Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer (original) (raw)
Given the limited number of animals and reagents available to conduct immunological studies in large-animal models such as hemophilia B dogs, we designed experiments in mice that would allow us to investigate reduced incidence of inhibitor formation following hepatic gene transfer.
Sustained systemic expression of hF.IX in immunocompetent mice by hepatic gene transfer. Vectors AAV-EF1α-hF.IX and AAV-ApoE/hAAT-hF.IX were produced for expression of hF.IX from the ubiquitous EF1α promoter or a hepatocyte-specific ApoE enhancer/human α1-anti–trypsin promoter combination. These vectors were infused into the portal circulation via the spleen for efficient gene transfer to the liver. Recipients of gene transfer were male immunocompetent mice of three different inbred strains with defined MHC haplotypes: C57BL/6 (H-2b), BALB/c (H-2d), and C3H (H-2k). Following injection of AAV-EF1α-hF.IX (1011 vector genomes [vg’s] per animal), C57BL/6 and BALB/c mice showed sustained systemic expression of hF.IX at therapeutic levels (Figure 1, a and b). C3H mice transiently expressed low levels of hF.IX (< 15 ng/ml, 6 weeks), but developed anti-hF.IX (mostly IgG-1) starting 9 weeks after injection (Figure 1, c and g). No anti-hF.IX was measured in BALB/c mice (Figure 1f). C57BL/6 developed low-titer, non-neutralizing IgG-2b at late time points. In other experiments with the AAV-EF1α-hF.IX vector, we have also observed a late IgG-2b response in BALB/c mice (data not shown). After injection of 5 × 1011 vg, C3H mice also showed sustained expression (Figure 1d), either without anti-hF.IX (four of five) or a mixed IgG response that was not neutralizing to transgene expression (one of five; Figure 1, e and h). Following gene transfer with the AAV-ApoE/hAAT-hF.IX vector, all mice displayed sustained expression without any detectable anti-hF.IX formation for at least 20 weeks after gene transfer (Figure 2). Levels of expression were substantially higher with this vector compared with the EF1α promoter. For both vectors, C57BL/6 gave the highest expression levels followed by BALB/c, then by C3H mice. None of the injected mice had circulating IgA, IgE, or IgG3 anti-hF.IX (data not shown).
Plasma levels of hF.IX and anti-hF.IX (measured by ELISA or immuno-capture assay) in immune-competent mice as a function of time after liver-directed vector administration (AAV-EF1α-hF.IX vector). (a–d) hF.IX in ng/ml. (e–h) IgG anti-hF.IX in ng/ml. Red lines: IgG-1; blue lines IgG-2a; green lines: IgG-2b. (a and e) C57BL/6 mice (n = 4, 1 × 1011 vg/animal). (b and f) BALB/c mice (n = 4, 1 × 1011 vg/animal). (c and g) C3H mice (n = 4, 1 × 1011 vg/animal). (d and h) C3H mice (n = 5, 5 × 1011 vg/animal). Each line represents an individual animal. Symbols are identical for hF.IX and anti-hF.IX levels of the same animal (note that only one animal in f had an anti-hF.IX response). Vertical arrows indicate challenge by subcutaneous administration of 2 μg hF.IX formulated in CFA.
Plasma levels of hF.IX and anti-hF.IX (measured by ELISA or immuno-capture assay) in immunocompetent mice as a function of time after liver-directed vector administration (AAV-ApoE/hAAT-hF.IX vector, 1011 vg/animal). (a–c) hF.IX (ng/ml). (d–f) IgG anti-hF.IX (ng/ml). Red lines: IgG1; blue lines: IgG-2a; green lines: IgG-2b. (a and d) C57BL/6 mice (n = 3). (b and e) BALB/c mice (n = 3). (c and f) C3H mice (n = 4). Each line represents an individual animal. Symbols are identical for hF.IX and anti-hF.IX levels of the same animal. Vertical arrows indicate challenge by subcutaneous administration of 2 μg hF.IX formulated in CFA.
