identified CEA epitope of particular interest for the development of antitumor vaccines.

Adoptive protection from experimental myasthenia gravis with T cells from mice treated nasally with acetylcholine receptor epitopes.

Nasal administration of synthetic CD4(+) epitopes of the acetylcholine receptor (AChR) prevents experimental myasthenia gravis (EMG) in C57Bl/6 mice, but not in IL4-deficient C57Bl/6 (IL4(-/-)) mice. Here we verify that nasal tolerance requires IL4, by showing that CD4(+) cells from C57Bl/6 mice treated nasally with a pool of AChR CD4(+) epitopes protected IL4(-/-) mice from EMG and caused a reduced production of anti-AChR antibody. CD4(+) cells from C57Bl/6 mice treated with unrelated peptides or sham-treated did not induce protection. CD4(+) cells from C57Bl/6 mice treated with just one AChR peptide protected IL4(-/-) mice from EMG without affecting antibody synthesis.

Distribution of Th1- and Th2-induced anti-factor VIII IgG subclasses in congenital and acquired hemophilia patients.

Development of antibodies (Ab) that inhibit the procoagulant function of factor VIII (fVIII) seriously complicates the treatment of hemophilia A patients. It also causes acquired hemophilia, a rare yet serious autoimmune disease. The design of effective fVIII-specific tolerizing procedures will require lucidation of the role of the different CD4(+) T cell subsets that drive inhibitor synthesis. To examine the contribution of Th1 and Th2 cells in the anti-fVIII Ab response, we measured the concentration of Th1- and Th2-driven anti-fVIII IgG subclasses in 17 patients with severe hemophilia A and 18 patients with acquired hemophilia. We found that both congenital and acquired hemophilia patients had similar and comparable proportions of Th1- and Th2-induced anti-fVIII Ab, suggesting a more important role of Th1 cells in the immune response to fVIII than previously appreciated. The distribution of anti-fVIII IgG subclasses was stable for periods of up to six months. More intense anti-fVIII Ab responses and higher inhibitor titers correlated with a predominance of Th2-driven subclasses. In contrast, Th1-driven anti-fVIII Ab were predominant in patients who had low anti-fVIII Ab concentrations, even when this was the result of successful immune tolerance or immunosuppressive therapy, which had caused drastic reduction or disappearance of inhibitors. Thus, synthesis of Th2-driven inhibitors occurs when the anti-fVIII Ab response is intense, while Th1 cells may be involved in the long-term maintenance of anti-fVIII Ab synthesis.

Cryptic determinants and promiscuous sequences on human acetylcholine receptor: HLA-dependent dichotomy in T-cell function.

Experimental autoimmune myasthenia gravis can be induced in some strains of mice and rats by immunizing with acetylcholine receptor. Also, epidemiologic studies demonstrate an MHC linkage of myasthenia gravis in the man. In order to obtain direct experimental evidence for the influence of the genes of the MHC complex in the development of myasthenia gravis, we used mice transgenic to individual HLA molecules. We observed an increased susceptibility to the disease in HLA DQ8 transgenic mice compared to HLA DQ6 transgenic mice ( J. Immunol. 160:4169; 1998). These mice lacked endogenous mouse class II molecules. In the present study we mapped the cryptic and dominant sequences on the extra cellular region of human acetylcholine receptor. Although some epitopes (e.g., alpha11-30, alpha141-160, alpha171-190) were common between DQ8 and DQ6 transgenic mice, several others were disparately recognized. We also found a functional dichotomy in T cells from mice differing by one MHC molecule (HLA DQ8 or DQ6) when primed by sequences immunodominant in DQ8 and DQ6 tg mice. Differential disease manifestation in the two different HLA transgenic mice could be explained not only by differential recognition of peptides by these antigen presenting molecules, but also by the difference in the functional profile of T cells generated when primed by promiscuous sequence regions.

