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. 1994 Mar;93(3):1020–1028. doi: 10.1172/JCI117050

Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

L Moiola 1, P Karachunski 1, M P Protti 1, J F Howard Jr 1, B M Conti-Tronconi 1
PMCID: PMC294026  PMID: 7510715

Abstract

We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle 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 beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beeson D., Brydson M., Newsom-Davis J. Nucleotide sequence of human muscle acetylcholine receptor beta-subunit. Nucleic Acids Res. 1989 Jun 12;17(11):4391–4391. doi: 10.1093/nar/17.11.4391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Besinger U. A., Toyka K. V., Hömberg M., Heininger K., Hohlfeld R., Fateh-Moghadam A. Myasthenia gravis: long-term correlation of binding and bungarotoxin blocking antibodies against acetylcholine receptors with changes in disease severity. Neurology. 1983 Oct;33(10):1316–1321. doi: 10.1212/wnl.33.10.1316. [DOI] [PubMed] [Google Scholar]
  3. Conti-Tronconi B., Tzartos S., Lindstrom J. Monoclonal antibodies as probes of acetylcholine receptor structure. 2. Binding to native receptor. Biochemistry. 1981 Apr 14;20(8):2181–2191. doi: 10.1021/bi00511a017. [DOI] [PubMed] [Google Scholar]
  4. DeLisi C., Berzofsky J. A. T-cell antigenic sites tend to be amphipathic structures. Proc Natl Acad Sci U S A. 1985 Oct;82(20):7048–7052. doi: 10.1073/pnas.82.20.7048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Del Prete G. F., De Carli M., Ricci M., Romagnani S. Helper activity for immunoglobulin synthesis of T helper type 1 (Th1) and Th2 human T cell clones: the help of Th1 clones is limited by their cytolytic capacity. J Exp Med. 1991 Oct 1;174(4):809–813. doi: 10.1084/jem.174.4.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Demotz S., Lanzavecchia A., Eisel U., Niemann H., Widmann C., Corradin G. Delineation of several DR-restricted tetanus toxin T cell epitopes. J Immunol. 1989 Jan 15;142(2):394–402. [PubMed] [Google Scholar]
  7. Engel A. G. Myasthenia gravis and myasthenic syndromes. Ann Neurol. 1984 Nov;16(5):519–534. doi: 10.1002/ana.410160502. [DOI] [PubMed] [Google Scholar]
  8. Gammon G., Sercarz E. Does the presence of self-reactive T cells indicate the breakdown of tolerance? Clin Immunol Immunopathol. 1990 Sep;56(3):287–297. doi: 10.1016/0090-1229(90)90150-o. [DOI] [PubMed] [Google Scholar]
  9. Gorin M. B., Yancey S. B., Cline J., Revel J. P., Horwitz J. The major intrinsic protein (MIP) of the bovine lens fiber membrane: characterization and structure based on cDNA cloning. Cell. 1984 Nov;39(1):49–59. doi: 10.1016/0092-8674(84)90190-9. [DOI] [PubMed] [Google Scholar]
  10. Gotti C., Conti-Tronconi B. M., Raftery M. A. Mammalian muscle acetylcholine receptor purification and characterization. Biochemistry. 1982 Jun 22;21(13):3148–3154. doi: 10.1021/bi00256a018. [DOI] [PubMed] [Google Scholar]
  11. Granato D. A., Fulpius B. W., Moody J. F. Experimental myasthenia in Balb/c mice immunized with rat acetylcholine receptor from rat denervated muscle. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2872–2876. doi: 10.1073/pnas.73.8.2872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guerder S., Matzinger P. A fail-safe mechanism for maintaining self-tolerance. J Exp Med. 1992 Aug 1;176(2):553–564. doi: 10.1084/jem.176.2.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heinrikson R. L., Meredith S. C. Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem. 1984 Jan;136(1):65–74. doi: 10.1016/0003-2697(84)90307-5. [DOI] [PubMed] [Google Scholar]
  14. Ho P. C., Mutch D. A., Winkel K. D., Saul A. J., Jones G. L., Doran T. J., Rzepczyk C. M. Identification of two promiscuous T cell epitopes from tetanus toxin. Eur J Immunol. 1990 Mar;20(3):477–483. doi: 10.1002/eji.1830200304. [DOI] [PubMed] [Google Scholar]
  15. Hohlfeld R., Conti-Tronconi B., Kalies I., Bertrams J., Toyka K. V. Genetic restriction of autoreactive acetylcholine receptor-specific T lymphocytes in myasthenia gravis. J Immunol. 1985 Oct;135(4):2393–2399. [PubMed] [Google Scholar]
