Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2011 Mar 8;19(7):1263–1272. doi: 10.1038/mt.2011.33

Nonredundant Roles of IL-10 and TGF-β in Suppression of Immune Responses to Hepatic AAV-Factor IX Gene Transfer

Brad E Hoffman 1, Ashley T Martino 1, Brandon K Sack 1, Ou Cao 1, Gongxian Liao 2, Cox Terhorst 2, Roland W Herzog 1
PMCID: PMC3129563  PMID: 21386826

Abstract

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce immunological tolerance to a variety of proteins. Regulatory T-cells (Treg) induced by this route suppress humoral and cellular immune responses against the transgene product. In this study, we examined the roles of immune suppressive cytokines interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) in the development of tolerance to human coagulation factor IX (hF.IX). Interestingly, IL-10 deficient C57BL/6 mice receiving gene transfer remained tolerant to hF.IX and generated Treg that suppressed anti-hF.IX formation. Effects of TGF-β blockade were also minor in this strain. In contrast, in C3H/HeJ mice, a strain known to have stronger T-cell responses against hF.IX, IL-10 was specifically required for the suppression of CD8+ T-cell infiltration of the liver. Furthermore, TGF-β was critical for tipping the balance toward an regulatory immune response. TGF-β was required for CD4+CD25+FoxP3+ Treg induction, which was necessary for suppression of effector CD4+ and CD8+ T-cell responses as well as antibody formation. These results demonstrate the crucial, nonredundant roles of IL-10 and TGF-β in prevention of immune responses against AAV-F.IX-transduced hepatocytes.

Introduction

Hepatic gene transfer using adeno-associated viral (AAV) vectors has been shown to efficiently induce systemic immunological tolerance to a variety of proteins in various preclinical models. The success of tolerance induction is significantly influenced by vector design, dose, target tissue, and route of administration.1,2,3,4 Other important factors include the strain/animal model, the transgene product, and the tissue-specific microenvironment associated with expression.3,5,6,7,8,9 Previously, we have demonstrated that hepatocyte-derived transgene expression induces a state of immunological tolerance. This tolerance is driven by antigen-specific regulatory CD4+CD25+FoxP3+ T-cells (Treg), which suppress humoral and cellular immune responses against the transgene product.3,8,10,11,12 In general, CD4+CD25+FoxP3+ Treg can be further differentiated based on their origin. Naturally occurring Treg (nTreg) emerge from the thymus and play a critical role in preventing autoimmunity and maintaining tolerance to self-antigens. They express additional molecules important for their suppressive phenotype, including CTLA-4, TGF-β1, and glucocorticoid-induced tumor necrosis factor receptor (GITR).

Immunological tolerance can also be achieved through peripheral mechanisms. Several studies have shown that Foxp3 may also be induced in CD4+Foxp3− T-cells upon engagement of the T-cell receptor (TCR) with antigen in the presence of TGF-β, thereby generating induced Treg (iTreg).13 iTreg have been shown to produce increased amounts of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), and are capable of suppressing T-cell proliferation in both contact-dependent and -independent pathways. Thus far, studies on the role of these suppressive cytokines in tolerance induction by hepatic gene transfer have been very limited, in particular for TGF-β.

AAV vectors have been successfully utilized to induce tolerance to a variety of protein antigens in several inbred strains of immunocompetent mice with different major histocompatibility complex (MHC) haplotypes. Treg induced by sustained hepatocyte-restricted transgene expression not only suppress CD4+ and CD8+ inflammatory T-cell responses against the liver but can also protect against responses directed toward the transgene product in other tissues.7,11,14 However, mouse strain specific factors clearly influence the ability to induce tolerance by liver gene transfer.3,5,6,8 In this study, we used C3H/HeJ (H-2Kk) and C57BL/6 (H-2Kb) mice to examine the role of IL-10 and TGF-β in the development of antigen-specific tolerance to the human coagulation factor IX (hF.IX). We have previously reported that long-term stable expression of hF.IX (without the formation of antibodies against hF.IX) can be achieved in both strains of mice following hepatic delivery of an AAV2 vector with a liver specific promoter.3,8,11 However, C3H/HeJ mice have substantially stronger B- and T-cell responses to hF.IX, and are therefore more difficult to tolerize.3,15 Here, we report that IL-10 and TGF-β are critical to control immune responses against AAV-hF.IX transduced hepatocytes in the strain with stronger T-cell responses (C3H/HeJ mice).

