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• W2083756553 abstract "We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP+ cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-γ (IFN-γ), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols. We investigated whether transgene expression levels influence the immunogenicity of transduced hematopoietic grafts upon transplantation into partially myeloablated mice. To this aim, bone marrow cells (BMCs) transduced with retroviral vectors driving green fluorescent protein (GFP) expression either at high (high-EGFP) or low levels (low-EGFP) were transplanted into congenic recipients conditioned with sublethal doses of total body irradiation (TBI) or busulfan. Virtually all recipients showed evidence of donor engraftment 4 weeks after transplantation. However, as opposed to recipients receiving low-EGFP transduced grafts, the risk of rejecting the EGFP+ cells by 30 days after transplantation was significantly higher in mice conditioned with busulfan and receiving high-EGFP transduced grafts. Anti-EGFP cellular immune responses were demonstrated in high-EGFP-treated mice conditioned with busulfan by interferon-γ (IFN-γ), enzyme-linked immunospot assay (ELISPOT), and cytotoxic T lymphocyte (CTL) assays, in contrast to that observed in mice transplanted with low-EGFP BMC. These results show for the first time that transgene expression levels can be critical for the immunogenicity of gene-modified hematopoietic grafts, especially in immunocompetent or in partially immunosuppressed recipients. These results have profound implications in vector choice and in the design of gene therapy (GT) protocols. One of the major challenges gene therapists are faced with is the potential immunogenicity of vectors and transgene products. For ex vivo strategies, such as those used in hematopoietic stem cell (HSC) gene therapy (GT), transgene products constitute the major source of potential antigens because the proteins encoded by the therapeutic genes may contain peptides and epitopes that the host immune system has never “seen” before. Indeed, immune responses to transgene products or to cells expressing transgenes have been reported in several preclinical settings1Yuasa K Sakamoto M Miyagoe-Suzuki Y Tanouchi A Yamamoto H Li J et al.Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product.Gene Ther. 2002; 9: 1576-1588Crossref PubMed Scopus (113) Google Scholar,2Latta-Mahieu M Rolland M Caillet C Wang M Kennel P Mahfouz I et al.Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression.Hum Gene Ther. 2002; 13: 1611-1620Crossref PubMed Scopus (96) Google Scholar,3Ge Y Powell S Van Roey M McArthur JG Factors influencing the development of an anti-factor IX (FIX) immune response following administration of adeno-associated virus-FIX.Blood. 2001; 97: 3733-3737Crossref PubMed Scopus (83) Google Scholar,4Mount JD Herzog RW Tillson DM Goodman SA Robinson N McCleland ML et al.Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.Blood. 2002; 99: 2670-2676Crossref PubMed Scopus (284) Google Scholar,5Gao G Lebherz C Weiner DJ Grant R Calcedo R McCullough B et al.Erythropoietin gene therapy leads to autoimmune anemia in macaques.Blood. 2004; 103: 3300-3302Crossref PubMed Scopus (127) Google Scholar as well as in GT clinical trials.6Traversari C Marktel S Magnani Z Mangia P Russo V Ciceri F et al.The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies.Blood. 2007; 109: 4708-4715Crossref PubMed Scopus (179) Google Scholar,7Herzog RW Dobrzynski E Immune implications of gene therapy for hemophilia.Semin Thromb Hemost. 2004; 30: 215-226Crossref PubMed Scopus (40) Google Scholar,8Zaldumbide A Hoeben RC How not to be seen: immune-evasion strategies in gene therapy.Gene Ther. 2008; 15: 239-246Crossref PubMed Scopus (31) Google Scholar Immunogenicity of transduced cells is determined or influenced by many factors, including the route of entry, the molecular structure of the transgene product, antigen dose, and host factors such as the level of immunocompetence or immune suppression of the host, the genetic background, the histocompatibility molecules, the repertoire of immune cells, or the presence of danger signals at the time and site of antigen presentation by antigen-presenting cells (APCs). Ex vivo HSC gene transfer constitutes a unique situation, because after full or partial myeloablative conditioning the engrafted gene-modified HSCs contribute to rebuilding a lymphohematopoietic system anew, in which the transgene product is presented by transduced immature APC in a tolerogenic manner.9Heim DA Hanazono Y Giri N Wu T Childs R Sellers SE et al.Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model.Mol Ther. 2000; 1: 533-544Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,10Bagley J Bracy JL Tian C Kang ES Iacomini J Establishing immunological tolerance through the induction of molecular chimerism.Front Biosci. 2002; 7: d1331-d1337Crossref PubMed Scopus (28) Google Scholar,11Gunthart M Kearns-Jonker M Gene therapy for the induction of chimerism and transplant tolerance.Curr Gene Ther. 2007; 7: 411-420Crossref PubMed Scopus (7) Google Scholar,12Kang E Giri N Wu T Sellers S Kirby M Hanazono Y et al.In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting.Hum Gene Ther. 2001; 12: 1663-1672Crossref PubMed Scopus (33) Google Scholar Nevertheless, immune responses to transgene products have been reported after transplantation of gene-modified hematopoietic cells in normal, nonmyeloablated mice,13Bubnic SJ Nagy A Keating A Donor hematopoietic cells from transgenic mice that express GFP are immunogenic in immunocompetent recipients.Hematology. 2005; 10: 289-295Crossref PubMed Scopus (26) Google Scholar partially and nonmyeloablated dogs,14Lutzko C Kruth S Abrams-Ogg AC Lau K Li L Clark BR et al.Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency.Blood. 1999; 93: 1895-1905PubMed Google Scholar and even after full (i.e., lethal) myeloablation in nonhuman primates.15Morris JC Conerly M Thomasson B Storek J Riddell SR Kiem HP Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning.Blood. 2004; 103: 492-499Crossref PubMed Scopus (59) Google Scholar This can be a critical issue in clinical GT protocols where transduced hematopoietic grafts are transplanted or infused into weakly or nonimmunosuppressed recipients. In this study, we demonstrate that partially myeloablated mice transplanted with hematopoietic bone marrow cells (BMCs) expressing high levels of enhanced green fluorescent protein (EGFP) are at a much higher risk to develop anti-EGFP immune responses and to reject the transduced cells than those receiving BMC transduced with vectors driving lower levels of EGFP expression, indicating a key role of transgene expression levels in the immunogenic potential and the outcome of the transduced cells. Two retroviral vectors, SF1-EGFP [in which EGFP expression is driven by the retroviral long-terminal repeat, named high-EGFP hereon] and SF91-IiMOG-IRES-EGFP [where EGFP expression is driven by the internal ribosome entry site (IRES), named low-EGFP hereon] were used to transduce BMC (Figure 1). We analyzed the extent to which these two vectors drove different EGFP expression levels in BMC. The multiplicity of infection used was similar for both vectors, and the percentages of transduced cells using both vectors were comparable in all experiments (Table 1). However, high-EGFP transduced BMC yielded a threefold higher mean fluorescence intensity (MFI) values, on average, than their low-EGFP transduced counterparts (Figure 2; Table 1).Table 1Transduction efficiency was assessed by flow cytometry before transplantation, 24 hours after the second transduction cycleLow-EGFP vectorHigh-EGFP vectorExperiment% TransductionMFI ratio% TransductionMFI ratioExperiment I19.396.517.2445.7Experiment II17.350.326.4365.8Experiment III20.2570.616.41,335Experiment IV44.962250.72,076Mean ± SD25.4 ± 13.0334.9 ± 303.2*P < 0.05.27.7 ± 16.01,056.0 ± 809.7*P < 0.05.Abbreviations: EGFP, enhanced green fluorescent protein; MFI, mean fluorescence intensity.MFI ratio was calculated relative to the negative control of each independent assay. Data from four experiments are shown. A one-tail one-way ANOVA test was applied to compare the MFI ratios.