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December 2000, Volume 7, Number 23, Pages 2036-2040
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Acquired Diseases
Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope
A Gambotto1,2, G Dworacki3, V Cicinnati3, T Kenniston1, J Steitz4, T Tüting4, P D Robbins1 and A B DeLeo3,5

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

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

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

4Department of Dermatology, University of Mainz, Germany

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

Correspondence to: A Gambotto, Department of Surgery, University of Pittsburgh, School of Medicine, Biotech Center, Room 208, 300 Technology Drive, Pittsburgh, PA 15219, USA

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 IFNbold gamma-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. Gene Therapy (2000) 7, 2036-2040.

Keywords

EGFP; BALB/c; epitope; H2-Kd

Genetic immunization represents a novel strategy for the experimental development of antigen-specific vaccines against infectious diseases and cancer.1,2 Several nonviral as well as viral gene transfer systems are currently being investigated and compared with other vaccine vehicles consisting of synthetic peptides or recombinant proteins.2,3 The use of beta-galactosidase, chicken ovalbumin, hen egg lysozyme, and viral antigens such as influenza hemaglutinin or ECMV glycoprotein as model antigens in murine studies has greatly improved our understanding of cellular immune responses at the molecular level. The identification of peptide epitopes derived from these antigens which bind to MHC class I and MHC class II molecules and are recognized by CD8+ and CD4+ T cells, respectively, allows for the assessment of the efficacy of various vaccine strategies in stimulating antigen-specific cellular immunity in these experimental models. This knowledge has also been applied in the field of tumor immunology. For example, it has been shown that immunization of mice against the model antigen beta-galactosidase or chicken ovalbumin using various gene delivery systems can protect against a subsequent challenge with tumor cells stably transduced with the same model antigen.2,3,4,5,6,7,8,9 These experimental systems are currently employed for the comparison and continued development of effective vaccine strategies particularly as a therapy of established tumors.

Recently, cDNA encoding a green fluorescent protein (GFP) was isolated from the jellyfish, Aequorea victoria. GFP absorbs blue light (maximum 395 nm) and emits green light (maximum 509 nm) due to its particular protein structure.10 The intrinsic fluorescence of GFP does not require any additional proteins, substrates, or cofactors. Moreover, GFP fluorescence is stable, species-independent, and can be monitored noninvasively using techniques of fluorescence microscopy, flow cytometry, and macroscopic imaging.10,11,12,13 For these reasons, GFP is now routinely used as a reporter molecule to monitor gene expression.14 Enhanced green fluorescent protein (EGFP) is a red shifted variant, which fluoresces much more intensely than wild-type GFP. Importantly, EGFP can readily be detected by flow cytometry because it efficiently absorbs light at 488 nm emitted by the FACS laser.15

One potential problem with the use of EGFP as marker is that it may be immunogenic, thus limiting its use in clinical studies. However, if EGFP is immunogenic it could be useful as a model antigen for development of tumor-specific vaccines. To this end, the immunogenic BALB/c sarcoma CMS4 was stably transduced with DFG-EGFP-IRES-Zeo, a retrovirus encoding EGFP and the zeocin resistance gene. Interestingly, expression of EGFP, but not the zeocin resistance gene, abrogated growth of CMS4 tumors following subcutaneous injection of 106 cells in immunocompentent BALB/c (Figure 1) but not in SCID mice (data not shown). This indicates that endogenous expression of EGFP enhances the immunogenicity of CMS4 sarcoma cells. Our results agree with recently published observations demonstrating that constitutive expression of EGFP in the pre-B cell lymphoma BM185 inhibits leukemia development in immunocompetent BALB/c but not in nude mice.16