Results

IL-10 deficient C57BL/6 mice fail to form antibodies to hF.IX and remain tolerant after challenge with antigen

IL-10 is an essential anti-inflammatory cytokine able to regulate the immune system by controlling inflammatory and autoimmune responses.16,17 Hepatic gene transfer in wild-type C57BL/6 (C57BL/6WT) mice with AAV2-ApoE/hAAT-hF.IX (AAV2-hFIX) results in long-term expression of the hF.IX transgene due to the induction of T- and B-cell tolerance.8,11,18 Moreover, this antigen-specific induced tolerance is sustained when the mice are subsequently challenged with hF.IX in complete Freund's adjuvant (CFA). To determine if this tolerogenic state is dependent on the presence of IL-10, we performed hepatic gene transfer in C57BL/6 mice homozygous for the Il10tm1Cgn targeted mutation (C57BL/6IL10−) using 1011 vector genomes (vg) AAV2-hFIX per mouse. These mice completely lack the ability to produce IL-10. Six weeks later, mice were challenged subcutaneously with 1 IU of rhF.IX emulsified in CFA. Beginning at 2 weeks postgene transfer, all animals showed circulating levels of hF.IX (800–2,700 ng/ml, similar to expression in wild-type mice). In addition, there was no evidence of antibody formation against hF.IX as determined by enzyme-linked immunosorbent assay (ELISA) (Figure 1a,b and data not shown). Systemic transgene expression and lack of antibody formation was sustained until the termination of the experiment at 9 weeks, even after immunogenic challenge at 6 weeks. To verify that the IL-10 deficient mice are indeed immune competent and are capable of producing antibodies, naive C57BL/6IL10− and C57BL/6WT mice were subjected to the identical subcutaneous hF.IX/CFA challenge. As shown in Figure 1c, both groups of mice rapidly produced an anti-hF.IX IgG1 response. In sum, there was no evidence of a requirement for IL-10 expression in tolerance to hF.IX in C57BL/6 mice.

Figure 1.

Figure 1

Open in a new tab

Interleukin-10 (IL-10) deficient C57BL/6 mice fail to form antibodies to hF.IX and remain tolerant after challenge with antigen. (a–b) IL-10 deficient C57BL/6 (n = 4) received hepatic gene transfer with 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX via injection into the splenic capsule. They subsequently received s.c challenge 6 weeks later with 5 µg rhF.IX emulsified in CFA. (a) Plasma levels of hF.IX measured by enzyme-linked immunosorbent assay and (b) immunoglobulin (Ig) G1 anti-hF.IX measured by immuno-capture assay as a function of time after. (c) Control C57BL/6WT and C57BL/6IL10− mice (n = 3 per group) were immunized with 5 µg rhF.IX/CFA. Anti-hF.IX IgG1 was determined beginning 2 weeks later. All data are mean ± SEM.

Adoptively transferred Tregs from tolerized C57BL/6WT and C57BL/6IL10− mice equally suppressed the immune response in recipient mice

In the following experiment, the immunomodulatory contribution of IL-10 expression by hepatic gene transfer-induced CD4+CD25+ Tregs was evaluated. C57BL/6IL10− and C57BL/6WT mice were tolerized to hF.IX as described above. Six weeks later, 106 CD4+CD25+ T-cells were adoptively transferred into naive C57BL/6WT mice via tail-vein injection (Figure 2a). Transferred cells had been isolated and pooled from the spleens of either naive or tolerized C57BL/6IL10− mice or from naive or tolerized C57BL/6WT mice (controls). Twenty-four hours later, mice were challenged by subcutaneous injection of hF.IX in CFA. Two weeks later, plasma samples were analyzed for anti-hF.IX formation (Figure 2b). As expected, mice from both groups that received Tregs from naive donors formed high-titer hF.IX-specific immunoglobulin (Ig) G1. On the other hand, mice that had received CD4+CD25+ T-cells from vector-tolerized animals had a significant reduction (~50%) in antibody titers. Notably, there was no difference in the antibody levels in the cohort that had received tolerized IL-10 deficient Treg and those that had received tolerized wild-type Tregs. Taken together, the data imply that in C57BL/6 mice, the induction and functionality of Treg capable of suppressing antibody formation is independent of IL-10.