  16. Hohlfeld R., Kalies I., Kohleisen B., Heininger K., Conti-Tronconi B., Toyka K. V. Myasthenia gravis: stimulation of antireceptor autoantibodies by autoreactive T cell lines. Neurology. 1986 May;36(5):618–621. doi: 10.1212/wnl.36.5.618. [DOI] [PubMed] [Google Scholar]
  17. Horton R. M., Manfredi A. A., Conti-Tronconi B. M. The 'embryonic' gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle. Neurology. 1993 May;43(5):983–986. doi: 10.1212/wnl.43.5.983. [DOI] [PubMed] [Google Scholar]
  18. Killen J. A., Hochschwender S. M., Lindstrom J. M. The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype. J Neuroimmunol. 1985 Aug;9(3-4):229–241. doi: 10.1016/s0165-5728(85)80021-7. [DOI] [PubMed] [Google Scholar]
  19. Levinson A. I., Zweiman B., Lisak R. P. Immunopathogenesis and treatment of myasthenia gravis. J Clin Immunol. 1987 May;7(3):187–197. doi: 10.1007/BF00915723. [DOI] [PubMed] [Google Scholar]
  20. Lindstrom J. An assay for antibodies to human acetylcholine receptor in serum from patients with myasthenia gravis. Clin Immunol Immunopathol. 1977 Jan;7(1):36–43. doi: 10.1016/0090-1229(77)90027-7. [DOI] [PubMed] [Google Scholar]
  21. Lindstrom J., Shelton D., Fujii Y. Myasthenia gravis. Adv Immunol. 1988;42:233–284. doi: 10.1016/s0065-2776(08)60847-0. [DOI] [PubMed] [Google Scholar]
  22. Lisak R. P., Laramore C., Levinson A. I., Zweiman B., Moskovitz A. R., Witte A. In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood cells: role of suppressor T cells in normal subjects. Neurology. 1984 Jun;34(6):802–805. doi: 10.1212/wnl.34.6.802. [DOI] [PubMed] [Google Scholar]
  23. Luther M. A., Schoepfer R., Whiting P., Casey B., Blatt Y., Montal M. S., Montal M., Linstrom J. A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671. J Neurosci. 1989 Mar;9(3):1082–1096. doi: 10.1523/JNEUROSCI.09-03-01082.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Manfredi A. A., Protti M. P., Dalton M. W., Howard J. F., Jr, Conti-Tronconi B. M. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits. J Clin Invest. 1993 Aug;92(2):1055–1067. doi: 10.1172/JCI116610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Manfredi A. A., Protti M. P., Wu X. D., Howard J. F., Jr, Conti-Tronconi B. M. CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides. Neurology. 1992 May;42(5):1092–1100. doi: 10.1212/wnl.42.5.1092. [DOI] [PubMed] [Google Scholar]
  26. Martin R., Jaraquemada D., Flerlage M., Richert J., Whitaker J., Long E. O., McFarlin D. E., McFarland H. F. Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals. J Immunol. 1990 Jul 15;145(2):540–548. [PubMed] [Google Scholar]
  27. Melms A., Malcherek G., Gern U., Wiethölter H., Müller C. A., Schoepfer R., Lindstrom J. T cells from normal and myasthenic individuals recognize the human acetylcholine receptor: heterogeneity of antigenic sites on the alpha-subunit. Ann Neurol. 1992 Mar;31(3):311–318. doi: 10.1002/ana.410310314. [DOI] [PubMed] [Google Scholar]
  28. Mosmann T. R., Cherwinski H., Bond M. W., Giedlin M. A., Coffman R. L. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986 Apr 1;136(7):2348–2357. [PubMed] [Google Scholar]
  29. Nelson S., Conti-Tronconi B. M. Adult thymus expresses an embryonic nicotinic acetylcholine receptor-like protein. J Neuroimmunol. 1990 Sep-Oct;29(1-3):81–92. doi: 10.1016/0165-5728(90)90150-l. [DOI] [PubMed] [Google Scholar]
  30. Noreen H. J., Davidson M. L., McCullough J., Bach F. H., Segall M. HLA class II typing by restriction fragment length polymorphism (RFLP) in unrelated bone marrow transplant patients. Transplant Proc. 1989 Feb;21(1 Pt 3):2968–2970. [PubMed] [Google Scholar]
  31. Ohno S. Many peptide fragments of alien antigens are homologous with host proteins, thus canalizing T-cell responses. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3065–3068. doi: 10.1073/pnas.88.8.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oldstone M. B. Molecular mimicry and autoimmune disease. Cell. 1987 Sep 11;50(6):819–820. doi: 10.1016/0092-8674(87)90507-1. [DOI] [PubMed] [Google Scholar]