* P < 0.05. Open table in a new tab Figure 2LTR drives EGFP expression at higher levels than IRES in BMC. Dot plots from representative samples of nontransduced BMC (left), BMC transduced with the high-EGFP (LTR) vector (center), and BMC transduced with the low-EGFP (IRES) vector (right). Plots show green fluorescence in events after appropriate gating on forward and side scatter parameters and 7-AAD labeling for dead cell exclusion. Note the different levels of EGFP expression driven by the two retroviral vectors. 7-AAD, 7-amino-actinomycin D; BMC, bone marrow cells; EGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; LTR, long-terminal repeat.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Abbreviations: EGFP, enhanced green fluorescent protein; MFI, mean fluorescence intensity. MFI ratio was calculated relative to the negative control of each independent assay. Data from four experiments are shown. A one-tail one-way ANOVA test was applied to compare the MFI ratios. To assess whether the differences in the levels of EGFP expression were important in establishing engraftment of the transduced cells after conditioning with busulfan, BMC from donor B6/SJL-PtprcaPep3b/BoyJ mice (CD45.1+) transduced with both retroviral vectors encoding EGFP were transplanted into partially myeloablated C57BL/6J recipients (CD45.2+) treated either with a sublethal dose of busulfan or with 3 Gy of total body irradiation (TBI). Four to six weeks after transplantation, donor chimerism and the proportion of EGFP+ cells were assessed by flow cytometry in the peripheral blood (PB) and/or in the bone marrow. Donor chimerism was detected in all 17 mice conditioned with TBI and in 57/68 mice conditioned with busulfan. Mice without donor engraftment (11 of 68 mice conditioned with busulfan) were considered primary graft failures and were not included in the analysis. Among the grafted recipients, levels of donor chimerism were similar between groups receiving the same conditioning regimen (TBI/low-EGFP: 11.00 ± 4.20%; TBI/high-EGFP: 8.40 ± 8.10%; P = 0.468. Busulfan/low-EGFP: 7.30 ± 9.60; busulfan/high-EGFP: 3.90 ± 4.80%; P = 0.072) (Figure 3a). Regarding transgene-expressing cells, they were detectable in 24/31 busulfan-treated mice that received low-EGFP BMC (mean levels: 4.0 ± 6.9%) in contrast to only in 3/26 mice receiving high-EGFP BMC (mean levels: 0.02 ± 0.05%; P < 0.0001) (Figure 3b,c). The absence of transduced cells in the high-EGFP treated animals may account for the lower rates of donor engraftment observed in the high-EGFP transplanted mice in comparison with their low-EGFP counterparts, because the transduced cells constituted a significant proportion of the grafts. Among recipients conditioned with TBI, EGFP-expressing cells were detected in 8/11 mice of the high-EGFP group versus 6/6 of their low-EGFP counterparts, although these differences did not reach statistical significance (P = 0.580) (Figure 3b,c). Mean percentages of EGFP+ cells in these groups, 4–6 weeks after transplantation were 6.5 ± 8.00% and 6.5 ± 2.30%, respectively. To assess the capability of the high-EGFP vector to transduce HSC with long-term repopulating ability, chimerism was analyzed 5 months after transplantation in a group of mice conditioned with 3 Gy of TBI and transplanted with high-EGFP BMC. EGFP+ cells were detected in the hematopoietic tissues of 3/5 TBI-treated mice (Table 2). In the remaining two animals, donor engraftment levels were very low and no EGFP+ cells were detected, probably due to a primary graft failure. In agreement with these observations in previous experiments, we had demonstrated that the high-EGFP vector allowed long-term (22 weeks) stable EGFP expression in mice conditioned with different doses of TBI.16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google ScholarTable 2Long-term engraftment data5 m post-BMTMice #%DCh%EGFP+119.06.229.32.831.10.0416.35.550.50.0Mean ± SD9.2 ± 8.52.9 ± 2.9Abbreviations: EGFP, enhanced green fluorescent protein; DCh, donor chimerism; PB, peripheral blood.To assess whether high-EGFP BMC allowed long-term engraftment, the cells were transplanted in five mice conditioned with 3 Gy of TBI and donor chimerism and engraftment of transgene-expressing cells were analyzed in the PB 5 