Figure 2.

Figure 2

Open in a new tab

Adoptively transferred regulatory T-cells from tolerized C57BL/6WT and C57BL/6IL10− mice equally suppress antibody formation against hF.IX. (a) CD4+CD25+ T-cells were isolated and pooled from the spleens of C57BL/6WT and C57BL/6IL10− that were either naive or immunized with 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX via injection into the splenic capsule (n = 3 per donor group), and adoptively transferred into naive C57BL/6WT via tail vein injection (n = 3 per recipient group). Twenty-four hours later recipient mice were subcutaneously challenged with 5 µg rhF.IX/CFA and (b) immunoglobulin (Ig) G1 titers against hF.IX (mean ± SEM) were determined by immuno-capture assay 2 weeks later. An unpaired two-tailed Student's t-test was used, **P < 0.01. IL, interleukin.

IL-10 deficiency in C3H/HeJ mice results in loss of transgene expression following hepatic AAV2-hF.IX gene transfer

Next, we wanted to investigate the effect of IL-10 deficiency in a strain of mice with stronger immune responses to hF.IX. Using the same vector construct as above, C3H/HeJIL10− and C3H/HeJWT mice were injected with 1011 vg of AAV2-hF.IX vector. Quantitative analysis of plasma samples obtained at 2 weeks postinjection revealed that C3H/HeJIL10− mice had a significant reduction (~10 fold) in circulating hF.IX as compared to C3H/HeJWT mice (Figure 3a). In fact, systemic hF.IX expression declined to nearly undetectable levels in C3H/HeJIL10− mice by 3 weeks. However, no antibodies against hF.IX were detected in either cohort (Figure 3b and data not shown). These results suggested that the loss of circulating hF.IX transgene in the IL-10 deficient mice was not caused by antibody formation.

Figure 3.

Figure 3

Open in a new tab

IL-10 deficiency in C3H/HeJ mice results in differential transgene expression following hepatic AAV2-hF.IX gene transfer. (a–d) C3H/HeJIL10− (n = 4) and C3H/HeJWT (n = 4) received hepatic gene transfer with 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX via injection into the splenic capsule. (a) Plasma levels of hF.IX measured by enzyme-linked immunosorbent assay and (b) immunoglobulin (Ig) G1 anti-hF.IX measured by immuno-capture assay were determined 2 and 3 weeks later. Data represent average ± SEM. (c) At 3 weeks, splenocytes isolated from individual AAV2-hF.IX transduced C3H/HeJIL10− and C3H/HeJWT mice were stimulated in vitro with p74 (SGGPHVTEVEGTSFL) or without (mock) and analyzed for INF-γ by ELISpot assay. Staphylococcal enterotoxin B (SEB) was used as a positive control. Data are average values for individual mice (n = 4, with each mouse assayed in quadruplicate). Horizontal bars indicated averages for each group (d). Representative immunofluorescent labeling of liver sections obtained from mice used in a–c at 3 weeks postgene transfer. Lack of hF.IX producing hepatocytes with infiltrating CD8+ T-cells is demonstrated in C3H/HeJIL10− (top panels). In contrast, the C3H/HeJWT (bottom panels) hepatocytes have well organized, characteristic hF.IX expression without CD8+ cells. (e) Amount of hF.IX from total liver protein that was purified from individual wild-type and IL-10 deficient AAV-F.IX transduced C3H/HeJ mice (n = 3 per group). Data are mean ± SEM. F test to compare variances from an unpaired two-tailed Student's t-test was used, ***P < 0.0001. Original magnification: ×100 and ×200. Arrows, CD8+ cells within the parenchyma; arrowhead, CD8+ cell located in a blood vessel.

