Specific immunotherapeutic strategy for myasthenia gravis: targeted antigen-presenting cells

The pathogenesis of myasthenia gravis (MG) involves a T cell-dependent antibody-mediated autoimmune response directed against acetylcholine receptors (AChR). Inactivation of AChR-specific T cells should interrupt the immune response, resulting in therapeutic benefit. Since each individual's repertoire of T cells responds to a heterogeneous and unique spectrum of AChR epitopes presented in association with self-major histocompatibility complex (MHC) class II, an individualized approach is required to target all relevant AChR-specific T cells. The individual's own antigen-presenting cells (APC) can be used for this purpose, since they process and present the antigen appropriately, and express the correct MHC class II. A novel method of binding AChR to surface immunoglobulin with a heterobifunctional antibody conjugate allows us to use all B cells as APC. Conjugate-plus-AChR-treated B cells (AChR-APC) effectively targeted AChR-specific T cells, stimulating vigorous proliferative responses in a rat cell culture system. If APCs are 'fixed' with cross-linking reagents, they induce long-lasting or permanent 'anergy' of the specific T cells. We prepared AChR-APC, allowed them to process AChR in vitro, and fixed them with paraformaldehyde. Pre-culture of these fixed AChR-APC with AChR-specific T cells induced anergy: when restimulated with fresh AChR-APC, the T cells exhibited markedly reduced proliferative responses and IL-2 production, compared with responses of T cells pre-cultured with control fixed B cells. Implications for the design of antigen-specific therapeutic strategies for MG and other immune disorders will be discussed.     

Generation of acetylcholine receptor-specific human T cell lines using heterobifunctional antibody-targeted antigen presentation.

Characterizing AChR-specific T lymphocyte clones is an important step towards the ability to induce antigen-specific tolerance in myasthenia gravis (MG). However, the limited supply of relatively inefficient autologous antigen presenting cells (APCs) makes establishing AChR-specific T lymphocyte lines difficult. In this study we targeted AChR to autologous surface IgM+ (sIgM+) APCs using heterobifunctional antibodies (bi-Ab) consisting of anti-sIgM linked to anti-AChR antibodies. FACScan analysis and whole cell-based radioimmunoassay (RIA) showed binding of bi-Ab/AChR conjugates to sIgM+ APCs. Using antigen targeting, AChR-presentation to a well-characterized AChR-specific T cell clone, and to T cell lines raised de novo from MG thymocytes, was improved. Thus, antigen targeting using bi-Ab improved the efficiency of presentation of the scarce autoantigen AChR, suggesting that this method might allow the use of relatively impure antigen preparations and normally inefficient non-antigen-specific APCs, including those which can be immortalized, to accelerate the characterization of the AChR epitopes recognized by pathogenic T helper lymphocytes.     

Myasthenic thymus and thymoma are selectively enriched in acetylcholine receptor-reactive T cells

We compared T-cell proliferative responses to acetylcholine receptor (AChR) and to purified protein derivative (PPD) (of tuberculin) of hyperplastic thymus, thymoma, and blood cells from patients with myasthenia gravis (MG). Hyperplastic MG thymus cells gave significantly higher and more consistent responses to AChR than parallel cultures of autologous blood cells, whereas responses to PPD showed an opposite trend. Thus there was a preferential localization of AChR-reactive T cells in the hyperplastic MG thymus. Furthermore, there was a strong correlation between blood and thymus cell responses to PPD (but not to AChR), arguing that the hyperplastic MG thymus contains a sample of sensitized peripheral T cells. By contrast, both AChR- and PPD-responsive T cells were almost undetectable in thymus from nonmyasthenic patients, which is evidently much less receptive to circulating T cells. Cells from MG thymomas showed the highest stimulations by AChR but did not consistently react to PPD. However, the uninvolved thymus adjacent to these thymomas behaved almost identically to the hyperplastic samples described above. Our interpretation is that AChR-specific T cells are initially sensitized in the MG thymoma but are selectively trapped in the hyperplastic thymus after being primed elsewhere.     

