Advertisement

mRNA PCR-Based Epitope Chase Method

  • Jean-Daniel Doucet
  • Dominique Gauchat
  • Réjean Lapointe
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 969)

Abstract

The identification of specific viral and tumor antigen epitopes recognized by CD4+ or CD8+ T lymphocytes remains a challenge. Unfortunately, epitope mapping methods are generally costly and time-consuming. This chapter details a polymerase chain reaction (PCR)-based mRNA epitope identification method called mPEC, which is designed to rapidly and precisely identify relevant T cell epitopes recognized by previously isolated CD8+ or CD4+ T lymphocytes.

This method is based on the use of mRNA fragments synthesized from PCR-amplified cDNA with a variety of 3′end iterative deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to map the epitope in a given protein antigen. Considering mRNA’s sensitivity to degradation, we also insert a control define epitope at the mRNA’s 3′end to control for electroporated mRNA’s integrity and capacity to be translated.

Key words

Major histocompatibility complexes class I and II T cell epitope Epitope mapping mRNA transfection 

Notes

Acknowledgments

This work was supported by the Canadian Institutes of Health Research (CIHR). R.L. and J.-D.D. are recipients of scholarships from the Fonds de la recherche en santé du Québec (FRSQ). The authors would like to thank K. Lieber and L.-A. Hanafi for text editing.

References

  1. 1.
    Soethout EC, Meiring HD, de Jong AP, van Els C (2007) Identifying the epitope-specific T cell response to virus infections. Vaccine 25:3200–3203PubMedCrossRefGoogle Scholar
  2. 2.
    Pilla L, Rivoltini L, Patuzzo R, Marrari A, Valdagni R, Parmiani G (2009) Multipeptide vaccination in cancer patients. Expert Opin Biol Ther 9:1043–1055PubMedCrossRefGoogle Scholar
  3. 3.
    Liu J, Zhang S, Tan S, Zheng B, Gao GF (2011) Revival of the identification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, therapy and vaccine development. Exp Biol Med 236:253–267CrossRefGoogle Scholar
  4. 4.
    Doucet J-D, Gauchat D, Lapointe R (2011) Identification of T-cell epitopes by a Novel mRNA PCR-based epitope chase technique. J Immunother 34:183–186PubMedCrossRefGoogle Scholar
  5. 5.
    Bakker A, Hoppes R, Linnemann C, Toebes M, Rodenko B, Berkers CR, Reker HS, van Esch WJE, Heemskerk MHM, Ovaa H, Schumacher TNM (2008) Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Proc Natl Acad Sci USA 105:3825–3830PubMedCrossRefGoogle Scholar
  6. 6.
    Engelhard VH (2007) The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes. Int J Mass Spectrom 259:32–39PubMedCrossRefGoogle Scholar
  7. 7.
    Robbins P, el-Gamil M, Li Y, Topalian S, Rivoltini L, Sakaguchi K, Appella E, Kawakami Y, Rosenberg S (1995) Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes. J Immunol 154:5944–5950PubMedGoogle Scholar
  8. 8.
    Britten CM, Meyer RG, Graf C, Huber C, Wölfel T (2005) Identification of T cell epitopes by the use of rapidly generated mRNA fragments. J Immunol Methods 299:165–175PubMedCrossRefGoogle Scholar
  9. 9.
    Pelletier S, Tanguay S, Lee S, Gunaratnam L, Arbour N, Lapointe R (2009) TGF-alpha as a candidate tumor antigen for renal cell carcinomas. Cancer Immunol Immunother 58: 1207–1218PubMedCrossRefGoogle Scholar
  10. 10.
    Lapointe R, Royal RE, Reeves ME, Altomare I, Robbins PF, Hwu P (2001) Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100. J Immunol 167: 4758–4764PubMedGoogle Scholar
  11. 11.
    Lapointe R, Bellemare-Pelletier A, Housseau F, Thibodeau J, Hwu P (2003) CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells. Cancer Res 63:2836–2843PubMedGoogle Scholar
  12. 12.
    Schultze JL, Michalak S, Seamon MJ, Dranoff G, Jung K, Daley J, Delgado JC, Gribben JG, Nadler LM (1997) CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. J Clin Invest 100: 2757–2765PubMedCrossRefGoogle Scholar
  13. 13.
    Doucet J-D, Forget M-A, Grange C, Rouxel RN, Arbour N, von Messling V, Lapointe R (2011) Endogenously expressed matrix protein M1 and nucleoprotein of influenza A are efficiently presented by class I and class II major histocompatibility complexes. J Gen Virol 92:1162–1171PubMedCrossRefGoogle Scholar
  14. 14.
    Lepage S, Lapointe R (2006) Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments. Cancer Res 66: 2423–2432PubMedCrossRefGoogle Scholar
  15. 15.
    Niazi KR, Ochoa M-T, Sieling PA, Rooke NE, Peter AK, Mollahan P, Dickey M, Rabizadeh S, Rea TH, Modlin RL (2007) Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences. Immunology 122:522–531PubMedCrossRefGoogle Scholar
  16. 16.
    Wang R-F (2003) Identification of MHC class II-restricted tumor antigens recognized by CD4+ T cells. Methods 29:227–235PubMedCrossRefGoogle Scholar
  17. 17.
    Wang RF, Wang X, Atwood AC, Topalian SL, Rosenberg SA (1999) Cloning genes encoding MHC class II-restricted antigens: mutated CDC27 as a tumor antigen. Science 284: 1351–1352PubMedCrossRefGoogle Scholar
  18. 18.
    Qian J, Xie J, Hong S, Yang J, Zhang L, Han X, Wang M, Zhan F, Shaughnessy JDJ, Epstein J, Kwak LW, Yi Q (2007) Dickkopf-1 (DKK1) is a widely expressed and potent tumor-associated antigen in multiple myeloma. Blood 110:1587–1594PubMedCrossRefGoogle Scholar
  19. 19.
    Contreras CE, Ploton IN, Siliciano RF, Karp CL, Viscidi R, Kumar N (1998) Mapping of specific and promiscuous HLA-DR-restricted T-cell epitopes on the Plasmodium falciparum 27-kilodalton sexual stage-specific antigen. Infect Immun 66:3579–3590PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jean-Daniel Doucet
    • 1
  • Dominique Gauchat
    • 1
  • Réjean Lapointe
    • 1
  1. 1.Research Centre, Centre Hospitalier de l’Université de Montréal (CRCHUM), Hôpital Notre-Dame, Université de Montréal and Institut du Cancer de Montréal (ICM)MontrealCanada

Personalised recommendations