CD8+ T-cell responses target AAV2-hF.IX transduced liver in IL-10 deficient C3H/HeJ mice

We next wanted to determine if the loss of circulating hF.IX in the C3H/HeJIL10− mice correlated with induction of hF.IX-specific CD8+ T-cells. ELISpot assay for INF-γ was performed using splenocytes isolated from individual AAV2-hF.IX transduced C3H/HeJIL10− and C3H/HeJWT mice (n = 3 per group). Splenocytes were restimulated in vitro with the previously identified H2-Kk-restricted hF.IX CD8+ T-cell epitope (p74) or mock-stimulated (Figure 3c).3 Surprisingly, the frequency of hF.IX-specific T-cells was not elevated in IL-10 deficient compared to wild-type mice.

In order to resolve these conflicting results, we explored the possibility of an organ-specific response that differed from the systemic response. To do so, liver tissue was collected, and cryosections were immunofluorescently stained with α-hF.IX (red) and α-CD8a (green). The resulting images showed substantially fewer hF.IX expressing hepatocytes in the livers of the C3H/HeJIL10− mice as compared to the wild-type mice (Figure 3d). Wild-type mice, as expected for use of an AAV2 vector, exhibited uniform clusters of hF.IX expressing hepatocytes. In contrast, livers of IL-10 deficient mice showed scattered single hF.IX positive hepatocytes.3 More importantly, the reduction of expressing hepatocytes seen in the C3H/HeJIL10− mice was accompanied by substantial CD8+ T-cell infiltrates (arrows). Very few, if any, CD8+ cells (arrow head) could be visualized in sections from C3H/HeJWT mice. To further quantify the loss of hepatic transgene expression, hF.IX levels were determined by ELISA using protein extracts from liver tissue dissected from individual mice. The results demonstrated an approximately fourfold reduction in hepatic hF.IX production in IL-10 deficient compared to wild-type C3H/HeJ mice (Figure 3e). The data presented here suggests that in the less tolerogenic C3H/HeJ strain, the protective, immunosuppressant role of IL-10 was crucial in the microenvironment of the liver.

TGF-β blockade reduces systemic hF.IX expression in wild-type C3H/HeJ mice

TGF-β is another cytokine that has been linked to the immunosuppressive function of Tregs in vivo in various models.19 Knockout mice for this cytokine die within 3 weeks of age because of severe lymphoproliferative disorder. Therefore, to define the specific contributions of TGF-β in tolerance induction, we transiently administered a monoclonal antibody (clone 1D11) that reacts with both TGF-β1 and TGF-β2 and inhibits their biological activities. C3H/HeJ mice received hepatic gene transfer with 1 × 1011 vg AAV2-hF.IX. Half of these animals were additionally injected intraperitoneal with a 25 mg/kg dose of α-TGF-β 2 hours before gene transfer, and every 3rd day thereafter for 2 weeks. Beginning 2 weeks after gene transfer, plasma samples from mice that had been receiving the α-TGF-β antibody exhibited a significant reduction in circulating transgene product compared to the control mice. This low systemic expression level was sustained until termination of the experiment (Figure 4a,d). Using at least two pieces of liver tissue dissected from different lobes of individual mice, we were able to demonstrate a significant loss of hF.IX produced by hepatocytes as a result of TGF-β blockade (Figure 4c). Interestingly, there was also an IgG1 antibody response against hF.IX, which, however, declined after stopping of the TGF-β blockade (Figure 4b).

Figure 4.

Figure 4

Open in a new tab

Transforming growth factor-β (TGF-β) blockade reduces systemic hF.IX expression in wild-type C3H/HeJ mice but not C57Bl/6. (a–d) C3H/HeJWT mice (n = 4–7 per group) or (e–f) C57BL/6 (n = 4 per group) received 25 mg/kg α-TGF-β (clone: 11D1) or phosphate-buffered saline (PBS) by intraperitoneal injection prior to hepatic gene transfer and every third day for 2 weeks (gray region in a–b & e–f). Hepatic gene transfer consisted of 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX, administered via injection into the splenic capsule. (a,e) Plasma levels of hF.IX measured by enzyme-linked immunosorbent assay (ELISA) and (b,f) IgG1 anti-hF.IX measured by immuno-capture assay as a function of time after. (c) Amount of hF.IX, as determined by ELISA, from total liver protein that was purified and pooled from 2 different lobes of individual AAV-F.IX transduced C3H/HeJWT mice that received either blocking antibody or PBS (n = 3 per group). Data are mean ±SEM. An unpaired two-tailed Student's t-test was used, *P < 0.05. (d) Representative immunofluorescent labeling of liver sections obtained from mice treated as above at 2.5 weeks postgene transfer. Original magnification: ×100.