Myasthenia gravis: stimulation of antireceptor autoantibodies by autoreactive T cell lines

We studied autoreactive acetylcholine receptor (AChR)-specific T cell lines from two patients with myasthenia gravis. Anti-AChR autoantibody production by peripheral blood mononuclear cells (PBM) from the donors of the T cell lines was measured with an enzyme-linked immunoadsorbent assay, using purified human AChR as antigen. Freshly isolated PBM produced barely detectable amounts of anti-AChR autoantibodies. If, however, autologous AChR-specific T cells were added to the cultures, the production of anti-AChR autoantibodies, and of total IgM and IgG, was markedly stimulated, depending on the number of T line cells and on the amount of AChR present in the cultures. AChR-specific functional helper T-lymphocytes may have a role in the immunoregulation of myasthenia gravis.     

Thymic abnormalities in patients with myasthenia gravis

Thymic abnormalities were first noticed at autopsies of patients with myasthenia gravis (MG) more than 100 years ago. The thymus is believed to play an important role in the pathogenesis of MG, an autoimmune disease mediated by antibodies against the acetylcholine receptor (AChR) of skeletal muscles. Production of these antibodies in B cells is T cell dependent. T cells potentially specific for AChR are probably generated in the thymus via nontolerogenic thymopoiesis by an aberrant function of thymic epithelial cells. However, generation of these AChR-specific T cells is not the cause of MG, because these cells are also found in healthy individuals. The pathogenetic step in MG involves the activation of these potentially AChR-specific T cells; this activation is the trigger to develop the disease and a therapeutic target. The intra-thymic activation of AChR-specific T cells is probably limited to particular types of MG patients: those with early-onset MG in whom the thymus exhibits lymphofollicular hyperplasia (TLFH) and a few patients in whom MG is associated with a thymoma. The majority of thymomas and atrophic thymuses of patients with late-onset MG, an increasingly common condition, do not exhibit this T cell-activation process. In this paper, we review the available literature on thymic changes (TLFH, thymoma, and atrophic thymus) and the relationship of these changes to the pathogenesis of MG.     

重症肌无力患者胸腺细胞经AChR刺激后T淋巴细胞亚群Bcl-2的表达

目的研究重症肌无力(myasthenia gravis,MG)患者胸腺细胞经乙酰胆碱受体(AChR)刺激后T淋巴细胞亚群Bcl-2的表达水平和临床意义。方法应用流式细胞仪测定11例MG患者胸腺T淋巴细胞亚群经AChR刺激后Bcl-2表达水平。结果经AChR刺激后,MG组增生胸腺CD4、Bcl-2双阳性细胞明显高于对照组(P<0.01),CD8、Bcl-2双阳性细胞与对照组相比差异无统计学意义(P>0.05)。结论MG患者增生胸腺中AChR特异性辅助T细胞上Bcl-2表达增高,Bcl-2表达升高可能和AChR的诱导有关。     

CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR alpha subunit. Several epitopes were identified. Only the most severely affected patients recognized alpha subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several alpha subunit peptide sequences could be detected when CD(8+)-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any alpha subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48-67, 101-137, 293-337, and 308-437).     

Synthesis of anti-acetylcholine receptor antibodies by CD5- B cells from peripheral blood of myasthenia gravis patients

An increased frequency of CD5⁺ B cells (or, according to a new nomenclature, B 1 cells) has been detected in the peripheral blood of a proportion of patients with myasthenia gravis (MG), as in some other autoimmune diseases. To elucidate the pathogenic significance of this B-cell subset in myasthenia gravis, mononuclear cells from the peripheral blood of six MG patients were separated into T and B lymphocytes by a magnetic cell separation procedure employing superparamagnetic microbeads (MACS). Subsequently, the B-cell fraction was depleted of CD5⁺ B cells in a second separation. The resulting purified CD5^– B-cell fraction was cultured alone or with the addition of autologous T cells. Anti-acetylcholine receptor (AChR) synthesis by CD5^– B cells in cultures with T cells was significantly increased by pokeweed mitogen (176 ±130 fmol/ml per week/2 × 10⁵ B cells) compared with unfractionated cells (75 ± 101) or CD5^– B cells alone (19 ± 4). These results demonstrate that in MG anti-AChR are synthesized, at least in part, by CD5^– B cells which are dependent on T cells. Although this does not exclude the existence of AChR-specific CD5⁺ B cells, it provides evidence against a pivotal role of this B-cell subset in anti-AChR synthesis.     

