BM stromal cells ameliorate experimental autoimmune myasthenia gravis by altering the balance of Th cells through the secretion of IDO

In addition to their capacity to differentiate, BM stromal cells (BMSC) have immunosuppressive qualities that make them strong candidates for use in cell therapy against human autoimmune diseases. We studied the immunoregulatory activities of BMSC on experimental autoimmune myasthenia gravis (EAMG) in vitro and in vivo. Intravenous administration of syngenic BMSC to EAMG‐model rats on the day of their second immunization was effective in ameliorating the pathological features of the disease. In vitro, the proliferative ability of T cells or B cells from EAMG rats was inhibited when they were cocultured with BMSC at proper ratios. This inhibitory effect was at least partially dependent on the secretion of IDO. We also determined that the development of EAMG is accompanied by an imbalance among the Th1, Th2, Th17, and Treg cell subsets, and that this can be corrected by the administration of BMSC, which leads to an increase of Th2 (IL‐4) and Treg (Foxp3) cells, and a reduction of Th1 (IFN‐γ) and Th17 (IL‐17) cells, through an IDO‐dependent mechanism. These results provide further insights into the pathogenesis of MG, EAMG, and other immune‐mediated diseases, and support a potential role for BMSC in their treatment.     

Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.     

Myasthenia Gravis and its Animal Model: T Cell Receptor Expression in an Antibody Mediated Autoimmune Disease

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease. Since the primary target antigen of the autoimmune response is known and a well-characterized animal model is available, MG is often considered an excellent situation for the application of novel specific immunotherapies, many of which are directed at T lymphocytes. CD4⁺ helper T cells are required for the development of the animal model, experimental autoimmune MG (EAMG). Even though the target antigen, acetylcholine receptor (AChR) is immunologically complex, the T cell response to AChR in mice is dominated by recognition of a single peptide by about 50% of the T cells. These T cells, in turn, utilize a restricted set of TCR gene elements and conserved CDR3 regions. While specific therapy directed at the immunodominant T cells is capable of reducing the magnitude of the anti-AChR response, considerable flexibility is apparent and reveals the ability of additional T cells to provide the requisite B cell help. In human MG patients, AChR-specific T cells have been identified but in many studies the frequencies were surprisingly low. In a very few cases, AChR-specific T cells have been cloned from MG patients. Analysis reveals heterogeneity in epitope recognition and MHC restriction. Little information on TCR structure is available. Our own studies using antigen-specific as well as non-specific methods for examining clonal T cell expansions in MG have led to an alternative hypothesis concerning T-B collaboration in MG.     

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.     

'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.     

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 co-stimulation 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.     

Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN-γ and IL-4

CDS⁺ T cells can perform both Th1 - and Th2-like functions by producing cytokines such as interferonγ (IFN-γ) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-β (TGF-β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8⁺ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-γ secreting Th1-like cells. To identify the involvement of IFN-γ, IL-4 and TGF-β in the development of EAMG after CD8⁺ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8⁺ T cells resulted in decreased levels of IFN-7 and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8⁺ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).     

Experimental Autoimmune Myasthenia Gravis (EAMG): From immunochemical characterization to therapeutic approaches

Myasthenia Gravis (MG) is an organ-specific autoimmune disease. In high percentage of patients there are autoantibodies to the nicotinic acetylcholine receptor (AChR) that attack AChR on muscle cells at the neuromuscular junction, resulting in muscle weakness. Experimental Autoimmune Myasthenia Gravis (EAMG) is an experimental model disease for MG. EAMG is induced in several animal species by immunization with acetylcholine receptor (AChR), usually isolated from the electric organ of electric fish, which is a rich source for this antigen. Our lab has been involved for several decades in research of AChR and of EAMG. The availability of an experimental autoimmune disease that mimics in many aspects the human disease, provides an excellent model system for elucidating the immunological nature and origin of MG, for studying various existing treatment modalities and for attempting the development of novel treatment approaches. In this review in honor of Michael Sela and Ruth Arnon, we report first on our early pioneering contributions to research on EAMG. These include the induction of EAMG in several animal species, early attempts for antigen-specific treatment for EAMG, elicitation and characterization of monoclonal antibodies and anti-idiotypic antibodies, measuring humoral and cellular AChR-specific immune responses in MG patient and more. In the second part of the review we discuss more recent studies from our lab towards developing and testing novel treatment approaches for myasthenia. These include antigen-dependent treatments aimed at specifically abrogating the humoral and cellular anti-AChR responses, as well as immunomodulatory approaches that could be used either alone, or in conjunction with antigen-specific treatments, or alternatively, serve as steroid-sparing agents.     