When similar experimental conditions were applied to a cohort of wild-type C57BL/6 mice, there were no significant differences in the levels of circulating hF.IX between α-TGF-β treated and control mice at vector doses 1 × 1011 vg/mouse (Figure 4e). However, transient IgG1 formation against hF.IX was detectable in mice treated with α-TGF-β (albeit at lower titer compared to α-TGF-β C3H/HeJ mice; Figure 4f).

TGF-β blockade prevents suppression of effector T-cell responses upon hepatic gene transfer to C3H/HeJ mice

It has been shown that TGF-β inhibits antigen-specific CD8+ T-cell effector function directly as well as through the suppressive actions of Treg.20,21 Therefore, following hepatic gene transfer with the AAV2 vector, we evaluated the effect of TGF-β blockade by comparing the phenotype of splenocytes isolated from wild-type C3H/HeJ mice. These mice received α-TGF-β only, AAV2-hF.IX only, and the combined treatment of α-TGF-β/AAV2-hF.IX at ~2.5 weeks postgene transfer. Cell suspensions from individual mice were labeled with monoclonal antibodies to CD3 and CD4 surface proteins and analyzed by flow cytometry (Figure 5a). The CD3+CD4− population was classified de facto as CD8+ T-cells. The combined data revealed a significant increase of nearly twofold in both CD4+ and CD8+ T-cell frequencies in the group of mice receiving α-TGF-β and gene transfer compared to all other groups (Figure 5b–d). Gene transfer alone had little effect on these T-cell frequencies. Treatment with α-TGF-β only caused a minor increase in CD4+ T-cell frequency. The relative proportion of CD4+ to CD8+ T-cells was unchanged in all groups. Together, these findings indicated a heightened T-cell response to gene transfer upon blockage of TGF-β.

Figure 5.

Figure 5

Open in a new tab

TGF-β blockade prevents suppression of effector T-cell responses upon hepatic gene transfer to C3H/HeJ mice. (a–b) C3H/HeJWT mice (n = 3 per group) received 25 mg/kg α-TGF-β (clone: 11D1) or phosphate-buffered saline by intraperitoneal injection prior to hepatic gene transfer and every third day for 2 weeks (gray region in a and b). Hepatic gene transfer consisted of 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX, administered via injection into the splenic capsule. Splenocytes isolated at ~2.5 weeks were labeled with α-CD3 and α-CD4 and analyzed by flow cytometry. (a) Representative contour plots indicating the percentage of cells pregated for lymphocyte populations based on forward versus side scatter. (b) Combined analysis of the above groups (mean ± SEM). One-way ANOVA with Bonferroni's post-test was used for statistical analysis *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

TGF-β is required to tip the balance of the response toward FoxP3+ Treg

Next, we investigated whether TGF-β blockade would alter the ratio of CD4+CD25hiFoxP3+ Treg to FoxP3− effector cells, a potential mechanism for prevention of tolerance. Splenocytes from individual mice that had been injected with 1 × 1010 vg of AAV2-hF.IX and that received α-TGF-β treatment, as well as those from control animals, were analyzed for intracellular expression of FoxP3 (Figure 6a). There was a significant increase in the regulatory:effector T-cell ratio for AAV2-hF.IX transduced mice in favor of Treg (Figure 6b). The combination of α-TGF-β and AAV2-hF.IX abrogated the Treg response otherwise seen following gene transfer (Figure 6d). This effect promoted activation of effector T-cells (Teff) (Figure 6e,f) and created a ratio of CD4+ Teff:Treg and of CD8+ T-cell:Treg that tipped the balance from regulatory toward immune responses (Figure 6b,c). In contrast, we did not see any differences in the frequencies of CD4+CD25hiFoxp3+ Treg or FoxP3— Teff cells between unmanipulated C3H/HeJ mice and those only receiving α-TGF-β (Figure 6d,e). Taken together, these results suggest that in the presence of TGF-β signaling, the balance of the immune response is in favor of Treg, while in the absence, Teff responses can proceed and drive the CD8+ T and B cell responses documented in Figures 4 and 5.