months after BMT. Open table in a new tab Abbreviations: EGFP, enhanced green fluorescent protein; DCh, donor chimerism; PB, peripheral blood. To assess whether high-EGFP BMC allowed long-term engraftment, the cells were transplanted in five mice conditioned with 3 Gy of TBI and donor chimerism and engraftment of transgene-expressing cells were analyzed in the PB 5 months after BMT. The disappearance of EGFP+ cells, though not of untransduced donor-derived cells in mice conditioned with busulfan and transplanted with high-EGFP BMC, reasonably rules out a primary graft failure and points to a specific immune rejection of these cells. To investigate anti-EGFP humoral immune responses, we used a sensitive enzyme-linked immunosorbent assay for anti-EGFP immunoglobin G Ab. Among the mice conditioned with busulfan, the number of animals developing anti-EGFP Ab was significantly higher in the high-EGFP group than in the low-EGFP-treated mice (10/26 versus 0/31, P < 0.0001; Figure 4a). However, when mice clearing the EGFP+ cells (10/33) were compared with those that did not (6/41), regardless of the conditioning and the vector used, no significant differences were found in the percentages of positive mice (P = 0.155; Figure 4b), indicating a lack of impact of the Ab detected on the outcome of the transduced cells. Anti-EGFP cellular immune responses were assessed using interferon-γ (IFN-γ) enzyme-linked immunospot assay (ELISPOT) and cytotoxic T lymphocyte (CTL) assays. For the ELISPOT assays, because the assessment of engraftment requires relatively long observation periods and the cellular immune responses tend to weaken over time, in one experiment the recipients were conditioned with busulfan and killed 10 days after transplantation. In these mice, the frequency of splenocytes producing IFN-γ upon EGFP challenge was significantly higher in high-EGFP treated mice than in the controls (311 ± 174.2 ELISPOTs per 106 splenocytes in the high-EGFP group versus 30.1 ± 32.8 in the low-EGFP and 5.1 ± 6.8 in the nontransplanted animals) (Figure 5a). As a source of syngeneic APCs, we used different clones of irradiated EGFP+ EL4 cells expressing different levels of EGFP (MFI: 2,151, 7,023 and 36,699, respectively). It is noteworthy that only the exposure to the EL4-EGFP clone with the highest level of EGFP expression was able to discriminate between the immune reactivity of high- and low-EGFP groups (data not shown). For the CTL assays, we used thawed splenocytes from mice that were killed 30 days after transplantation of the transduced grafts, and EL4-EGFP as target cells. Results showed a significant antigen-specific cytolytic activity in the mice transplanted with the high-EGFP transduced grafts (all animals had rejected the EGFP+ cells) as opposed to that observed in the low-EGFP recipients (that had not rejected the EGFP+ cells) (Figure 5b). In a series of experiments aimed to investigate minimally myeloablative regimens allowing stable engraftment of gene-modified HSCs, marrow grafts from CD45.1+ mice were transduced with retroviral vectors encoding EGFP (either high-EGFP or low-EGFP) and transplanted into partially myeloablated congenic recipients. This model renders a precise and simultaneous quantification of donor chimerism and transgene-expressing cells in PB samples by flow cytometry allowing to discriminate between the lack of donor cells (which would suggest a primary graft failure) and a lack of transgene-expressing donor cells but not of untransduced cells (which would suggest either an inefficient transduction, gene silencing or a transgene-specific immune rejection). In our study, donor chimerism was detectable in the PB of most recipient mice after transplantation. However, EGFP+ cells were detected in the majority of mice receiving low-EGFP transduced grafts but not in those transplanted with high-EGFP transduced grafts. Inefficient gene transfer can be ruled out because some animals receiving high-EGFP BMC from the same pool had evidence of long-term engraftment of the transduced cells. Gene silencing of high-EGFP vector expression can also be reasonably ruled out because it was not observed when a more stringent conditioning was used.16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google Scholar This inconsistency prompted us to carry out additional experiments to determine whether this was a consistent phenomenon and whether it was a consequence of a transgene-specific immune rejection. In these prospective experiments, we also included groups of mice conditioned with low dose TBI, as a positive control, because in previous experiments we had observed that this partially myeloablative regimen allowed, in our experimental conditions, long-term transgene expression.16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google Scholar Because both vectors used contained the same EGFP sequence and BMC transductions were performed using the same multiplicity of infection, we hypothesized that the most critical factor determining the outcome of EGFP+ cells under our experimental conditions was a differential level of EGFP expression on a per cell basis. In this regard, we first analyzed the extent to which these two vectors drove different transgene expression levels in BMC due to their different transcriptional regulatory elements. The high-EGFP vector clearly resulted in higher EGFP+ expression levels than the low-EGFP vector. In a previous report, levels of EGFP expression driven by the SFβ1 vector long-terminal repeat, measured by flow cytometry, were shown to be five- to sixfold higher than those observed when transgene expression was driven by an IRES sequence placed in the same vector.17Flasshove M Bardenheuer W Schneider A Hirsch G Bach P Bury C et al.Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene.J Cancer Res Clin Oncol. 2000; 126: 391-399Crossref PubMed Scopus (27) Google Scholar Recently, reduced intensity conditioning regimens for hematopoietic transplantation have been developed as an attempt to reduce toxicity while allowing a therapeutic level of hematopoietic engraftment, which can be useful in allogeneic transplantation or GT applications. In this regard, busulfan at low or moderate doses is one of the most useful drugs that avoid the toxicities associated with TBI containing regimens.18Kang EM Hsieh MM Metzger M Krouse A Donahue RE Sadelain M et al.Busulfan pharmacokinetics, toxicity, and low-dose conditioning for autologous transplantation of genetically modified hematopoietic stem cells in the rhesus macaque model.Exp Hematol. 2006; 34: 132-139Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar,19Moayeri M Hawley TS Hawley RG Correction of murine hemophilia A by hematopoietic stem cell gene therapy.Mol Ther. 2005; 12: 1034-1042Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,20Andersson G Illigens BM Johnson KW Calderhead D LeGuern C Benichou G et al.Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice.Blood. 2003; 101: 4305-4312Crossref PubMed Scopus (48) Google Scholar The conditioning with busulfan used in this work is similar to the regimens used in many preclinical studies18Kang EM Hsieh MM Metzger M Krouse A Donahue RE Sadelain M et al.Busulfan pharmacokinetics, toxicity, and low-dose conditioning for autologous transplantation of genetically modified hematopoietic stem cells in the rhesus macaque model.Exp Hematol. 2006; 34: 132-139Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar and in some human hematopoietic GT clinical trials.21Aiuti A Slavin S Aker M Ficara F Deola S Mortellaro A et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (993) Google Scholar,22Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (988) Google Scholar The fact that mice conditioned with busulfan and transplanted with high-EGFP BMC were at much higher risk of rejecting the transduced cells than those conditioned with TBI, could be explained by the more potent immunosuppressive effect of the TBI used in comparison with that of busulfan. Most in vivo studies using gene-modified murine HSC reported so far have relied on conditioning regimens based on TBI, which is both a potent myelosuppressor and an immunosuppressor (as opposed to busulfan, which is nonimmunosuppressant23Yeager AM Shinn C Pardoll DM Lymphoid reconstitution after transplantation of congenic hematopoietic cells in busulfan-treated mice.Blood. 