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild- type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.      

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.     

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-γ 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.     

Impairment of regulatory T cells in myasthenia gravis: Studies in an experimental model

Myasthenia gravis (MG) is an antibody mediated, T cell dependent autoimmune disease characterized by muscle fatigability in which autoantibodies directed to the acetylcholine receptor (AChR) impair neuromuscular transmission. The identification of CD4⁺CD25⁺Foxp3⁺ Treg cells as important regulators of tolerance opened a major area of investigation raising the possibility that a dysfunction in the Treg compartment is involved in the etiology and pathogenesis of autoimmune diseases, including MG. In this paper we summarize shortly Treg abnormalities that were reported in MG patients and report on our studies of Treg in experimental autoimmune MG (EAMG). Hopefully these studies would pave the way towards the development of novel Treg-based treatment modalities that will restore self-tolerance in MG and other autoimmune diseases.In our previous studies in EAMG we have shown that Treg cells transferred from healthy rat donors to myasthenic rats suppress EAMG. However, Treg cells from sick animals do not have the same in vivo suppressive activity as those from healthy donors. The objective of the present study was to further characterize quantitative and qualitative alterations in Treg cells of rats with EAMG. We found that the frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells within the spleen and PBL was decreased in EAMG rats as compared to naïve and CFA-immunized healthy controls. Treg cells from myasthenic rats were less effective than Treg cells from controls in suppressing the proliferation of CD4⁺ T effector cells in response to ConA and of B cells in response to LPS. Moreover, CD4⁺CD25⁺ cells from EAMG rats exhibited an elevated extent of apoptosis and expressed upregulated levels of FAS and of Th17-associated cytokines. Since EAMG is an induced disease, these quantitative and qualitative alterations in Treg cells do not reflect predisposing impairments and seem to be associated with the specific autoimmune response resulting from AChR immunization.     

An immunodominant site of acetylcholine receptor in experimental myasthenia mapped with T lymphocyte clones and synthetic peptides

Myasthenia gravis (MG) is an autoimmune disease of man caused by antibodies directed against the acetylcholine receptor (AChR). In the experimental model of MG in mice, murine experimental autoimmune myasthenia gravis (EAMG), an anti-AChR immune response is induced by immunization with Torpedo AChR, and anti-AChR antibodies. AChR-sensitized T cells, and neuromuscular dysfunction result. The production of antibodies to AChR is thymus-dependent. In order to define the epitopes of the AChR identified by AChR-specific T cells, we generated T cell populations and T cell hybridoma clones and tested their reactivity to synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the alpha-chain of the AChR. The predominant reactivity of the T cell clones and the parent lines was to a peptide corresponding to residues 146-162 of Torpedo AChR. This data is consistent with a highly limited recognition of AChR determinants in murine EAMG by AChR-specific T cells.     

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell–dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.     

ACh receptor protein drives primary and memory autoantibody responses in chimeric human-SCID mice

The native antigen that drives the T-helper cells regulating production of muscle acetylcholine receptor (AChR) autoantibodies is unknown. Human T cell lines activated by autoantigens in vitro are of unproven relevance to B cell help. Here we report the functional interaction and unprecedented longevity of AChR-specific human T and B lymphocytes residing in SCID mice. Lymphoid cells from myasthenia gravis (MG) patients and healthy subjects were injected ip. Recombinant human AChR-alpha1-subunit-1-210 was injected after day 75. Human AChR-specific Ig was produced rapidly in MG-SCID mice challenged once. Only 1 of 32 control hu-SCID mice produced AChR-specific Ig. This required multiple immunizations, was initially cross-reactive with Torpedo AChR, and had a slow course. Thus, memory T and B lymphocytes specific for human AChR-alpha1-subunit are readily demonstrable in MG patients, interact to produce autoantibody of the same restricted specificity found in the donor's serum, and are long-lived without exogenous autoantigen challenge. In healthy subjects, AChR-specific lymphocytes are infrequent and exhibit naive response characteristics, including apparent affinity maturation of Ig specificity.     