Figure 6.

Figure 6

Open in a new tab

TGF-β is required to tip the balance of the response toward FoxP3+ regulatory T-cells (Tregs). (a–f) Identification of Treg phenotype by additional immuno-labeling of cells shown in Figure 5. Splenocytes isolated at ~2.5 weeks were additionally labeled with α-CD3, α-CD4, α-CD25, α-FoxP3 and analyzed by flow cytometry. (a) Representative contour plots of indicating the percentage of cells pregated for lymphocyte populations based on forward versus side scatter. (a) Representative density plots of CD4+ gated populations with percent indicated. (b) Ratio of CD4+FoxP3− (Teff) to CD25hiFoxP3+(Tregs), (c) Ratio of CD3+CD4− (CD8) to CD25hiFoxP3+(Tregs), (d) Percent of CD4+CD25hiFoxP3+(Tregs), (e) Percentage of CD4+FoxP3− (Teff), (f) Percentage of CD4+CD25hiFoxP3− (recently activated Teff). Data are mean ± SEM. One-way ANOVA with Bonferroni's post-test was used for statistical analysis *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

α-TGF-β treatment prevents induction of Helios− Treg: nTregs versus iTregs

Upregulation of the Ikaros family transcription factor Helios in nTreg has been reported by several groups.22,23 In a recent publication, Thorton et.al. developed a new monoclonal antibody against Helios (clone: 22F6). From a series of elegant experiments, the authors found that this novel antibody can discriminate between thymus-derived nTregs (FoxP3+Helios+) and peripherally induced iTregs (FoxP3+Helios−).22 Considering that TGF-β has been shown to differentially affect nTregs and iTregs, we utilized this new antibody to further characterize the phenotype of the Tregs in C3H/HeJ mice (Figure 7a). Consistent with data above, the mice that received only AAV-hF.IX hepatic gene transfer had a significant increase of CD4+FoxP3+Helios−, and CD4+CD25hiFoxP3+Helios− (iTregs). This was prevented in animals that underwent TGF-β blockade (Figure 7b,d). In addition, based on the coexpression of FoxP3 and Helios, there was a significant increase in the population of nTregs upon AAV-hF.IX gene transfer. This was less the case in the presence of α-TGF-β, which likely interfered with survival of nTreg (Figure 7c,e).

Figure 7.

Figure 7

Open in a new tab

α-TGF-β treatment results in a differential expression of Helios in regulatory T-cells (Treg) cells. (a–c) C3H/HeJWT mice (n = 3 per group) received 25 mg/kg α-TGF-β (clone: 11D1) or phosphate-buffered saline by intraperitoneal injection prior to gene transfer and every third day for 2 weeks. Hepatic gene transfer of 1011 vector genomes of AAV2-ApoE/hAAT-hF.IX was administered via injection into the splenic capsule. Intracellular labeling and flow cytometric analysis using α-CD4, α-FoxP3, and α-Helios of splenocytes obtained from individual mice at ~2.5 weeks postgene transfer. (a) Representative density plots of CD4+ gated populations with percent indicated. (b) Percent CD4+FoxP3+Helios− and (c) CD4+FoxP3+Helios+ cells. (d) Percent of iTregs identified as Helios-Treg (CD4+CD25hiFoxP3+Helios−) and (e) nTregs identified as Helios+ Treg (CD4+CD25hiFoxP3+Helios+). Data are mean ± SEM. An unpaired two-tailed Student's t-test was used, *P < 0.05, **P < 0.01.

