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Immune response to green fluorescent protein: implications for gene therapy

Abstract

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.

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Acknowledgements

We thank Lora Barsky for FACS-sorting the EGFP+ cells, Earl Leonard for the statistical analysis and Punam Malik and Kenneth Weinberg for critical reading of the manuscript. This work was supported by a translational grant from the Leukemia Society of America to DK (LSA Ref No. 6211-98). RS is a recipient of a career development fellowship from the Childrens Hospital Los Angeles and of a Special Fellow Award from the Leukemia Society of America. MCV is supported by an NIH grant (AI33314).

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Stripecke, R., del Carmen Villacres, M., Skelton, D. et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther 6, 1305–1312 (1999). https://doi.org/10.1038/sj.gt.3300951

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