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30 μg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (> 80% incidence). Although mice immunized with 10 μg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30 μg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.     

IL‐17‐producing CD4+ T cells contribute to the loss of B‐cell tolerance in experimental autoimmune myasthenia gravis

The role of Th17 cells in the pathogenesis of autoantibody‐mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR‐specific CD4⁺ T cells is producing IL‐17. IL‐17^ko mice developed fewer or no EAMG symptoms, although the frequencies of tAChR‐specific CD4⁺ T cells secreting IL‐2, IFN‐γ, or IL‐21, and the percentage of FoxP3⁺ T_reg cells were similar to WT mice. Even though the total anti‐tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL‐17^ko as compared to WT mice. Most importantly, pathogenic anti‐murine AChR antibodies were significantly lower in IL‐17^ko mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR β/δ^ko mice with WT or IL‐17^ko CD4⁺ T cells. In conclusion, we show that the level of IgG2b and the loss of B‐cell tolerance, which results in pathogenic anti‐murine AChR‐specific antibodies, are dependent on IL‐17 production by CD4⁺ T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody‐mediated autoimmunity.     

Myasthenia gravis and its animal model: T cell receptor expression in an antibody mediated autoimmune disease

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease. Since the primary target antigen of the autoimmune response is known and a well-characterized animal model is available, MG is often considered an excellent situation for the application of novel specific immunotherapies, many of which are directed at T lymphocytes. CD4+ helper T cells are required for the development of the animal model, experimental autoimmune MG (EAMG). Even though the target antigen, acetylcholine receptor (AChR) is immunologically complex, the T cell response to AChR in mice is dominated by recognition of a single peptide by about 50% of the T cells. These T cells, in turn, utilize a restricted set of TCR gene elements and conserved CDR3 regions. While specific therapy directed at the immunodominant T cells is capable of reducing the magnitude of the anti-AChR response, considerable flexibility is apparent and reveals the ability of additional T cells to provide the requisite B cell help. In human MG patients, AChR-specific T cells have been identified but in many studies the frequencies were surprisingly low. In a very few cases, AChR-specific T cells have been cloned from MG patients. Analysis reveals heterogeneity in epitope recognition and MHC restriction. Little information on TCR structure is available. Our own studies using antigen-specific as well as non-specific methods for examining clonal T cell expansions in MG have led to an alternative hypothesis concerning T-B collaboration in MG.     

A dual altered peptide ligand down-regulates myasthenogenic T cell responses and reverses experimental autoimmune myasthenia gravis via up-regulation of Fas-FasL-mediated apoptosis

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent, antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogues of two myasthenogenic peptides, p195-212 and p259-271, was demonstrated to down-regulate in vitro and in vivo MG-associated autoreactive responses. The aims of this study were to investigate the possible role of Fas-FasL-mediated apoptosis in the down-regulatory mechanism of the dual APL. We demonstrate here the effect of the dual APL on expression of key molecules involved in the Fas-FasL pathway, in a p195-212-specific T cell line, in mice immunized with Torpedo acetylcholine receptor and in mice afflicted with EAMG (induced with the latter). In vitro and in vivo results show that the dual APL up-regulated expression of Fas and FasL on the CD4 cells. Expression of the pro-apoptotic molecules, caspase 8 and caspase 3, was significantly up-regulated, while anti-apoptotic cFLIP and Bcl-2 were down-regulated upon treatment with the dual APL. The dual APL also increased phosphorylation of the mitogen-activated protein kinases, c-Jun-NH2-terminal kinase and p-38, known to play a role in the regulation of FasL expression. Further, in the T cell line incubated with the dual APL as well as in mice of the SJL inbred strain immunized with the myasthenogenic peptide and treated concomitantly with the dual APL, the percentage of apoptotic cells increased. Results strongly indicate that up-regulation of apoptosis via the Fas-FasL pathway is one of the mechanisms by which the dual APL reverses EAMG manifestations in C57BL/6 mice.