Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1995 Sep;86(1):116–121.

Presentation of endogenous acetylcholine receptor antigen to a specific CD4+ T-cell line by a transfected B-cell line.

A F Mulcahy 1, D M Beeson 1, N Willcox 1, A G Diamond 1
PMCID: PMC1383818  PMID: 7590870

Abstract

Currently, the limited supply and stability of some human autoantigens pose formidable difficulties in characterizing patients' T cells specific for them; recombinant preparations may contain bacterial contaminants, and synthetic peptides have arbitrarily chosen start and stop points. In order to provide a stable antigen source with naturally processed epitopes, a full-length acetylcholine receptor (AChR) alpha subunit construct was transfected into B-lymphoblastoid cell lines (B-LCL). Expression was much easier to detect at the mRNA level than the protein level. Nevertheless, this transfectant also stimulated a T-cell line that recognized the alpha 149-156 region in the context of HLA-DR4 at high sensitivity. The responses were specific both for the antigen transfected and for the presenting HLA-DR allele. This study thus confirms the potential of autologous B-LCL expressing natural epitopes in the context of HLA class II molecules for characterizing established T-cell lines, and perhaps also for initiating new ones.

Full text

PDF
116

Images in this article

117Figure 1
on p.117
118Figure 2
on p.118

Selected References

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

  1. Beeson D., Morris A., Vincent A., Newsom-Davis J. The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon. EMBO J. 1990 Jul;9(7):2101–2106. doi: 10.1002/j.1460-2075.1990.tb07378.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brocke S., Brautbar C., Steinman L., Abramsky O., Rothbard J., Neumann D., Fuchs S., Mozes E. In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes. J Clin Invest. 1988 Dec;82(6):1894–1900. doi: 10.1172/JCI113807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen B. P., Madrigal A., Parham P. Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule. J Exp Med. 1990 Sep 1;172(3):779–788. doi: 10.1084/jem.172.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Claudio T., Green W. N., Hartman D. S., Hayden D., Paulson H. L., Sigworth F. J., Sine S. M., Swedlund A. Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts. Science. 1987 Dec 18;238(4834):1688–1694. doi: 10.1126/science.3686008. [DOI] [PubMed] [Google Scholar]
  5. Claudio T., Paulson H. L., Green W. N., Ross A. F., Hartman D. S., Hayden D. Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant. J Cell Biol. 1989 Jun;108(6):2277–2290. doi: 10.1083/jcb.108.6.2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ewins D. L., Barnett P. S., Tomlinson R. W., McGregor A. M., Banga J. P. Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations. Autoimmunity. 1992;11(3):141–149. doi: 10.3109/08916939209035148. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Germain R. N., Margulies D. H. The biochemistry and cell biology of antigen processing and presentation. Annu Rev Immunol. 1993;11:403–450. doi: 10.1146/annurev.iy.11.040193.002155. [DOI] [PubMed] [Google Scholar]
  10. Guilhot S., Fowler P., Portillo G., Margolskee R. F., Ferrari C., Bertoletti A., Chisari F. V. Hepatitis B virus (HBV)-specific cytotoxic T-cell response in humans: production of target cells by stable expression of HBV-encoded proteins in immortalized human B-cell lines. J Virol. 1992 May;66(5):2670–2678. doi: 10.1128/jvi.66.5.2670-2678.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Harcourt G. C., Sommer N., Rothbard J., Willcox H. N., Newsom-Davis J. A juxta-membrane epitope on the human acetylcholine receptor recognized by T cells in myasthenia gravis. J Clin Invest. 1988 Oct;82(4):1295–1300. doi: 10.1172/JCI113729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harcourt G., Batocchi A. P., Hawke S., Beeson D., Pantic N., Jacobson L., Willcox N., Vincent A., Newsom-Davis J. Detection of alpha-subunit isoforms in human muscle acetylcholine receptor by specific T cells from a myasthenia gravis patient. Proc Biol Sci. 1993 Oct 22;254(1339):1–6. doi: 10.1098/rspb.1993.0118. [DOI] [PubMed] [Google Scholar]
  13. Hohlfeld R., Toyka K. V., Heininger K., Grosse-Wilde H., Kalies I. Autoimmune human T lymphocytes specific for acetylcholine receptor. Nature. 1984 Jul 19;310(5974):244–246. doi: 10.1038/310244a0. [DOI] [PubMed] [Google Scholar]