T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

Although the symptoms of myasthenia gravis (MG) and experimental MG (EAMG) are caused by autoantibodies, CD4(+) T cells specific for the target antigen, the nicotinic acetylcholine receptor, and the cytokines they secrete, have an important role in these diseases. CD4(+) T cells have a pathogenic role, by permitting and facilitating the synthesis of high-affinity anti-AChR antibodies. Th1 CD4(+) cells are especially important because they drive the synthesis of anti-AChR complement-fixing IgG subclasses. Binding of those antibodies to the muscle AChR at the neuromuscular junction will trigger the complement-mediated destruction of the postsynaptic membrane. Thus, IL-12, a crucial cytokine for differentiation of Th1 cells, is necessary for development of EAMG. Th2 cells secrete different cytokines, with different effects on the pathogenesis of EAMG. Among them, IL-10, which is a potent growth and differentiation factor for B cells, facilitates the development of EAMG. In contrast, IL-4 appears to be involved in the differentiation of AChR-specific regulatory CD4(+) T cells, which can prevent the development of EAMG and its progression to a self-maintaining, chronic autoimmune disease. Studies on the AChR-specific CD4(+) cells commonly present in the blood of MG patients support a crucial role of CD4(+) T cells in the development of MG. Circumstantial evidence supports a pathogenic role of IL-10 also in human MG. On the other hand, there is no direct or circumstantial evidence yet indicating a role of IL-4 in the modulatory or immunosuppressive circuits in MG.

C57BL/6 mice genetically deficient in IL-12/IL-23 and IFN-gamma are susceptible to experimental autoimmune myasthenia gravis, suggesting a pathogenic role of non-Th1 cells.

Immunization with Torpedo acetylcholine receptor (TAChR) induces experimental autoimmune myasthenia gravis (EAMG) in C57BL/6 (B6) mice. EAMG development needs IL-12, which drives differentiation of Th1 cells. The role of IFN-gamma, an important Th1 effector, is not clear and that of IL-17, a proinflammatory cytokine produced by Th17 cells, is unknown. In this study, we examined the effect of simultaneous absence of IL-12 and IFN-gamma on EAMG susceptibility, using null mutant B6 mice for the genes of both the IL-12/IL-23 p40 subunit and IFN-gamma (dKO mice). Wild-type (WT) B6 mice served as control for EAMG induction. All mice were immunized with TAChR in Freund's adjuvant. dKO mice developed weaker anti-TAChR CD4(+)T cells and Ab responses than WT mice. Yet, they developed EAMG symptoms, anti-mouse acetylcholine receptor (AChR) Ab, and CD4(+) T cell responses against mouse AChR sequences similar to those of WT mice. dKO and WT mice had similarly reduced AChR content in their muscles, and IgG and complement at the neuromuscular junction. Naive dKO mice had significantly fewer NK, NKT, and CD4(+)CD25(+)Foxp3(+) T regulatory (Treg) cells than naive WT mice. Treg cells from TAChR-immunized dKO mice had significantly less suppressive activity in vitro than Treg cells from TAChR-immunized WT mice. In contrast, TAChR-specific CD4(+) T cells from TAChR-immunized dKO and WT mice secreted comparable amounts of IL-17 after stimulation in vitro with TAChR. The susceptibility of dKO mice to EAMG may be due to reduced Treg function, in the presence of a normal function of pathogenic Th17 cells.

The susceptibility to experimental myasthenia gravis of STAT6-/- and STAT4-/- BALB/c mice suggests a pathogenic role of Th1 cells.