  33. Panina-Bordignon P., Tan A., Termijtelen A., Demotz S., Corradin G., Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol. 1989 Dec;19(12):2237–2242. doi: 10.1002/eji.1830191209. [DOI] [PubMed] [Google Scholar]
  34. Protti M. P., Manfredi A. A., Horton R. M., Bellone M., Conti-Tronconi B. M. Myasthenia gravis: recognition of a human autoantigen at the molecular level. Immunol Today. 1993 Jul;14(7):363–368. doi: 10.1016/0167-5699(93)90237-F. [DOI] [PubMed] [Google Scholar]
  35. Protti M. P., Manfredi A. A., Straub C., Howard J. F., Jr, Conti-Tronconi B. M. CD4+ T cell response to the human acetylcholine receptor alpha subunit in myasthenia gravis. A study with synthetic peptides. J Immunol. 1990 Feb 15;144(4):1276–1281. [PubMed] [Google Scholar]
  36. Protti M. P., Manfredi A. A., Straub C., Howard J. F., Jr, Conti-Tronconi B. M. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7792–7796. doi: 10.1073/pnas.87.19.7792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Protti M. P., Manfredi A. A., Wu X. D., Moiola L., Dalton M. W., Howard J. F., Jr, Conti-Tronconi B. M. Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor. J Clin Invest. 1992 Oct;90(4):1558–1567. doi: 10.1172/JCI116024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Protti M. P., Manfredi A. A., Wu X. D., Moiola L., Howard J. F., Jr, Conti-Tronconi B. M. Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor. J Immunol. 1991 Apr 1;146(7):2253–2261. [PubMed] [Google Scholar]
  39. Reimann J., Rudolphi A., Claesson M. H. Selective reconstitution of T lymphocyte subsets in scid mice. Immunol Rev. 1991 Dec;124:75–95. doi: 10.1111/j.1600-065x.1991.tb00617.x. [DOI] [PubMed] [Google Scholar]
  40. Romagnani S. Human TH1 and TH2 subsets: doubt no more. Immunol Today. 1991 Aug;12(8):256–257. doi: 10.1016/0167-5699(91)90120-I. [DOI] [PubMed] [Google Scholar]
  41. Roses A. D., Olanow C. W., McAdams M. W., Lane R. J. No direct correlation between serum antiacetylcholine receptor antibody levels and clinical state of individual patients with myasthenia gravis. Neurology. 1981 Feb;31(2):220–224. doi: 10.1212/wnl.31.2.220. [DOI] [PubMed] [Google Scholar]
  42. Salvetti M., Jung S., Chang S. F., Will H., Schalke B. C., Wekerle H. Acetylcholine receptor-specific T-lymphocyte clones in the normal human immune repertoire: target epitopes, HLA restriction, and membrane phenotypes. Ann Neurol. 1991 May;29(5):508–516. doi: 10.1002/ana.410290510. [DOI] [PubMed] [Google Scholar]
  43. Schoepfer R., Luther M., Lindstrom J. The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor. Cloning of the alpha-subunit cDNA. FEBS Lett. 1988 Jan 4;226(2):235–240. doi: 10.1016/0014-5793(88)81430-3. [DOI] [PubMed] [Google Scholar]
  44. Schönbeck S., Chrestel S., Hohlfeld R. Myasthenia gravis: prototype of the antireceptor autoimmune diseases. Int Rev Neurobiol. 1990;32:175–200. doi: 10.1016/s0074-7742(08)60583-8. [DOI] [PubMed] [Google Scholar]
  45. Sette A., Buus S., Colon S., Miles C., Grey H. M. Structural analysis of peptides capable of binding to more than one Ia antigen. J Immunol. 1989 Jan 1;142(1):35–40. [PubMed] [Google Scholar]
  46. Shibahara S., Kubo T., Perski H. J., Takahashi H., Noda M., Numa S. Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor. Eur J Biochem. 1985 Jan 2;146(1):15–22. doi: 10.1111/j.1432-1033.1985.tb08614.x. [DOI] [PubMed] [Google Scholar]
  47. Simpson E., Farrant J., Chandler P. Phenotypic and functional studies of human peripheral blood lymphocytes engrafted in scid mice. Immunol Rev. 1991 Dec;124:97–111. doi: 10.1111/j.1600-065x.1991.tb00618.x. [DOI] [PubMed] [Google Scholar]
  48. Sinigaglia F., Gotti C., Castagnoli R., Clementi F. Acetylcholine receptor-specific suppressive T-cell factor from a retrovirally transformed T-cell line. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7569–7573. doi: 10.1073/pnas.81.23.7569. [DOI] [PMC free article] [PubMed] [Google Scholar]

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