In subsequent experiments, the cytotoxic T cell response against EGFP was studied in vitro. BALB/c mice were immunized by a single intraperitoneal injection of 5 ´ 108 p.f.u. of Ad-EGFP, a recombinant adenovirus encoding EGFP. A control group of mice received the same amount of Ad-Psi5, a recombinant adenovirus without insert. Two or three weeks later, splenocytes were harvested from both groups of immunized mice, restimulated in vitro with irradiated CMS4-EGFP-Zeo for 5 days, and assessed for their cytotoxic activity against CMS4-EGFP-Zeo and CMS4-Zeo in standard chromium release assays (Figure 2). The results indicated that the effectors obtained from Ad-EGFP-immunized mice were cytotoxic to CMS4-EGFP-Zeo, but not the control CMS4-Zeo cell line, and that effectors obtained from control Ad-Psi-immunized mice displayed no cytotoxic reactivity against either target cell line tested. These experiments demonstrated the existence of cytotoxic effectors, presumably CTL, which recognize EGFP endogenously expressed in CMS4 sarcoma cells of BALB/c origin. A similar CTL response was observed in BALB/c mice after rejection of BM185 expressing EGFP.16

In order to utilize EGFP as a potential model tumor antigen, a peptide epitope recognized by EGFP-specific CTL was identified. The computer program developed by Parker et al17 was consulted for selection of potential MHC class I-binding epitopes. The 10 EGFP-encoded peptides with the highest predicted binding affinities for H2-Kd molecules were selected for synthesis. The algorithm did not yield any peptides with significant binding affinities for H-2Dd molecules. The selected peptides were first evaluated for their ability to stabilize the expression of H2-Kd molecules on the surface of the TAP-deficient cell line T2,18 stably transfected to express H-2Kd molecules. Only empty MHC class I molecules arrive on the surface of T2 cells due to lack of functional TAP. These are unstable at 37°C and rapidly disintegrate. However, MHC class I molecules are considerably more stable at room temperature and can be loaded with peptides externally. Peptide-MHC class I complexes remain stable when T2 cells are shifted to 37°C. The stabilization of MHC class I expression on the surface of T2 cells under these conditions therefore indirectly reflects the binding affinity of a given peptide to MHC class I molecules.19 As shown in Figure 3, two of the 10 candidate peptides, KFICTTGKL (EGFP46-54) and HYLSTQSAL (EGFP200-208), were able to stabilize expression of H2-Kd on T2-Kd cells.

The candidate EGFP-derived peptides were analyzed for recognition by cytotoxic effectors induced from Ad-EGFP-immunized BALB/c mice. Using the ELISPOT technique, each peptide was evaluated for its ability to stimulate the production of IFNgamma among splenocytes harvested from Ad-EGFP-immunized mice. As indicated in Figure 4, only the EGFP-derived peptide HYLSTQSAL (EGFP200-208), was recognized by these effectors. This peptide also displayed the highest binding affinity to H2-Kd molecules (see Figure 3). The reactivity to HYLSTQSAL (EGFP200-208) was strong compared with that observed with other CTL-defined peptides derived from beta-galactosidase or ovalbumin (data not shown). To confirm that EGFP-specific CTL recognize the EGFP200-208 epitope, EGFP-specific CTL generated by in vitro restimulation of splenocytes harvested from Ad-EGFP-immunized mice with irradiated CMS4-EGFP-Zeo were tested for cytotoxic reactivity against CMS4 cells pulsed with either EGFP46-54 or EGFP200-208 peptides. As shown in Figure 5, EGFP-specific CTL only recognized CMS4 cells pulsed with the EGFP200-208 peptide. These results demonstrate that the EGFP-derived peptide HYLSTQSAL (aa 200-208) represents an immunogenic H2-Kd-restricted CTL epitope.