1991; 78: 3312-3316Crossref PubMed Google Scholar), and this may help in explaining why this phenomenon had not been previously observed. EGFP is an intracellular protein that may induce strong CTL responses in rodents24Han WG Unger WW Wauben MH Identification of the immunodominant CTL epitope of EGFP in C57BL/6 mice.Gene Ther. 2008; 15: 700-701Crossref PubMed Scopus (22) Google Scholar,25Annoni A Battaglia M Follenzi A Lombardo A Sergi-Sergi L Naldini L et al.The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but not by CD4+CD25+ regulatory T cells.Blood. 2007; 110: 1788-1796Crossref PubMed Scopus (32) Google Scholar and in nonhuman primates.15Morris JC Conerly M Thomasson B Storek J Riddell SR Kiem HP Induction of cytotoxic T-lymphocyte responses to enhanced green and yellow fluorescent proteins after myeloablative conditioning.Blood. 2004; 103: 492-499Crossref PubMed Scopus (59) Google Scholar,26Rosenzweig M Connole M Glickman R Yue SP Noren B DeMaria 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: 1951-1959Crossref PubMed Scopus (136) Google Scholar Indeed, the immunodominant epitopes responsible for such immune response has been mapped both in BALB/c (H2-Kd) mice27Gambotto A Dworacki G Cicinnati V Kenniston T Steitz J Tüting T et al.Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope.Gene Ther. 2000; 7: 2036-2040Crossref PubMed Scopus (139) Google Scholar and C57BL6/J (H2-Kb) mice24Han WG Unger WW Wauben MH Identification of the immunodominant CTL epitope of EGFP in C57BL/6 mice.Gene Ther. 2008; 15: 700-701Crossref PubMed Scopus (22) Google Scholar as well as in macaques.26Rosenzweig M Connole M Glickman R Yue SP Noren B DeMaria 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: 1951-1959Crossref PubMed Scopus (136) Google Scholar To investigate anti-EGFP immune responses, we used IFN-γ ELISPOT assays, CTL assays, and a sensitive enzyme-linked immunosorbent assay for anti-EGFP Ab. Anti-EGFP Ab were detected only in high-EGFP-treated mice, especially in the group conditioned with busulfan, which had the highest prevalence and mean serum levels. However, Ab were observed only in a minor fraction of the animals and their presence was not predictive of the outcome of the EGFP+ cells. These results suggest that high-EGFP cells can trigger humoral immune responses at a higher rate than their low-EGFP counterparts, and also that most of these Ab are probably not pathogenic or do not play a major role in the rejection of the transduced cells. In a previous report, transgene-expressing hematopoietic cells were also rejected in six macaques after conditioning with 240 cGy of TBI and transplantation with autologous grafts transduced with a vector driving levels of EGFP expression that were equivalent to those driven by our high-EGFP vector. Interestingly, only three animals developed anti-EGFP Ab, although strong CTL responses were identified in five.26Rosenzweig M Connole M Glickman R Yue SP Noren B DeMaria 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: 1951-1959Crossref PubMed Scopus (136) Google Scholar In our experiments, the demonstration of significantly higher number of antigen-specific ELISPOTs and a higher CTL activity strongly supports a CTL-mediated rejection of the EGFP+ cells. It is noteworthy that only the exposure to the EL4-EGFP clone with the highest levels of EGFP expression in the ELISPOT assay was able to discriminate between the immune reactivity of high and low-EGFP groups, indicating that there is a threshold for antigen expression by APC which is required for optimal stimulation of T-cell responses in vitro, which is in accordance with the in vivo results. Antigen dose is a critical factor determining vaccine immunogenicity. In genetic vaccines, changes in the regulatory elements resulting in increased levels of transgene expression enhanced their immunogenicity,28Barouch DH Yang ZY Kong WP Korioth-Schmitz B Sumida SM Truitt DM et al.A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates.J Virol. 