Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Acquired Diseases
  • Published:

Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope

Gene Therapy volume 7, pages 2036–2040 (2000)Cite this article

Abstract

Enhanced green fluorescent protein (EGFP) is a novel marker gene product, which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we have examined the immunogenicity of EGFP in murine models. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein were chosen according to an epitope prediction program and synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNγ-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP200–208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Donnelly JJ, Ulmer JB, Shiver JW, Liu MA . DNA vaccines Ann Rev Immunol 1997 15: 617–648

    Article  CAS  Google Scholar 

  2. Tüting T, Storkus WJ, Falo LD . DNA immunization targeting the skin: molecular control of adaptive immunity J Invest Dermatol 1998 111: 183–188

    Article  PubMed  Google Scholar 

  3. Restifo NP . The new vaccines: building viruses that elicit antitumor immunity Curr Opin Immunol 1996 8: 658–663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang M et al. Active immunotherapy of cancer with a nonreplicating recombinant fowlpoxvirus encoding a model tumor-associated antigen J Immunol 1995 154: 4685–4692

    CAS  PubMed  Google Scholar 

  5. Chen PW et al. Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen J Immunol 1996 156: 224–231

    CAS  PubMed  Google Scholar 

  6. Rao JB et al. IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines J Immunol 1996 156: 3357–3365

    CAS  PubMed  Google Scholar 

  7. Condon C et al. DNA-based immunization by in vivo transfection of dendritic cells Nature Med 1996 2: 1122–1128

    Article  CAS  PubMed  Google Scholar 

  8. Tüting T, Gambotto A, Storkus WJ, De Leo AB . Co-delivery of T helper 1-biasing cytokine genes enhances the efficacy of gene gun immunization of mice: studies with the model tumor antigen β-galactosidase and the BALB/c Meth A p53 tumor-specific antigen Gene Therapy 1999 6: 629–636

    Article  PubMed  Google Scholar 

  9. Tüting T et al. Induction of tumor antigen-specific immunity using DNA immunization in mice Cancer Gene Ther 1999 6: 73–80

    Article  PubMed  Google Scholar 

  10. Chalfie M et al. Green fluorescent protein as a marker for gene expression Science 1994 263: 802–805

    Article  CAS  PubMed  Google Scholar 

  11. Zolotukhin S et al. A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells J Virol 1996 70: 4646–4654

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Heim R, Cubitt AB, Tsien RY . Improved green fluorescence Nature 1995 373: 663–664

    Article  CAS  PubMed  Google Scholar 

  13. Stearns T . Green fluorescent protein. The green revolution Curr Biol 1995 5: 262–264

    Article  CAS  PubMed  Google Scholar 

  14. Cheng L, Fu J, Tsukamoto A, Hawley RG . Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells Nat Biotechnol 1996 14: 606–609

    Article  CAS  PubMed  Google Scholar 

  15. Cormack BP, Valdivia RH, Falkow S . FACS-optimized mutants of the green fluorescent protein (GFP) Gene 1996 173: 33–38

    Article  CAS  PubMed  Google Scholar 

  16. Stripecke R et al. Immune response to green fluorescent protein: implications for gene therapy Gene Therapy 1999 6: 1305–1312

    Article  CAS  PubMed  Google Scholar 

  17. Parker KC, Bednarek MA, Coligan JE . Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains J Immunol 1994 152: 163–175

    CAS  PubMed  Google Scholar 

  18. Salter RD, Howell DN, Cresswell P . Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids Immunogenetics 1985 21: 235–246

    Article  CAS  PubMed  Google Scholar 

  19. Nijman HW et al. Identification of peptide sequences that potentially trigger HLA-A2.1-restricted cytotoxic T lymphocytes Eur J Immunol 1993 23: 1215–1219

    Article  CAS  PubMed  Google Scholar 

  20. Grabbe S, Beissert S, Schwarz T, Granstein RD . Dendritic cells as initiatiors of tumor immune responses: a possibe strategy for tumor immunotherapy? Immunol Today 1995 16: 116–120

    Google Scholar 

  21. Schuler G, Steinman RM . Dendritic cells as adjuvants for immune-mediated resistance to tumors J Exp Med 1997 186: 1183–1187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tüting T, De Leo AB, Lotze MT, Storkus WJ . Bone marrow-derived dendritic cells genetically modified to express tumor-associated antigens induce antitumor immunity in vivo Eur J Immunol 1997 27: 2702–2707

    Article  PubMed  Google Scholar 

  23. Tüting T et al. Dendritic cell-based genetic immunization in mice with a recombinant adenovirus encoding murine TRP2 induces effective anti-melanoma immunity J Gene Med 1999 6: 400–406

    Article  Google Scholar 

  24. Hardy S et al. Construction of adenovirus vectors through Cre–lox recombination J Virol 1997 71: 1842–1849

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institute of Health (NIH) Grants CA 64623 (ABD), CA59371 (PDR), a Telethon Grant (AG), and grant Tu 90/2–1 from the Deutsche Forschungsgemeinschaft (TT).

Author information

Authors and Affiliations

  1. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    A Gambotto, T Kenniston & PD Robbins

  2. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    A Gambotto

  3. Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    G Dworacki, V Cicinnati & AB DeLeo

  4. Department of Dermatology, University of Mainz, Germany

    J Steitz & T Tüting

  5. University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    AB DeLeo

Authors
  1. A Gambotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G Dworacki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V Cicinnati
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. T Kenniston
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Steitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. T Tüting
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. PD Robbins
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. AB DeLeo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gambotto, A., Dworacki, G., Cicinnati, V. et al. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther 7, 2036–2040 (2000). https://doi.org/10.1038/sj.gt.3301335

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301335

Keywords

This article is cited by

Search

Advanced search

Quick links