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Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.

The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

In myasthenia gravis (MG) there is an autoimmune response against muscle acetylcholine receptor (AChR). Embryonic and adult muscles express different AChRs; embryonic AChR contains a gamma subunit, instead of the homologous epsilon subunit that contributes to form adult AChR. We report propagation from the blood of MG patients of T helper (TH) cell lines specific for human embryonic AChR, by cycles of stimulation with a pool of synthetic peptides corresponding to the complete sequence of the gamma subunit (gamma pool). The TH lines strongly recognized AChR from embryonic mammalian muscle, and reacted less or not at all with adult muscle AChR. The existence of TH cells specific for embryonic AChR strongly suggests that the primary anti-AChR sensitization in MG occurs in a tissue other than the innervated skeletal muscle. This may be within the thymus, which expresses an AChR similar or identical to embryonic muscle AChR.

In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation.

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.