  14. Hohlfeld R., Toyka K. V., Tzartos S. J., Carson W., Conti-Tronconi B. M. Human T-helper lymphocytes in myasthenia gravis recognize the nicotinic receptor alpha subunit. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5379–5383. doi: 10.1073/pnas.84.15.5379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jalanko A., Kallio A., Ruohonen-Lehto M., Söderlund H., Ulmanen I. An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences. Biochim Biophys Acta. 1988 Feb 28;949(2):206–212. doi: 10.1016/0167-4781(88)90084-x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Martin A., Magnusson R. P., Kendler D. L., Concepcion E., Ben-Nun A., Davies T. F. Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells. I. Generation of human thyroid peroxidase-reactive T cells and their T cell receptor repertoire. J Clin Invest. 1993 Apr;91(4):1567–1574. doi: 10.1172/JCI116362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McIntosh R. S., Mulcahy A. F., Hales J. M., Diamond A. G. Use of Epstein-Barr virus-based vectors for expression of thyroid auto-antigens in human B-lymphoblastoid cell lines. J Autoimmun. 1993 Jun;6(3):353–365. doi: 10.1006/jaut.1993.1030. [DOI] [PubMed] [Google Scholar]
  20. Melms A., Malcherek G., Schoepfer R., Sommer N., Kalbacher H., Lindstrom J. Acetylcholine receptor-specific T cells are present in the normal immune repertoire. A study with recombinant polypeptides of the human acetylcholine receptor alpha-subunit. Ann N Y Acad Sci. 1993 Jun 21;681:310–312. doi: 10.1111/j.1749-6632.1993.tb22903.x. [DOI] [PubMed] [Google Scholar]
  21. Mullins R. J., Chernajovsky Y., Dayan C., Londei M., Feldmann M. Transfection of thyroid autoantigens into EBV-transformed B cell lines. Recognition by Graves' disease thyroid T cells. J Immunol. 1994 Jun 1;152(11):5572–5580. [PubMed] [Google Scholar]
  22. Nicolle M. W., Hawke S., Willcox N., Vincent A. Differences in processing of an autoantigen by DR4:Dw4.2 and DR4:Dw14.2 antigen-presenting cells. Eur J Immunol. 1995 Jul;25(7):2119–2122. doi: 10.1002/eji.1830250749. [DOI] [PubMed] [Google Scholar]
  23. Ong B., Willcox N., Wordsworth P., Beeson D., Vincent A., Altmann D., Lanchbury J. S., Harcourt G. C., Bell J. I., Newsom-Davis J. Critical role for the Val/Gly86 HLA-DR beta dimorphism in autoantigen presentation to human T cells. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7343–7347. doi: 10.1073/pnas.88.16.7343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. Sommer N., Harcourt G. C., Willcox N., Beeson D., Newsom-Davis J. Acetylcholine receptor-reactive T lymphocytes from healthy subjects and myasthenia gravis patients. Neurology. 1991 Aug;41(8):1270–1276. doi: 10.1212/wnl.41.8.1270. [DOI] [PubMed] [Google Scholar]
  29. Tzartos S. J., Seybold M. E., Lindstrom J. M. Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci U S A. 1982 Jan;79(1):188–192. doi: 10.1073/pnas.79.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vincent A., Jacobson L., Shillito P. Response to human acetylcholine receptor alpha 138-199: determinant spreading initiates autoimmunity to self-antigen in rabbits. Immunol Lett. 1994 Mar;39(3):269–275. doi: 10.1016/0165-2478(94)90168-6. [DOI] [PubMed] [Google Scholar]
  31. Wilkinson M. RNA isolation: a mini-prep method. Nucleic Acids Res. 1988 Nov 25;16(22):10933–10933. doi: 10.1093/nar/16.22.10933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Willcox N., Baggi F., Batocchi A. P., Beeson D., Harcourt G., Hawke S., Jacobson L., Matsuo H., Moody A. M., Nagvekar N. Approaches for studying the pathogenic T cells in autoimmune patients. Ann N Y Acad Sci. 1993 Jun 21;681:219–237. doi: 10.1111/j.1749-6632.1993.tb22888.x. [DOI] [PubMed] [Google Scholar]
  33. Willcox N. Myasthenia gravis. Curr Opin Immunol. 1993 Dec;5(6):910–917. doi: 10.1016/0952-7915(93)90105-2. [DOI] [PubMed] [Google Scholar]
  34. Yurin V. L., Rudensky A. Y., Mazel S. M., Blechman J. M. Immunoglobulin-specific T-B cell interaction. II. T cell clones recognize the processed form of B cells' own surface immunoglobulin in the context of the major histocompatibility complex class II molecule. Eur J Immunol. 1989 Sep;19(9):1685–1691. doi: 10.1002/eji.1830190924. [DOI] [PubMed] [Google Scholar]
  35. Zhang Y., Schluep M., Frutiger S., Hughes G. J., Jeannet M., Steck A., Barkas T. Immunological heterogeneity of autoreactive T lymphocytes against the nicotinic acetylcholine receptor in myasthenic patients. Eur J Immunol. 1990 Dec;20(12):2577–2583. doi: 10.1002/eji.1830201208. [DOI] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES