eGFP-transduced marrow cells) or in vitro CTL responses in the recipients. These findings, coupled with those of. Denaro et al7 (vide infra), suggest that eGFP is ...
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eGFP-transduced marrow cells) or in vitro CTL responses in the recipients. These findings, coupled with those of Denaro et al7 (vide infra), suggest that eGFP is not highly immunogenic in C57BL/6 mice. Clearly, eGFP is immunogenic in Balb/c mice and, as reported recently, in rhesus macaque monkeys.3 The lack of immunogenicity of this protein in C57BL/6 mice may be an exception rather than the rule. C57BL/6 mice have also been noted to have relatively weak immunologic responses to adenoviral vector proteins and human factor IX protein, compared with a number of other mouse strains.4–6 Our original caution pertaining to the use of eGFP as a selectable marker for in vivo studies, especially those intended for human subjects remains valid. We now propose the additional caution that eGFP may be a poor indicator of immune responses or induction of tolerance to transgene products in C57BL/6 mice. D Skelton1, N Satake1 and DB Kohn1,2,3 Division of Research Immunology/Bone Marrow Transplantation, John Connell Gene Therapy Program, Childrens Hospital Los Angeles, Los Angeles, CA, USA; 2 Department of Pediatrics, USC Keck School of Medicine, Los Angeles, CA, USA; and 3Department of Molecular Microbiology and Immunology, USC Keck School of Medicine, Los Angeles, CA, USA 1
EGFP-transduced EL-4 cells form tumors in C57BL/6 mice The immunogenicity of the enhanced green fluorescent protein (EGFP) has recently been demonstrated in mice.1,2 In particular, syngeneic tumor cells transduced with retroviral (RV) vectors expressing EGFP have been reported to be rejected following transplantation in immunocompetent C57BL/6 and BALB/c recipients.1 In the BALB/c mouse strain, the mechanism for rejection of CMS4 sarcoma and BM185 leukemia cells has been shown to be mediated by cytotoxic T-lymphocytes (CTLs) that recognize the foreign EGFP peptides.1,2 Furthermore, CTL responses have been observed in rhesus monkeys receiving a low nonmyeloablative dose of radiation and EGFP-transduced autologous hematopoietic cells.3 Attracted by the technical ease of using rejection in a solid tumor model for studying immunological tolerance, our laboratory has conducted a set of experiments using EL-4 T cell lymphoma cells growing in C57BL/6 mice, similar to those described by Stripecke et al.1 Briefly, EL4 cells (ATCC, Rockville, MD, USA) were transduced with a RV vector denoted GBiP. This vector contains an EGFP (G) cDNA followed by the human immunoglobulin heavy chain binding protein (BiP) which is an internal ribosomal entry site (IRES) inserted in a pLN4 backbone modified with a myeloproliferative sarcoma virus (MPSV) enhancer in the 3’LTR.5 The plasmid pGBiP-6 was deposited with the ATCC (No. PTA 2498). An EGFP+ EL-4 clone (EL4xEGFP) was established that Gene Therapy
References 1 Riddell SR et al. T cell-mediated rejection of gene-modified HIVspecific cytotoxic T lymphocytes in HIV-infected patients. Nat Med 1996; 2: 216–223. 2 Stripecke R et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Therapy 1999; 6: 1305–1312. 3 Rosenzweig M et al. Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells. Blood 2001; 97: 1951–1959. 4 Michou AI et al. Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Therapy 1997; 4: 473–482. 5 Barr D et al. Strain-related variations in adenovirally-mediated transgene expression from mouse hepatocytes in vivo comparisons between immunocompetent and immunodeficient strains. Gene Therapy 1995; 2: 151–155. 6 Fields PA et al. Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice. Gene Therapy 2001; 8: 354–361. 7 Denaro M et al. EGFP-transduced EL-4 cells form tumors in C57BL/6 mice. Gene Therapy 2001; 8: 1814–1815.
has stable EGFP expression with a mean fluorescence intensity of approximately 1000-fold above nontransduced wild-type (wt) cells. Additionally, EL-4 cells expressing the marker neo were established (EL4- neoR) by transduction with an amphotropic pLXSN4 vector and selection in 1 mg/ml G418. A neo-resistant pool of the EGFP+ EL-4 clone was established also by transduction with pLXSN (EL4xEGFP-neoR). In four separate experiments using a dose of 5 × 104 cells injected subcutaneously (s.c.) in the flank of female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA), EL4xEGFP develop into tumors as readily as wt EL-4 cells. Strong EGFP expression was still detectable in the cell suspension from tumors arising in the recipients of EL4xEGFP cells. Expression of MHC class I (H-2Db) in transduced cells was monitored using an anti-H-2Db antibody (Clone KH95; PharMingen, San Diego, CA, USA) and found to be equivalent to wt EL-4 cells. We reasoned that the detection of immunological rejection might depend on the size of the tumor cell inoculum. However, this did not appear to be a significant factor as a lack of growth inhibition of EGFP-transduced EL-4 cells, as compared with the wt cells was also seen at lower cell doses (Figure 1). In a separate experiment, neoR-transduced EL-4 cells, with or without co-transduction with EGFP, all formed tumors. Therefore, our results appear to be at variance with the previous findings of Stripecke et al1 who reported that three mice inoculated with EL-4 cells containing both neomycin resistance and EGFP genes failed to develop tumors. We would con-
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clude that the use of EL-4 T cell lymphoma cells implanted in C57BL/6 mice is not consistently applicable as a model for demonstrating immunological rejection due to expression of neomycin resistance and EGFP as foreign genes. A possible explanation for our negative results with this particular tumor and mouse strain is that the antigen presentation of EGFP and neoR-derived peptides by C57BL/6 H-2Db molecules may be deficient as compared with BALB/c H-2Kd molecules.2
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M Denaro1, B Oldmixon1, C Patience1, G Andersson2 and J Down1 1 BioTransplant Incorporated, Building 75, Charlestown Navy Yard, Charlestown, MA 02129, USA; and 2Present address: Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala BioMedical Center, Box 597, S-751 24 Uppsala, Sweden
References 1 Stripecke R et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Therapy 1999; 6: 1305–1312. 2 Gambotto A et al. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2Kd-restricted CTL epitope. Gene Therapy 2000; 7: 2036–2040. 3 Rosenzweig M et al. Induction of cytoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34+ hematopoietic cells. Blood 2001; 97: 1951–1959. 4 Miller AD, Rosen GJ. Improved retroviral vectors for gene transfer and expression. BioTechniques 1989; 7: 980–990. 5 Banerjee PT et al. A polycistronic retrovirus vector for expression of swine MHC class II DR␣/ heterodimers. Xenotransplantation 1997; 4: 161–173.
Figure 1 Tumor growth of EL-4 and EL4xEGFP in C57BL/6 mice at three cell doses as indicated. In each case, cells were injected s.c. on the right flank of female C57BL/6 mice. After tumor growth became palpable, growth was monitored every 1 to 2 days. Two measurements were made with calipers; the average size (mm) is shown. The results shown in panel (c) (at 5 × 104 cells per mouse) is representative of four experiments.
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