Sustained expression is associated with induction of immune tolerance. Since mice of different strains showed sustained expression of the hF.IX antigen and failed to mount a neutralizing anti-hF.IX response following hepatic gene transfer, we sought to investigate the nature of this immunological unresponsiveness to the transgene product. Immune-competent mice are not tolerant to the non–species-specific hF.IX antigen (despite approximately 80% homology with mF.IX) (18, 26). Unresponsiveness of the immune system may be the result of ignorance (e.g., due to lack of efficient antigen-derived peptide presentation following this route of administration). If the murine immune system was simply ignoring the hF.IX antigen, an immune response should occur given the proper immunological challenge. Alternatively, transgene expression may have induced immune tolerance. After challenge by subcutaneous injection of hF.IX (2 μg) in CFA, mice treated previously with hepatic gene transfer continued to express hF.IX without anti-hF.IX formation (Figures 1–1), while naive mice or mice injected with an AAV-GFP vector developed anti-hF.IX within 14 days after immunization. These mice developed IgG-1 anti-hF.IX, with some animals additionally synthesizing IgG-2a and IgG-2b (Figure 3 and data not shown). A limited number of mice that failed to produce anti-F.IX after the first adjuvant challenge (n = 2 per strain, n = 6 for C57BL/6 mice) were followed for several weeks after the second adjuvant boost and also did not show anti-hF.IX at these later time points (data not shown). Since mice lacked immune responses after stringent immunological challenge, unresponsiveness cannot be explained by ignorance. Thus, hepatic gene transfer does not simply avoid immune responses, but induces tolerance to the hF.IX transgene product.
Plasma levels of IgG1 anti-hF.IX on day 14 after immunological challenge by subcutaneous administration of 2 μg hF.IX formulated in CFA. Mice were naive or had received hepatic gene transfer with AAV-EF1α-hF.IX (EF1α), AAV-ApoE/hAAT-hF.IX (hAAT), or AAV-GFP (GFP) vector 2–3 months earlier at the indicated vector doses. Each bar represents an individual animal. (a) C57BL/6, (b) BALB/c, (c) C3H, (d) CD-1 mice. Note that (in contrast to naive mice) none of the vector-treated animals had detectable IgGa after challenge (not shown). *IgG-1 anti-hF.IX levels are shown as increase over levels before CFA challenge for those mice that had IgG-1 anti-hF.IX after gene transfer.
Higher levels of transgene expression favor tolerance. When we performed hepatic gene transfer in the outbred CD-1 strain, three of four mice did not have detectable hF.IX plasma levels 2 weeks after injection of 1011 AAV-EF1α-hF.IX, and four of the mice developed anti-hF.IX by week 4 (Table 1). Ab subclass analyses revealed primarily IgG-2a production (indicating a primarily Th1-driven response) and, additionally, IgG-1 and IgG-2b anti-hF.IX. Based on results obtained with C3H mice (see above), we hypothesized that Ab formation could be avoided by an increase in vector dose. Subsequent injection of CD-1 mice with increasing vector doses (4 × 1011 vg/animal or 2 × 1012 vg/animal) confirmed this hypothesis. The mid-dose cohort showed mixed results with mice showing IgG-1 or IgG-2a anti-hF.IX or sustained expression without anti-hF.IX (Table 1). In the high-dose cohort, sustained expression was achieved in four of four mice (three of four animals without anti-hF.IX, one of four animals with non-neutralizing IgG-1; Table 1). Injection of the more powerful AAV-ApoE/hAAT-hF.IX vector gave sustained subtherapeutic levels of expression (10–20 ng/ml; lower than in the three strains described above) at a dose of 1011 vg/animal, while anti-hF.IX formation without detectable expression was observed at lower vector doses (Table 1). Therapeutic levels of expression were measured in the high-dose cohort (5 × 1011 vg/animal, again three to four animals without anti-F.IX, one of four animals with non-neutralizing IgG-1; Table 1). Therefore, levels of expression as determined by vector dose and promoter strength mainly determined incidence of Ab formation. Mice treated with 1011 AAV-ApoE/hAAT-hF.IX produced low-titer anti-hF.IX after immunological challenge with hF.IX in CFA, whereas mice in the high-dose cohorts of either vector generally did not produce anti-hF.IX after challenge (see Figure 3d and Table 1). Those mice with IgG-1 anti-hF.IX before CFA injection, one in four in each high-dose cohort, continued to express hF.IX, but showed an increase in Ab titer (Figure 3d).
Outcome of liver-directed hF.IX gene transfer in CD-1 mice as a function of promoter and vector dose
Tolerance induction is antigen specific. To test antigen specificity of tolerance induction, we challenged C57BL/6 mice that had received AAV-hF.IX vector by subcutaneous injection of the closely related serine protease hF.X formulated in CFA. These mice formed anti-hF.X at titers (8.5 ± 3.5 μg IgG-1/ml at day 14 after immunization) similar to naive control mice (10.7 ± 3.3 μg IgG-1/ml, data not shown). Next, we performed subcutaneous injections of a mix of hF.IX and hF.X (5 μg each per mouse) in CFA in C57BL/6 mice that had received hepatic AAV-hF.IX gene transfer. Naive control mice formed high-titer anti-hF.IX (21.5 ± 10.3 μg IgG-1/ml) and anti-hF.X (8.6 ± 4 μg IgG-1/ml) by day 14, as expected, while mice with hepatocyte-derived hF.IX expression formed only high-titer anti-hF.X (6 ± 2.2 μg IgG-1/ml anti-hF.X versus 0.5 ± 0.4 μg IgG-1/ml anti-hF.IX; data not shown), illustrating antigen specificity of tolerance induction.
Unresponsiveness to hF.IX on the T cell level. Anti-F.IX formation in protein therapy, as well as in gene therapy, is a Th cell–dependent process (26, 27). To investigate whether induction of immune tolerance is reflected in CD4+ Th cell responses, we challenged tolerized mice a second time with hF.IX in IFA and sacrificed the animals 5 days later for in vitro restimulation of lymphocytes with hF.IX antigen. As compared with naive mice challenged in parallel, lymphocytes from AAV-EF1α-hF.IX–transduced mice showed no (C57BL/6 and BALB/c) or reduced (C3H) proliferation following in vivo challenge with hF.IX/iFA and in vitro restimulation with hF.IX antigen, whereas the identical experiment resulted in a proliferative response to hF.IX after immunization of naive mice of these strains (Figure 4).
Lymphocyte proliferation following in vitro restimulation with hF.IX protein. Naive or AAV-EF1α-hF.IX–treated mice (portal infusion of 1011 vg/animal for C57BL/6 and BALB/c mice and 5 × 1011 vg/animal for C3H mice) were boosted twice with hF.IX formulated in adjuvant (1.5 months after gene transfer for vector-treated mice for the first challenge with hF.IX/cFA and 1 month later with hF.IX/iFA) and sacrificed on day 5 after the second boost (animals were identical to those used in Figure 1). Total pooled splenocytes and inguinal lymph node cells (n = 3/strain) were cultured in the presence or absence (mock, white bars) of hF.IX antigen (10 μg/ml media, gray bars) for 5 days prior to pulse with 3T-thymidine. 3T-thymidine incorporation was measured by scintillation counting. All lymphocyte cultures were set up in quadruplicate. Average counts per minute ± 90% confidence interval are shown. Numbers above bars are stimulation indexes (ratio of counts per minute for antigen versus mock-stimulated cells).
Evidence for CD4+ regulatory T cells. If tolerance induction involves regulatory or suppressor lymphocytes, we should be able to transfer unresponsiveness to the hF.IX antigen from tolerized animals to naive mice of the same strain. To address this question, we adoptively transferred pooled splenocytes from C57BL/6 mice that had received hepatic gene transfer or from naive C57BL/6 mice (controls) to naive C57BL/6 mice (5 × 107 of total splenocytes were injected into the tail vein). Mice were challenged by subcutaneous injection of hF.IX in CFA 24 hours after receiving splenocytes and plasma samples analyzed for anti-hF.IX 2 weeks after the challenge. As compared with controls, mice that had received cells from vector-treated animals produced, on average, four- to eightfold lower IgG-1 anti-hF.IX levels (Figure 5). This result was similar for splenocyte transfer from AAV-EF1α-hF.IX– and AAV-ApoE/hAAT-hF.IX–treated mice. When purified CD4+ T cells were transferred (107 cells/animal), an identical result was obtained, whereas CD4+ T cell–depleted splenocytes (5 × 107 cells/animal) failed to transfer unresponsiveness (Figure 5).
Plasma levels of IgG-1 anti-hF.IX on day 14 after immunological challenge by subcutaneous administration of 2 μg hF.IX formulated in CFA in C57BL/6 mice that had received adoptive transfer of splenocytes from naive or vector-treated C57BL/6 mice. Each bar is average Ab titer for four to five animals ± 90% confidence interval. Adoptive transfer was by tail vein injection 24 h before challenge. Vector-treated mice had received hepatic gene transfer with 1011 vg/animal of AAV-EF1α-hF.IX (EF1α) or AAV-ApoE/hAAT-hF.IX (hAAT) vector 1.5 months before the experiment. Total splenocytes (5 × 107, bars 1–4), CD4+ T cell–depleted splenocytes (1.5 × 107, bar 7), or MACS-purified CD4+ T cells (bars 5 and 6) were transferred. *Difference in anti-hF.IX titer between mice that received splenocytes from naive versus AAV-ApoE/hAAT-hF.IX–treated animals was statistically different (P < 0.01). **Difference in anti-hF.IX titer between mice that received CD4+ cells from naive versus AAV-ApoE/hAAT-hF.IX–treated animals was statistically different (P < 0.05).
Requirements for tolerance induction. To test requirements for tolerance induction by hepatic F.IX gene transfer, we performed injections of AAV-EF1α-hF.IX vector in several knockout strains (C57BL/6 genetic background) deficient in molecules that affect immune function. CD4+ and CD8+ T cells express a T cell receptor composed of α and β subunits. Previous work has shown that in mice deficient in T cells expressing the rarer γδ-T cell receptor it is more difficult to induce oral tolerance to antigens (28). Literature on oral tolerance also describes induction of regulatory CD8+ T cells secreting TGF-β cytokine (29). Both γδ-T cell receptor–deficient mice and CD8+ T cell–deficient mice, however, showed sustained expression of hF.IX without anti-hF.IX formation following hepatic gene transfer (Table 2). Immunological unresponsiveness was upheld after challenge with hF.IX/cFA (Table 2).
Liver-directed gene transfer with AAV-EF1α-hF.IX vector in knockout mice deficient in γδ-T cell receptor, CD8+ T cells, IL-4 cytokine expression, or Fas
In previous studies on muscle-directed gene transfer with AAV-F.IX vector, we found a predominantly Th2-driven anti-F.IX response. Since results documented above show a predominant Th1 response in the context of low levels of hF.IX expression in liver-directed gene transfer, one could hypothesize that transduced liver is prone to produce a Th1 response, but at higher expression levels this Th1 response is suppressed by regulatory Th2 cells. To test this interpretation, we transduced IL-4–deficient mice, which cannot produce Th2-dependent Ab’s, but form IgG-2a anti-hF.IX after IM injection of vector (26). These mice also showed sustained expression without evidence for anti-hF.IX (Table 2). In particular, no IgG-2a was detected, indicating that tolerance induction cannot be explained by suppression of an imminent Th1 response by Th2 cells.
To evaluate a potential requirement for apoptotic cell death mediated by the Fas-Fas ligand pathway, we performed hepatic gene transfer in Fas-deficient C57BL/6 mice. These mice also did not develop anti-hF.IX during the first month after vector administration (Figure 6b). At this time point, mice were challenged with hF.IX/cFA. Subsequently, Fas-deficient mice produced IgG1 anti-hF.IX (six of six) mice within 2 weeks after challenge. This immune response neutralized expression in only two of six mice, however, while four of six Fas-deficient animals continued to show circulating hF.IX levels (Figure 6a). Mice with a neutralizing response had high-titer anti-hF.IX, while the other four animals developed only low-titer anti-hF.IX (Figure 6b). As described above for other experiments, normal C57BL/6 controls (n = 4) continued to express hF.IX without Ab formation when challenged 1 month after vector administration, and Fas-deficient mice not challenged with hF.IX/cFA continued to express hF.IX without anti-hF.IX formation (n = 6; data not shown).
Liver-directed gene transfer with AAV-EF1α-hF.IX vector (2 × 1011 vg/animal) in C57BL/6 mice deficient in cell death receptor Fas (n = 6). Shown are plasma levels of hF.IX and anti-hF.IX (measured by ELISA or immuno-capture assay) as a function of time after vector administration. (a) Plasma levels of hF.IX (ng/ml). (b) IgG1 anti-hF.IX. Vertical arrows indicate challenge by subcutaneous administration of 2 μg hF.IX formulated in CFA. Symbols are identical for hF.IX and anti-hF.IX levels of the same animal.
Treatment of hemophilia B mice with large F.IX gene deletion. To test tolerance induction to F.IX by hepatic gene transfer in animal models of hemophilia B, we bred F.IX knockout mice onto three different genetic backgrounds, BALB/c, C3H, and CD-1 (sustained expression of F.IX transgenes from different viral vectors following intravenous or portal infusion is already well documented in the literature for C57BL/6 mice) (5, 6, 14). As summarized in Table 3, sustained expression of mF.IX was obtained after hepatic gene transfer in three of five BALB/c mice treated with the AAV-EF1α-mF.IX and in four of four BALB/c mice treated with the AAV-ApoE/hAAT-mF.IX vector (3 × 1011 vg of either vector per mouse for all hemophilic mice injected). In CD-1 mice, expression was achieved in three of five mice injected with AAV-ApoE/hAAT-mF.IX vector, while none of the AAV-EF1α-mF.IX–injected mice showed mF.IX expression in the circulation (zero of five). Those mice that did not express mF.IX had developed inhibitory anti-mF.IX (Table 3). Inhibitory anti-mF.IX included IgG-1 and IgG-2a subclasses (data not shown).
Immunity versus tolerance in hemophilia B mice of different strain backgrounds receiving hepatic gene transfer with AAV-EF1α-mF.IX or AAV-ApoE/hAAT-mF.IX vectorA
Interestingly, there was one hemophilic BALB/c mouse with transient expression at 1 month after AAV-EF1α-mF.IX gene transfer, followed by inhibitor formation at later time points. This animal synthesized IgA and IgG-2b anti-mF.IX at 1 month, which was not neutralizing to mF.IX expression or partial correction of coagulation (data not shown). Immune deviation toward this Th3-type, TGF-β–dependent response shifted to a Th1 response with neutralizing IgG-2a by month 4 (data not shown), however. In contrast, expression was sustained in all other mice that had mF.IX levels at a 1-month time point.
Some mice with sustained mF.IX expression were challenged by subcutaneous administration of mF.IX in CFA (2–4 months after vector administration) and were assayed 1.5 months later for transgene expression and inhibitor formation. Of eight mice challenged, seven continued to express mF.IX (without evidence for inhibitor formation) at a level identical to that prior to challenge, while the hemophilia B CD-1 mouse developed an inhibitor after challenge (Table 3). This mouse had the lowest level of transgene expression (approximately 30 ng/ml), whereas all mice expressing more than 50 ng/ml did not form inhibitors after challenge. While the success rate of tolerance induction for these vector/strain combinations was as predicted from experiments with hF.IX in hemostatically normal mice (see above; i.e., higher levels of expression such as in BALB/c mice versus CD-1 mice or with the ApoE/hAAT versus the EF1α promoter gave a higher success rate), C3H mice gave a much lower rate of success than predicted (only one in five mice injected with AAV-ApoE/hAAT-mF.IX; Table 3).