Epitopes on the f Subunit of Human Muscle Acetylcholine Receptor Recognized by CD4+ Cells of Myasthenia Gravis Patients and Healthy Subjects Lucia Moiola, Peter Karachunski, Maria Pia Protti, James F. Howard, Jr.,* and Bianca M. Conti-Tronconi Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108; Department of Pharmacology, University ofMinnesota, Minneapolis, Minnesota 55455; and *Department ofNeurology, University ofNorth Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7025 Abstract We investigated the sequence regions of the human muscle ace- tylcholine receptor (AChR) ( subunit forming epitopes recog- nized by T helper cells in myasthenia gravis (MG), using over- lapping synthetic peptides, 20 residues long, which screened the sequence of the AChR (3 subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of (3 subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimula- tion of blood lymphocytes with the pooled peptides correspond- ing to the (3 subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for sev- eral months. Three lines were tested with purified bovine mus- cle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the (3 subunit sequence. Severalf( subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides ,(181-200, ,(271-290, and the overlapping peptides (3316-335 and (3331-350) were recognized by most MG lines. The (3 subunit-specific T lines from controls could be propa- gated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above. (J. Clin. Invest. 1994. 93:1020-1028.) Key words: synthetic peptides * T cell lines * CD4 + subsets Introduction In myasthenia gravis (MG)' an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) occurs Address correspondence to Bianca M. Conti-Tronconi, Department of Biochemistry, College of Biological Sciences, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108. Lucia Moiola and Maria Pia Protti's present address is H. San Raffaele, Via Olgettina 60, 20132 Milan, Italy. Receivedfor publication I December 1992 and in revisedform 12 July 1993. 1. Abbreviations used in this paper: AChR, muscle nicotonic acetylcho- line receptor; a, (3, y, or 6 pool, a pool of synthetic peptides correspond- ing to the complete sequence of human AChR a, ,B, y, or 6 subunit; APC, antigen-presenting cells; BAChR, bovine muscle AChR; MG, myasthenia gravis; TAChR, Torpedo californica AChR; TCM, tissue culture medium; Th, T helper. (for reviews see references 1-5), with synthesis of AChR anti- bodies and sensitization of AChR-specific T helper (Th, CD4+) cells. The AChR of mammalian muscle is a complex transmembrane protein, formed by four homologous subunits, a, f3, e (,y in embryonic muscle), and a (for review see refer- ence 6). Since Th cells are necessary for synthesis of high affinity anti-AChR IgG antibodies (7) and possibly for maintenance of B cell tolerance by mechanisms of clonal anergy (8), it is im- portant to define and catalog the epitope repertoire of the au- toimmune anti-AChR Th cells in MG patients and of poten- tially autoreactive Th cells able to recognize AChR sequences in healthy subjects. Several sequence regions forming DR-re- stricted epitopes recognized by AChR Th cells in MG patients have been identified on the a, y, and a AChR subunits (for review see reference 9). Some such regions, 20 residues long, are recognized by most MG patients, irrespective of their MHC class II haplotype ( 10-12). In this study we investigated whether the AChR ,3 subunit is involved in the sensitization of Th cells and we sought identifi- cation of sequence regions of the fl subunit forming epitopes recognized by CD4' cells of MG patients. Since CD4' cells able to recognize autologous antigens, including the AChR, have been described in healthy subjects (e.g., see references I 1-16), we also investigated the existence of anti-AChR f3 sub- unit CD4+ cells in healthy controls and their epitope specific- ity. We used a panel of 32 synthetic peptides, 20 residues long and overlapping each other by 5 residues, corresponding to the complete sequence of the human AChR f3 subunit (17). We first used these peptides to test the response of unselected CD4+-enriched, CD8+-depleted blood cells. No response could be detected in our patients, who had mild or moderate symptoms. This agrees with previous studies which indicated that only unselected blood cells from severely affected MG pa- tients showed a measurable in vitro response to synthetic AChR antigens (13, 18). To obtain a CD4+ population enriched in f3 subunit-specific cells, we attempted propagation of T cell lines by cycles of stimulation with the pooled peptides corresponding to the fl subunit sequence. Anti-,B subunit CD4+ T lines were obtained from four of six patients and from three of seven controls. The sequence regions of the f3 subunit forming epitopes recognized by the T lines were identified by challenging the lines in microproliferation assays with the indi- vidual synthetic peptides. Methods Patients and controls. We used 12 MG patients, all suffering from mild or moderate generalized symptoms. In all but one patient, we tested the response of unselected CD8+-depleted, CD4+-enriched blood mono- nuclear cells to pools of synthetic peptides corresponding to the com- plete sequence of the human AChR a, f3, 'y, and 6 subunits ( 17, 19-21 ) and to individual peptides screening the human ,B subunit sequence, as described below. 1020 Moiola, Karachunski, Protti, Howard, and Conti-Tronconi J. Clin. Invest. ©0 The American Society for Clinical Investigation, Inc. 002 1-9738/94/03/1020/09 $2.00 Volume 93, March 1994, 1020-1028