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.     

T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits

We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.     

Molecular biology and immunology of acetylcholine receptor in relation to myasthenia gravis

In search for the myasthenogenic sites in the molecular structure of acetylcholine receptor (AChR) alpha-subunit, the conformation-dependent B-cell epitopes, and the MHC class II-restricted, immune cofactors-modified T-cell epitopes were studied. Using the peptides synthesized corresponding to AChR amino acid sequence, the alpha 183-200 (as an antigen to raise "blocking antibody") and the alpha 70-90, alpha 125-147 and alpha 67-76 with alpha 107-116 (as antigens to raise "binding antibody") were found immunogenic in the induction of the disease in animals. Phenotypic changes in the T-cell lineages in the thymus were discussed. An impairment of excitation-contraction coupling in some of myasthenic muscles was attributed to a defect caused by antibodies raised against ryanodine receptor protein. Myasthenia gravis patients' sera containing anti-ryanodine receptor antibodies inhibited the calcium-induced release of calcium in response to caffeine in human rhabdomyosarcoma cell line. Buffalo/Mna rats with spontaneous benign thymoma showed (1) ryanodine receptor expressed in the thymic epithelial cells, (2) anti-ryanodine receptor antibodies in serum, and (3) reduced twitch and tetanic force without abnormality in synaptic transmission and muscle membrane properties. It is suggested that thymic epithelial cell and skeletal muscle share common ryanodine receptor antigen. The finding seen in this rat strain can be a counterpart of the feature reflecting an immune attack directed against a compartment of skeletal muscle in myasthenia gravis.     

Early-onset myasthenia gravis: a recurring T-cell epitope in the adult-specific acetylcholine receptor epsilon 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 to alpha, gamma and epsilon subunits; the gamma and epsilon distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the epsilon, rather than the alpha or gamma, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide epsilon201-219 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 epsilon201-219 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.     

Abnormal T cell receptor V gene usage in myasthenia gravis: prevalence and characterization of expanded T cell populations

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5-6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die.     

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.     

Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates, and diagnostic value

Elevated amounts of antibodies specific for acetylcholine receptors were detected in 87 percent of sera from 71 patients with myasthenia gravis but not in 175 sera from individuals without myasthenia gravis, including those with other neurologic or autoimmune diseases. Antireceptor antibodies were not directed at the acetylcholine binding site of the receptor. Presence or titer of antibody did not appear to correlate with age, sex, steroid therapy, or duration of symptoms. Myasthenia gravis patients with only ocular symptoms had lower antibody titers, while the majority of titers in myasthenia gravis patients with thymoma exceeded the median titer of the myasthenia gravis group as a whole. Assay of antireceptor antibody should prove a useful test in the diagnosis of myasthenia gravis.     

Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis

The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG.     

Lewis rats given antibodies against denatured acetylcholine receptor become resistant to induction of experimental autoimmune myasthenia gravis

The embryotoxic and dysmorphogenic effects of mercuric chloride (HgCl2) have been studied in mouse embryos cultured in vitro. In addition, the alterations induced in the developing branchial nerves and ganglia were analyzed. Mouse embryos with 6-8 pairs of somites were exposed for 26 hr to increasing concentrations (0, 12.5, 25, 50 microM) of HgCl2. After this period, a first set of embryos was removed and a second set of embryos transferred to culture medium without HgCl2 and remained in culture for an additional 22 hr. Both sets of embryos were examined for (1) survival, (2) presence of external dysmorphogenesis, (3) growth, and (4) differentiation. Dose-related alterations of these parameters were observed. The main target was the cephalic neural tube (mainly the forebrain), but several other systems were also affected (e.g., the turning of the embryos, the optic system). The 48-hr cultured embryos were immunostained using a monoclonal antineurofilament antibody to visualize defects in the development of branchial nerves and ganglia. HgCl2 induced a pronounced retardation in the differentiation of ganglion/nerve V and a slight retardation in the differentiation of ganglia/nerves VII and IX. The ganglia/nerves VIII and X were not retarded. In addition, hight percentages of abnormalities of ganglion/nerve V and fusions between ganglia/nerves IX and X were observed in these embryos. Disorganized fibers between ganglia/nerves VII-VIII and IX and between ganglia/nerves IX and X were also more frequently observed. At the highest concentration, asymmetric defects were induced by HgCl2 with a more pronounced effect observed on the right side of the embryos. These results demonstrate the usefulness of this approach in evaluating the susceptibility of the developing branchial nerves to the adverse effects of developmental toxicants.     