The identification of EGFP200-208 as an H-2Kd-restricted CTL epitope facilitates the use of EGFP as a model tumor antigen for the continued development of vaccine strategies for tumor immunotherapy. Importantly, all novel gene delivery methods are currently being evaluated for their transduction efficiency in vitro and in vivo using EGFP cDNA. These methods can now readily be assessed for their ability to stimulate EGFP-specific CTL using the EGFP200-208 epitope in BALB/c mice. Furthermore, the purified recombinant protein is available, allowing for detection of EGFP-specific T helper cell responses as well as antibodies. The utility of EGFP as a model antigen can best be demonstrated in the development of dendritic cell-based genetic immunization. Dendritic cells are bone marrow-derived leukocytes, which are critical antigen presenting cells for the induction of cellular immunity.20,21 Dendritic cells can be generated in culture, transduced with antigen ex vivo, and applied as a vaccine. Transduction efficiency and dendritic cell phenotype and function are key parameters for vaccine efficacy. Using EGFP as a model antigen, transduction efficiency and dendritic cell phenotype can easily be monitored using flow cytometry for a given vaccine strategy.22,23 Using the EGFP200-208 peptide epitope and recombinant EGFP, we have been able to assess simultaneously the induction of CTL and antibody responses in BALB/c mice following genetic immunization with recombinant adenovirus either injected directly or with cultured dendritic cells (data not shown). Novel gene delivery systems for antigen expression in dendritic cells both in vitro or directly in vivo are currently being developed for vaccine purposes. Our results will allow the use of EGFP not only as a marker for DC gene transfer but as a model tumor antigen.

These results are also of considerable value for the continued development of gene delivery systems able to mediate long-term transgene expression in vivo. This is a requirement for many applications of gene therapy such as gene correction or replacement. A major obstacle of current research is the induction of immune responses to transgene products using available gene transfer technologies, severely limiting the duration of gene expression in vivo. An ideally suited strategy for long-term gene transfer using EGFP as a marker could involve induction of tolerance to the EGFP200-208 peptide without detectable CTL or antibody responses.

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).

References

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

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, MEDLINE

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, MEDLINE

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

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

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 beta-galactosidase and the BALB/c Meth A p53 tumor-specific antigen. Gene Therapy 1999; 6: 629-636, MEDLINE

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

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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, MEDLINE

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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, MEDLINE

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

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

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, MEDLINE

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

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, MEDLINE

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,

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

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, MEDLINE

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,

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Figures

Figure 1 Expression of EGFP in the BALB/c CMS4 sarcoma abrogates in vivo tumor growth. The murine methylcholanthrene-induced BALB/c CMS4 sarcoma was stably transduced with EGFP using the retroviral vector DFG-EGFP-IRES-Zeo. As a control, CMS4 was also stably transduced with MFG-Zeo. Supernatants containing recombinant retroviruses were produced by transient calcium phosphate transfection of the BOSC23 packaging cell line according to standard protocols. Transduction of CMS4 was performed in the presence of 8 mug/ml polybrene. Stably transduced cell lines were selected with 100 mug/ml zeocin and expression of EGFP was verified using fluorescence microscopy. Groups of five female BALB/c mice (6-10 weeks old, obtained from Jackson Laboratory, Bar Harbor, ME, USA) were injected with 106 cells subcutaneously in the flank. Tumor size was determined by measuring perpendicular tumor diameters with a vernier caliper. Results are reported as mean tumor area (mm2) ± s.e.m. Data are representative of three independent experiments.

Figure 2 EGFP is recognized by cytotoxic lymphocytes in vitro. BALB/c mice were immunized by intraperitoneal injection of 5 ´ 108 p.f.u. Ad-EGFP, a recombinant E1- and E3-deleted adenoviral vector expressing EGFP. A control group of mice was immunized with Psi5, the corresponding recombinant adenovirus without insert. Ad-EGFP was constructed through Cre-lox recombination as previously described,24 propagated on CRE8 or 293 cells, and purified by cesium chloride density gradient centrifugation followed by dialysis according to standard protocols. Titers of viral particles were determined by optical densitometry, and recombinant viruses were stored in 3% sucrose at -80°C. Splenocytes were harvested 2-3 weeks after immunization and red blood cells depleted. 106 Cells were restimulated with 4 ´ 104 irradiated (20 kRad) CMS4-EGFP-Zeo tumor cells in 2 ml of complete medium per well of a 24-well plate containing 20 IU/ml recombinant human IL-2 (Chiron, Emeryville, CA, USA). In vitro-restimulated lymphocytes were tested for their cytolytic reactivity against CMS4-EGFP-Zeo and CMS4-Zeo in standard 4-h 51Cr release assays using 96-well round-bottom plates. 2 ´ 106 Target cells were radiolabeled with 100 muCi Na2-51CrO4 (NEN-Dupont, Bedford, MA, USA) for 1 h at 37°C. The percentage of specific 51Cr release was calculated as 100 ´ (experimental release - spontaneous release)/(maximum release - spontaneous release). Target cells incubated in medium alone or in medium containing 5% Triton-X 100 were used to determine spontaneous and maximum 51Cr release, respectively. Data represent one experiment of four.