2005; 79: 8828-8834Crossref PubMed Scopus (131) Google Scholar which can be useful in cancer applications. In GT for transplantation tolerance, it has been suggested that the level of transgene expression (using non-self major histocompatibility complex molecules) can be critical for tolerance induction.29Bagley J Tian C Sachs DH Iacomini J Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy.Blood. 2002; 99: 4394-4399Crossref PubMed Scopus (67) Google Scholar As for hereditary diseases, GT must minimize the risk of immune sensitization while providing long-term therapeutic expression. In view of these scenarios, vector choice must balance effectiveness and risks, including immunogenicity. The results presented here clearly identify for the first time that the expression level is a very critical risk factor for transgene immunogenicity of transduced hematopoietic grafts, at least when transplanted into partially myeloablated recipients. This work supports the notion that there is a threshold in transgene expression levels that may turn an otherwise theoretically safe procedure into a high-risk intervention, which can not only make the therapeutic attempt useless, but also sensitize the recipient and hinder future gene or replacement therapies. We believe that this concept is highly relevant for vector design and should be taken into account when assessing the safety of human GT. We propose that this issue should be carefully investigated in preclinical studies prior to human GT protocols. Retroviral vector construction and producer cell lines. The generation of pSF1-EGFP (high-EGFP vector) has been described previously.30Limón A Briones J Puig T Carmona M Fornas O Cancelas JA et al.High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.Blood. 1997; 90: 3316-3321PubMed Google Scholar The pSF91-IiMOG-IRES-EGFP (low-EGFP vector) was constructed from the pSF91 backbone31Hildinger M Abel KL Ostertag W Baum C Design of 5′ untranslated sequences in retroviral vectors developed for medical use.J Virol. 1999; 73: 4083-4089Crossref PubMed Google Scholar and has been described elsewhere.32Eixarch H Espejo C Gómez A Mansilla MJ Castillo M Mildner A et al.Tolerance induction in experimental autoimmune encephalomyelitis using non-myeloablative hematopoietic gene therapy with autoantigen.Mol Ther. 2009; 17: 897-905Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar High-titer stable clones of vector producing cells were generated using the NX-e packaging cell line as described elsewhere.16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google Scholar Bone marrow transduction and transplantation. Six to eight-week-old female mice C57Bl/6J (CD45.2) were purchased from Harlan (Barcelona, Spain), and female B6/SJL-PtprcaPep3b/BoyJ congenic mice (CD45.1) were bred in our animal facility. BMC were obtained and transduced as previously described,16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google Scholar using a multiplicity of infection of two. Up to 106 cells transduced with both vectors were injected intravenously into recipients conditioned with two consecutive doses of 20 mg/kg of busulfan (Sigma, St Louis, MO) given on days −3 and −2 pretransplantation, or with a single dose of 3 Gy of TBI given on the day before transplantation. Flow cytometric analysis. Prior to transplantation, transduced BMC were analyzed for EGFP fluorescence using a FACSAria and a FACSCalibur flow cytometer and the DIVA software (BD Biosciences, San Jose, CA). MFI values were considered as indicators of the transgene expression levels. The MFI ratio was calculated by dividing the MFI of the EGFP+ cell population by that of the negative control for each experiment. Donor engraftment and EGFP expression were analyzed by flow cytometry in the PB 3–4 weeks after transplantation and/or in the BM on the day of killing as described elsewhere.16Puig T Kádár E Limón A Cancelas JA Eixarch H Luquín L et al.Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.Gene Ther. 2002; 9: 1472-1479Crossref PubMed Scopus (17) Google Scholar Briefly, donor chimerism and transduction efficiency in vivo (measured as the % of CD45.1+ events and EGFP+ events within the total CD45+ cell population, respectively) were analyzed in the hematopoietic samples using PE-conjugated anti-CD45.1 and allophycocyanin-conjugated anti-CD45.2 (eBioscience, San Diego, CA). Graft failure was defined as the complete absence of donor-derived cells and rejection of EGFP+ cells was suspected when donor-derived cells were present but no EGFP expression was detected. Animals with abnormally low percentages of EGFP+ cells (within the CD45.1+ cell population) in the PB but with no EGFP+ cells in the BM 4–6 weeks after transplantation were considered to be in the process of rejecting the EGFP+ cells and counted as rejectors. Anti-EGFP Ab detection by enzyme-linked immunosorbent assay. Serum samples obtained at different times after transplantation were analyzed for the presence of anti-EGFP Ab. Diluted (1:50) samples were added to EGFP-coated 96-well plates and bound Ab were revealed with horseradish peroxidase–conjugated goat anti-mouse immunoglobin G (H+L) (Caltag Laboratories, Burlingame, CA) and TMB Substrate Reagent Set (BD Biosciences). IFN-γ ELISPOT. Murine splenocytes (0.5 × 106 per ml) were cultured with 5 × 104 irradiated (40 Gy) syngeneic EL4 cells (derived from a murine lymphoma) or with EL4-EGFP transduced cell clones expressing different fluorescence intensities (low, medium, or high; MFI values 2,151, 7,023 and 36,699, respectively) as a source of APC. We adapted a previously described ELISPOT method,25Annoni A Battaglia M Follenzi A Lombardo A Sergi-Sergi L Naldini L et al.The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but not by CD4+CD25+ regulatory T cells.Blood. 2007; 110: 1788-1796Crossref PubMed Scopus (32) Google Scholar using the mouse IFN-γ ELISPOT PLUS kit (Mabtech AB, Nacka Strand, Sweden). Fluorolysometric-CTL assay. To measure antigen-specific cytotoxicity, as the antigen was the fluorescent protein EGFP and we had a transduced EL4 cell clone expressing high levels of EGFP, we used these cells as targets and a flow cytometry based assay as previously described,33Chen K Chen L Zhao P Marrero L Keoshkerian E Ramsay A et al.FL-CTL assay: fluorolysometric determination of cell-mediated cytotoxicity using green fluorescent protein and red fluorescent protein expressing target cells.J Immunol Methods. 2005; 300: 100-114Crossref PubMed Scopus (20) Google Scholar but with some modifications. Briefly, 1:1 mixtures of irradiated (80 Gy) EL4 cells (reference cells, R) and EL4 cells of the clone expressing the highest levels of EGFP (target cells, T) were washed and cocultured with thawed splenocytes (effector cells) at various effector:target cell (E:T) ratios in 200 µl of RPMI 1640 with 20% fetal calf serum in 5% CO2 for 18 hours at 37 °C. Cells were transferred to wells or tubes that contained 100 µl of phosphate-buffered saline with 2% bovine serum albumin, and 3 µl of 7-amino actinomycin D (Molecular Probes, Eugene, OR), final concentration 30 µg/ml, to discriminate between viable and nonviable cells. Samples were acquired on a FACSCanto cytometer (BD Biosciences). The percentage of antigen-specific killing was calculated as (1 − number of target cells in experimental T-R ratio/control T-R ratio) × 100%. This work has been supported by the “Fondo de Investigación Sanitaria” (FIS, PI020205, and 05/1441) and the VI Framework Program of the European Union (CONSERT, LSHB CT2004-005242). We thank the Red Española de Esclerosis Múltiple (REEM) sponsored by the Fondo de Investigación Sanitaria (FIS), Ministry of Science and Innovation, Spain, and the Ajuts per donar Suport als Grups de Recerca de Catalunya (SGR 2005-1081), sponsored by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Spain. H.E., E.K., and M.G. were supported by the VI Framework Program of the European Union (CONSERT, LSHB CT2004-005242). J.B., C.E., J.P., and R.G. are partially supported by the Miguel Servet program from the FIS, Ministry of Science and Innovation, Spain. A.G. is supported by AGAUR. We thank Joseph Graells for help with language edition of the manuscript. This work has been done in Barcelona (Spain)." @default.
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• W2083756553 title "Transgene Expression Levels Determine the Immunogenicity of Transduced Hematopoietic Grafts in Partially Myeloablated Mice" @default.
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