Immune response to green fluorescent protein: implications for gene therapy

Gene Ther. 1999 Jul;6(7):1305-12. doi: 10.1038/


Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • B7-1 Antigen / immunology
  • Gene Transfer Techniques*
  • Genetic Therapy*
  • Granulocyte-Macrophage Colony-Stimulating Factor / immunology
  • Green Fluorescent Proteins
  • Leukemia, Experimental / immunology*
  • Leukemia, Experimental / therapy
  • Luminescent Proteins / genetics*
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • T-Lymphocytes, Cytotoxic / immunology*
  • Vaccines, DNA


  • B7-1 Antigen
  • Luminescent Proteins
  • Vaccines, DNA
  • Green Fluorescent Proteins
  • Granulocyte-Macrophage Colony-Stimulating Factor