Human muscle acetylcholine receptor reactive T and B lymphocytes in the peripheral blood of patients with myasthenia growth

The prevalence of T and B cells reactive with the acetylcholine receptor (AChR) of human skeletal muscle was studied in 33 patients with myasthenia gravis (MG), 18 patients with other neurological diseases (OND) or autoimmune disorders (AD) and 27 age- and sex-matched healthy controls. T cell stimulation was estimated by enumerating cells secreting interferon (IFN)-γ and interleukin (IL)-2 in response to the AChR, whereas B cell reactivity was estimated by enumerating cells secreting IgG antibodies binding to the AChR. AChR-reactive T cells were increased in the peripheral blood of patients with MG as compared to patients with OND, AD and healthy individuals. Of the patients with MG, 29/33 (87.7%) had numbers of IFN-γ secreting cells higher than the mean ± 2 SD of the mean of controls as compared to 4/18 (22.2%) of patients with OND or AD and 2/27 (7.4%) of the controls. The mean value of the numbers of AChR-reactive T cells in the patients with MG was 19.6/10⁵ PBMC, corresponding to 1/5100 PBMC. Comparable results were obtained also for IL-2-secreting cells. Anti-AChR IgG antibody-secreting cells were detected in the blood of 30/33 (91%) of the patients with MG, 3/18 (16.7%) of the patients with OND or AD and 2/25 (8%) of the controls. The mean value of the antibody-secreting cells in MG was 11.7 cells/10⁶ PBMC corresponding to 1/70400 PBMC in the patients with MG, compared to a mean value of antibody-secreting cells in the patients with OND or AD of 0.33 and controls of 0.16 cells/10⁶ PBMC.     

重组白细胞介素2及T细胞亚群对重症肌无力患者AChR—ab的影响

对１９例全身型重症肌无力（ＭＧ）患者周围血Ｂ细胞分化能力进行了研究，并观察重组白细胞介素２（ｒＩＬ－２）及Ｔ细胞亚群对其影响。方法分离外周血纯Ｂ细胞，体外与电鳗乙酰胆碱受体（ＡＣｈＲ）一起培养，并分别加入去ＣＤ４＋Ｔ细胞，去ＣＤ８＋Ｔ细胞及ｒＩＬ－２。用酶联免疫－生物素法检测其上清液的乙酰胆碱受体抗体（ＡＣｈＲ－ａｂ）。同时以１０例健康人为对照组。结果去ＣＤ８＋Ｔ细胞后ＡＣｈＲ－ａｂ的产生无明显增加；去ＣＤ４＋Ｔ细胞加入ｒＩＬ－２后ＡＣｈＲ－ａｂ产生减少。结论提示ｒＩＬ－２对ＭＧ患者有潜在治疗价值。     

Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease

We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.     

The thymus in myasthenia gravis: Site of “innate autoimmunity”?

Myasthenia gravis (MG) is an autoimmune disorder caused, in most cases, by autoantibodies against components of the neuromuscular junction, frequently the acetylcholine receptor (AChR), and less often the muscle-specific kinase receptor. The thymus plays a major role in the pathogenesis of MG with anti-AChR antibodies: it shows marked pathologic alterations (hyperplastic or tumoral) in most AChR-positive patients and contains the elements required to initiate and sustain an autoimmune reaction (AChR autoantigen, AChR-specific T cells, and autoantibody-secreting plasma cells). In this study we review early and more recent findings implicating the thymus as site of AChR autosensitization in MG and briefly discuss the therapeutic role of thymectomy. We also summarize data showing that the MG thymus is in a state of chronic inflammation, and we review emerging evidence of a viral contribution to the onset and maintenance of the thymic autoimmune response.     