Autoantibodies to the muscle acetylcholine receptor (AChR) cause the symptoms of human and experimental myasthenia gravis (EMG). AChR-specific CD4+ T cells permit development of these diseases, but the role(s) of the Th1 and Th2 subsets is unclear. The STAT4 and STAT6 proteins, which mediate intracellular cytokine signaling, are important for differentiation of Th1 and Th2 cells, respectively. Wild-type (WT) BALB/c mice, which are prone to develop Th2 rather than Th1 responses to Ag, are resistant to EMG. We have examined the role of Th1 and Th2 cells in EMG using STAT4 (STAT4-/-)- or STAT6 (STAT6-/-)-deficient BALB/c mice. After AChR immunization, STAT6-/- mice were susceptible to EMG: they developed more serum anti-AChR Ab, and had more complement-fixing anti-AChR IgG2a and 2b and less IgG1 than WT or STAT4-/- mice. The susceptibility to EMG of STAT6-/- mice is most likely related to the Th1 cell-induced synthesis of anti-AChR Ab, which trigger complement-mediated destruction of the neuromuscular junction. CD4+ T cells of the STAT6-/- mice had proliferative responses to the AChR comparable to those of WT and STAT4-/- mice, and recognized similar AChR epitopes. STAT6-/- mice had abundant AChR-specific Th1 cells, which were nearly absent in WT and STAT4-/- mice. Spleen and lymph nodes from STAT6-/- mice contained cells that secreted IL-4 when cultured with AChR: these are most likely STAT6-independent cells, stimulated in a non-Ag-specific manner by the cytokines secreted by AChR-specific Th1 cells.

Absence of IL-4 facilitates the development of chronic autoimmune myasthenia gravis in C57BL/6 mice.

Myasthenia gravis (MG) is a T cell-dependent, Ab-mediated autoimmune disease. Ab against muscle acetylcholine receptor (AChR) cause the muscular weakness that characterizes MG and its animal model, experimental MG (EMG). EMG is induced in C57BL6 (B6) mice by three injections of Torpedo AChR (TAChR) in adjuvant. B6 mice develop anti-TAChR Ab that cross-react with mouse muscle AChR, but their CD4+ T cells do not cross-react with mouse AChR sequences. Moreover, murine EMG is not self-maintaining as is human MG, and it has limited duration. Several studies suggest that IL-4 has a protecting function in EMG. Here we show that B6 mice genetically deficient in IL-4 (IL-4-/-) develop long-lasting muscle weakness after a single immunization with TAChR. They develop chronic self-reactive Ab, and their CD4+ T cells respond not only to the TAChR and TAChR subunit peptides, but also to several mouse AChR subunit peptides. These results suggest that in B6 mice, regulatory mechanisms that involve IL-4 contribute to preventing the development of a chronic Ab-mediated autoimmune response to the AChR.

Identification of immunodominant regions among promiscuous HLA-DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3.

The molecular characterization of the CD4(+) T-cell epitope repertoire on human tumor antigens would contribute to both clinical investigation and cancer immunotherapy. In particular, the identification of promiscuous epitopes would be beneficial to a large number of patients with neoplastic diseases regardless of their HLA-DR type. MAGE-3 is a tumor-specific antigen widely expressed in solid and hematologic malignancies; therefore, is an excellent candidate antigen. We used a major histocompatability complex (MHC) class II epitope prediction algorithm, the TEPITOPE software, to predict 11 sequence segments of MAGE-3 that could form promiscuous CD4(+) T-cell epitopes. In binding assays, the synthetic peptides corresponding to the 11 predicted sequences bound at least 3 different HLA-DR alleles. Nine of the 11 peptides induced proliferation of CD4(+) T cells from both healthy subjects and melanoma patients. Four immunodominant regions (residues 111-125, 146-160, 191-205, and 281-295), containing naturally processed epitopes, were recognized by most of the donors, in association with 3 to 4 different HLA-DR alleles, thus covering up to 94% of the alleles expressed in whites. On the contrary, the other promiscuous regions (residues 161-175 and 171-185) contained epitopes not naturally processed in vitro. The immunodominant epitopes identified will be useful in the design of peptide-based cancer vaccines and in the study of the functional state of tumor-specific CD4(+) T cells in patients bearing tumors expressing MAGE-3.