Figure 3 Identification of two H2-Kd-binding peptides derived from EGFP. A search for H-2Kd-binding peptides derived from EGFP was performed using the algorithm developed by Dr K Parker. The following peptides were chosen for further analysis: LFTGVVPIL (EGFP8-16), GVVPILVEL (EGFP11-19), KFICTTGKL (EGFP46-54), CFSRYPDHM (EGFP71-79), GYVQERTIF (EGFP92-100), DFKEDGNIL (EGFP130-138), EYNYNSHNV (EGFP143-151), NYNSHNVYI (EGFP145-153), VLLPDNHYL (EGFP194-202) and HYLSTQSAL (EGFP200-208). These peptides were synthesized by standard F-moc chemistry and purified by HPLC by the Peptide Synthesis facility of the University of Pittsburgh Cancer Institute (shared resource). The binding-activity of candidate peptides to H2-Kd molecules was determined by their ability to stabilize the expression of Kd on the TAP-deficient T2 cells expressing H2-Kd (T2-Kd cells). T2-Kd cells were incubated at RT for 12-18 h to increase surface expression of MHC molecules. Subsequently, 2 ´ 105 cells were incubated in 200 mul of medium containing the various peptides at concentrations of 10-4 to 10-10 M for 3 h at RT. Cells were then shifted to 37°C. After 3 h, surface expression of H2-Kd was analyzed by flow cytometry using the FITC conjugated anti-H2-Kd antibody (Pharmingen, San Diego, CA, USA; clone SF1-1.1). A FITC-conjugated anti-H2-Kb antibody (Pharmingen; clone AF6-88.5) served as negative control. Data represent one experiment of two.

Figure 4 Splenocytes harvested from EGFP-immune mice secrete IFNgamma in response to the EGFP-derived peptide HYLSTQSAL200-208. Mice were immunized by intraperitoneal injection of 5 ´ 108 p.f.u. Ad-EGFP. Splenocytes were harvested 2-3 weeks after immunization and red blood cells depleted. Splenocytes were seeded in Millipore HA 96-well plates (Bedford, MA, USA) (2 ´ 105 cells per well) which had been coated overnight with 10 mug/ml (50 mul per well) of anti-mIFNgamma mAb (R4-6A2; Pharmingen) in PBS. After 48 h cells were washed out of the plates and bound cytokines visualized by incubation with 2.5 mug/ml (50 mul per well) of biotinylated anti-mIFNgamma mAb (XMG1.2; Pharmingen) for 1.5 h at 37°C, followed by 100 ml per well streptavidin-peroxidase (Boehringer Mannheim, Indianapolis, IN, USA; 1:1500 dilution in PBS containing 1% BSA, and 0.05% Tween20) for 30 min at RT, and premixed peroxidase substrate kit DAB (Vector, Burlingame, CA, USA). The number of spots were automatically determined by computer-assisted video image analysis (CVIA) using an autoimager software (KS Elispot 4.1 version; Zeiss- Kontron, Jena, Germany). Data are representative of three independent experiments.

Figure 5 The EGFP-derived peptide HYLSTQSAL200-208 is recognized by cytotoxic lymphocytes in vitro. Mice were immunized by intraperitoneal injection of 5 ´ 108 p.f.u. Ad-EGFP; splenocytes were harvested and restimulated in vitro with CMS4-EGFP-Zeo as described in Figure 2. In vitro-restimulated lymphocytes were tested for their cytolytic reactivity against CMS4 cells pulsed with 1 mug/ml of the EGFP-derived peptides KFICTTGKL46-54 and HYLSTQSAL200-208 in standard 4-h 51Cr release assays, as described in Figure 2. Data represent one experiment of three.

Received 21 June 2000; accepted 14 September 2000
December 2000, Volume 7, Number 23, Pages 2036-2040
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