重症肌无力胸腺单个核细胞白细胞介素-2、白细胞介素-10及其mRNA水平的变化

目的　研究重症肌无力 ( myasthenia gravis,MG)患者胸腺是否存在辅助性 T细胞 ( Th)亚群的免疫活性紊乱。方法　利用亲和层析法自人腓肠肌纯化获得乙酰胆碱受体 ( ACh R)并进行了鉴定 ,然后用其作为特异性抗原 ,经体外培养刺激 5例 MG患者胸腺和外周血单个核细胞 ,酶联免疫吸附分析 ( ELISA)法检测上清液中白细胞介素 -2 ( IL-2 )、白细胞介素 -1 0 ( IL-1 0 )水平 ,狭缝印迹杂交法检测 MG胸腺及外周血 IL-1 0 m RNA的表达。结果　MG胸腺和外周血经 ACh R刺激后 ,其上清液 IL-2水平略高于对照组 ( P>0 .0 5 ) ,经 ELISA法和狭缝印迹杂交法发现 MG外周血 IL-1 0及其 m RNA表达水平均略高于对照组 ( P>0 .0 5 ) ,胸腺则明显高于对照组( P<0 .0 5 )。结论　 MG发病过程中 IL-1 0的异常增高可能发生于转录或转录以上水平 ,其异常表达的机制有待于进一步研究。     

Suppression of experimental autoimmune myasthenia gravis by nasal administration of acetylcholine receptor

Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non-specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freund's adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-γ-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR.     

Dendritic cells activate autologous T cells and induce IL-4 and IL-10 production in myasthenia gravis

Dendritic cells (DC), as initiators and orchestrators of immune responses, control both naive and primed T cell responses. Depending on their maturation stage, DC promote immunity or tolerance. Here we investigated (1) the phenotype and cytokine secretion patterns of IL-10-modulated immature DC (IL-10-DC) and lipopolysaccharide (LPS)-driven mature DC (LPS-DC) in comparison with unmodulated immature DC (imDC) and (2) the effects of IL-10-DC, and of LPS-DC, vs. imDC on autologous T cell responses in patients with myasthenia gravis (MG) compared with healthy controls (HC). All three types of DC derived from MG significantly increased the levels of CD4+CD25+ T cells and of their subfraction expressing CD69, when compared to DC derived from HC. IL-10-DC induced production of IL-10 and IL-4 by T cells from MG patients, but only IL-10 production from HC. LPS-DC activated autologous T cells as reflected by augmented CD25, CD69 and CTLA-4 expression on CD4+ T cells, without differences between MG and HC. This was associated with increased production of both Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines by T cells. These results indicate that DC-induced activation of autologous T cells is more pronounced in MG than in HC. In addition, DC-induced T cell responses in MG vs. HC are more Th2-prone.     

Specific immunotherapy of experimental myasthenia gravis in vitro and in vivo: The Guided Missile strategy

Current immunotherapy of myasthenia gravis (MG) is often effective, but entails risks of infection and neoplasia. The "Guided Missile" strategy described here is designed to target and eliminate the individual's unique AChR-specific T cell repertoire, without otherwise interfering with the immune system. We genetically engineered dendritic cells to present AChR epitopes and simultaneously express Fas ligand in an ongoing EAMG model. In both in vitro and in vivo experiments, these engineered cells specifically killed AChR-responsive T cells without otherwise damaging the immune system. AChR antibodies were markedly reduced in the treated mice. Translation of this method to treat human MG is possible.     