Anti-idiotypic antibodies against the receptor antibodies in myasthenia gravis

Anti-idiotypic antibodies were prepared against purified acetylcholine receptor antibodies from two patients with myasthenia gravis. The purified idiotypes did not cross-react. Reaction with idiotypes from other patients were found in 8% and 37%, respectively, which suggests that shared idiotypic specificities occur. The anti-idiotypic IgG fractions had no receptor-like activity and did not bind cholinergic ligands. Receptor antibodies from two mothers and their newborn children with neonatal myasthenia gravis showed marked differences in the reactions with an anti-idiotypic antibody. This suggests that not passive transfer of maternal antibodies but a transient synthesis of a receptor antibody with a different specificity is an important factor in the pathogenesis of neonatal myasthenia gravis.     

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.     

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.     

Pure red cell aplasia and myasthenia gravis with thymoma: a case report and review of the literature

In women, osteoporosis with fractures can happen between 50 years, the age of the menopause, and 80 years. Over these three decades, the condition of bone metabolism is not the same. The years after menopause present with high bone turnover. During these years, the administration of antiresorptive drugs is promising: Estrogens (plus progestagens), calcitonins, bisphosphonates. About 10 years after the menopause bone turnover slows down to low turnover. Now the administration of bone-formation stimulating agents is to be preferred: fluorides in the first line, perhaps anabolics in single cases. The prominent fracture of women older than 75 years is the hip fracture (type II osteoporosis). Bone turnover mostly is accelerated again, now due to secondary hyperparathyroidism as a consequence of long-lasting calcium and vitamin D deficiency. For treatment, calcium plus vitamin D is recommended. The exact diagnosis of the metabolic status of the skeleton is a prerequisite for the choice of the optimal therapeutic principle.     

Expression of human-Torpedo hybrid acetylcholine receptor (AChR) for analysing the subunit specificity of antibodies in sera from patients with myasthenia gravis (MG)

The nicotinic AChR, a pentamer composed of alpha2betagamma(or epsilon)delta subunits, is the autoantigen in the human autoimmune disease MG. Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore determination of their specificities requires the use of intact AChR. Indirect antibody competition studies have suggested that most MG antibodies are inhibited from binding to AChR by MoAb to the main immunogenic region (MIR) on the alpha-subunits. More recently, based on the knowledge that MG antibodies show little detectable cross-reaction with Torpedo AChR, we have shown, using mouse-Torpedo hybrid AChR, that most MG antibodies that detectably cross-react with the mouse AChR bind to the alpha-subunit. To analyse the whole anti-AChR antibody repertoire in MG sera, we expressed on stably transfected fibroblasts a novel human alpha+ Torpedo betagammadelta AChR and compared the antibody titres against human, Torpedo, and the hybrid AChR. Direct information was provided for the subunit specificity of several MoAbs and sera from 50 MG patients. On average, at least 48% of the anti-AChR antibodies in the sera were directed against the alpha-subunit. Interestingly, the anti-alpha-subunit antibodies predominated in low titre (0.6-7.4 nM) but not in high titre (10-386 nM) sera, where they comprised on average 68% versus 23% of the antibodies, respectively. Finally, the directly determined anti-alpha-subunit antibodies and the anti-MIR antibodies defined by antibody competition were significantly correlated, thus suggesting that at least a significant fraction of the anti-MIR antibodies in MG sera bind to the alpha-subunit.     

Termination of peripheral tolerance to a T cell epitope by heteroclitic antigen analogues

Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC(88-103)) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC(88-103) in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC(88-103)-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC(88-103), were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.     

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.     

B cell population and its autoimmune activity in the thymus of patients associated with myasthenia gravis

Immunohistochemical studies using antibodies to B lymphocyte and to immunoglobulin G bearing cell were carried out for resected thymus-specimens of 10 patients associated with myasthenia gravis. In each case, abundant B cells (L26 positive cell) resided in the follicles and the medulla of thymus, especially, were congregated in the follicles developing germinal center and around Hassall's corpuscles. Amount of B cell population was various among each case regardless of type of myasthenia gravis or age. B cells were greatly increased in the thymus of patients with values of anti-acetylcholine receptor antibody titers over 100 nmol/l. Although numerous B cells were present in the thymus of these patients, IgG bearing cells were extremely rare. In the most cases, B cells lacked IgG expression. From the results, numerous B cells pre-activating autoimmune antibody production were accumulated in the thymus of patients associated with myasthenia gravis.     

Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.