T-helper epitopes on human nicotinic acetylcholine receptor in myasthenia gravis.
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)
Moiola
L
L
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Protti
M P
MP
Manfredi
A A
AA
Yuen
M H
MH
Howard
J F
JF
Conti-Tronconi
B M
BM
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D000939:Epitopes; D005260:Female; D006801:Humans; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009132:Muscles / Q000196:embryology; D009157:Myasthenia Gravis / Q000276:immunology*; D010455:Peptides / Q000276:immunology; D011978:Receptors, Nicotinic / Q000276:immunology*; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology; D013950:Thymus Gland / Q000276:immunology; D013997:Time Factors; D014101:Torpedo
D001323:Autoantibodies; D000939:Epitopes; D010455:Peptides; D011978:Receptors, Nicotinic