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

Enhanced green fluorescent protein (EGFP) is a novel marker gene product, w hich is readily detectable using techniques of fluorescence microscopy, flo w cytometry, or macroscopic imaging. In the present studies, we have examin ed the immunogenicity of EGFP in murine models. A stable transfectant of th e transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Z eo, was generated. Splenocytes harvested from mice immunized with a recombi nant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with C MS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against GM S4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A numbe r of candidate H2-K-d-binding peptides derived from the EGFP protein were c hosen according to an epitope prediction program and synthesized. These pep tides were tested for their ability to bind to H2-K-d molecules and stimula te IFN gamma -production by splenocytes harvested from Ad-EGFP-immunized mi ce. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP(2 00-208)) which strongly binds to H2-Kd molecules, was identified as a natur ally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.