The B-cell repertoire in myasthenia gravis includes all four acetylcholine receptor subunits

By enumerating cells secreting IgG antibodies of particular specificities using an enzyme-linked immunospot (ELISPOT) assay, the B-cell responses to Torpedo acetylcholine receptor (AChR) and its α-, β-, γ- and δ-subunits in peripheral blood from patients with myasthenia gravis (MG), and controls with other neurological diseases (OND) as well as healthy subjects were determined. Compared to controls, the patients with MG had elevated numbers of B cells secreting antibodies against AChR and its α-, β-, γ- and δ-subunits in peripheral blood in parallel. The mean numbers of anti-AChR antibody secreting cells were about 17 per 10⁵ blood MNC, and for the subunits 10 to 15 in MG patients, compared to between 0.8 and 1.9 per 10⁵ blood MNC in OND patients, and 0.1 to 0.3 in healthy controls. Such B cells detected in controls probably represent naturally occurring B cells responded to AChR and its subunits. The finding that most (60%) MG patients had B cells predominantly recognizing the α-subunit may be an indirect argument for the existence of a main immunogenic region (MIR). In the remaining 40% of patients with MG the predominant B-cell responses were directed to β-, γ- or δ-subunit. The data suggest that all four AChR subunits may function as strong immunogens in MG, though the α-subunit may be the major immune target in a substantial proportion of MG patients.     

Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells

We investigated immune property of a myoid cell line, established from Fisher rat thymus. Immunization of syngeneic rats with the myoid cells induced anti-rat acetylcholine receptor (AChR). Implantation of them into the thymus failed to induce typical thymic pathology of human myasthenia gravis (MG) or anti-AChR responses. We also demonstrated that the myoid cells were able to present exogenous antigens to T cells and induce antigen-specific T cell proliferation. These results suggest that myoid cells have the potential antigenicity to induce anti-AChR and the functions of antigen-presenting cells, but their expansion in the thymus may not directly cause MG.     

Interleukin 10 aggravates experimental autoimmune myasthenia gravis through inducing Th2 and B cell responses to AChR

The damage of acetylcholine receptor (AChR) at neuromuscular junctions of experimental autoimmune myasthenia gravis (EAMG), an animal model of human MG, is mediated by B cells which require T cell help. The Th2 associated cytokine IL-10 suppresses production of cytokines released by Th1 cells and is considered for treatment of human autoimmune diseases. To evaluate the role of IL-10 in EAMG, rhIL-10 was administered daily to Lewis rats by the subcutaneous route starting at the day of immunization and continued for 7 weeks. IL-10 failed to abrogate EAMG at low dose (0.1 or 1 microg/day) and at the dose of 3 microg/day caused earlier onset and aggravated clinical signs of EAMG when compared to EAMG rats injected with PBS only. Although Th1 responses reflected by AChR-induced lymphocyte proliferation and levels of IFN-gamma secreting cells, as well as AChR-induced Th1 cytokine mRNA expression was suppressed, augmented IL-4 mRNA expression and AChR-specific B cell responses may play an important role in the failure of IL-10 to abrogate EAMG. This study implicates a critical precaution in planning immunotherapy of IL-10 in antibody-mediated autoimmune diseases, e.g. MG.     

Early-onset myasthenia gravis: A recurring T-cell epitope in the adult-specific acetylcholine receptor ε subunit presented by the susceptibility allele HLA-DR52a

No immunodominant T-cell epitopes have yet been reported in the human acetylcholine receptor (AChR), the target of the pathogenic autoantibodies in myasthenia gravis (MG). We have selected and characterized T cells from MG patients by restimulation in culture with recombinant human AChR α, γ, and ε subunits; the γ and ε distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the ε, rather than the α or γ, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide ε201–219 and to low concentrations of adult but not fetal AChR. Moreover, although using different T-cell receptor genes, they were all restricted to HLA-DR52a (DRB3*0101), a member of the strongly predisposing HLA-A1-B8-DR3 haplotype. This apparently immunodominant ε201–219 epitope (plus DR52a) was also recognized by clones from an elderly patient whose MG had recently been provoked by the drug D -penicillamine. In all 4 cases, however, the serum antibodies reacted better with fetal than adult AChR and may thus be end products of determinant spreading initiated by adult AChR-specific T cell responses. Furthermore, as these T cells had a pathogenic Th1 phenotype, with the potential to induce complement-activating antibodies, they should be important targets for selective immunotherapy. Ann Neurol 1999;45:224–231