Effects of α-GITR administration on immune tolerance induction in C3H/HeJ mice

Previously, we have shown that Treg are required for tolerance induction to hF.IX by hepatic AAV2 gene transfer in C57BL/6 mice.12 To address this point in the C3H/HeJ strain, the GITR-GITRL costimulatory pathway was targeted. GITR is a type I transmembrane protein with homology to other tumor necrosis factor receptor family members.24,25 Under homeostatic conditions, GITR is expressed at low levels on resting T lymphocytes while being constitutively expressed at high levels on CD4+CD25+Foxp3+ Tregs. Interaction of GITR with its ligand (GITRL) has been shown to render effector T-cells resistant to the suppressive effects of Tregs. Administration of α-GITR mAb abrogates Treg-mediated suppression, and is therefore a suitable approach to test for a requirement of Treg in immune tolerance.24,25,26

To examine the potential role that GITR expressing Tregs have on the initial induction of tolerance, we injected wild-type C3H/HeJ with a α-GITR antibody (clone: DTA-1) at the time of hepatic AAV2-hF.IX gene transfer and once every 2 weeks for a total of four injections (n = 5 per experimental group). Control mice received gene transfer and isotype control antibody. All mice were challenged with hF.IX protein in CFA at 6 weeks postgene transfer (Supplementary Figure S1a). Comparative analysis of plasma samples obtained before the protein challenge (week 6) with those obtained 2 weeks later (week 8) revealed a slight reduction in the circulating hF.IX levels in the α-GITR treated mice (Supplementary Figure S1b). However, there was a significant increase in hF.IX-specific antibody formation in this group compared to the control mice, which remained tolerant (Supplementary Figure S1c). Next, we considered if the above results were dependent on the timing of the α-GITR treatment. The experiment was modified by shifting the administration of α-GITR timeline to begin at 6 weeks after AAV-hF.IX hepatic gene transfer (Supplementary Figure S1d). A reduction in hF.IX levels in the α-GITR treated mice was seen before and after protein challenge (Supplementary Figure S1e). After challenge, the difference in systemic hF.IX expression compared to control mice became statistically significant. These changes in transgene expression corresponded with formation of antibodies against hF.IX in the mice treated with α-GITR, which increased in titer after adjuvant challenge (Supplementary Figure S1f). These results are in agreement with other published reports showing that stimulation of GITR by the DTA-1 mAb decreases Treg functionality and is accompanied by antibody formation, and support the hypothesis that Treg are required for induction and maintenance tolerance to hF.IX upon hepatic AAV gene transfer24

Discussion

The ability to induce antigen-specific immune tolerance is multifactorial, involving both intrinsic and extrinsic factors. Hepatic gene transfer with AAV vectors can efficiently induce systemic tolerance to an assortment of proteins in various strains of mice.27 It is widely acknowledged that the microenvironment of the liver favors immune tolerance via a combination of tolerogenic antigen-presenting cells (Kupffer cells, liver sinusoidal endothelial cells, dendritic cells) and cytokines (IL-10, IL-4, TGF-β). Consequently, multiple factors and interlinked mechanisms influence induction of tolerance. In an attempt to better define some of the key factors, we evaluated the role of two potentially critical immunosuppressive cytokines, TGF-β, and IL-10. To do so, we established a system that utilized two different strains of mice that we have previously shown to differ in their response to hF.IX.8C57BL/6 (H-2Kb) mice more easily establish and maintain antigen-specific tolerance following hepatic gene transfer with an AAV2-hF.IX or other viral vectors.8,9 In contrast, the C3H/HeJ (H-2Kk) strain less amendable to tolerance induction, requiring an optimized liver gene transfer protocol.8,9 For example, suboptimal gene transfer results in antibody formation and CD8+ T-cell-mediated loss of hF.IX expression in F.IX deficient C3H/HeJ mice.3 In general, C3H/HeJ mice generally exhibit much more robust CD8+ and T helper cell responses and antibody responses to hF.IX than C57BL/6 mice.3,18,26,28,29,30

IL-10 plays a strain-dependent immunosuppressive role in the liver that prevents CD8+ T-cell responses against hF.IX-transduced hepatocytes

In vivo, both IL-10 and TGF-β have been associated with the maintenance of peripheral tolerance by CD4+CD25+ Treg. Accordingly, systemic blockade of these immunosuppressive cytokines, either by targeted mutation or neutralizing antibody, would potentially result in abrogation of tolerance. This could occur directly or indirectly by suppressing the function or generation of Tregs.31 IL-10 is a potent anti-inflammatory cytokine that inhibits Th1-, Th-17- and T helper (Th) 2 mediated immune responses. Its importance in regulating immune responses has been demonstrated in human infectious diseases such as tuberculosis and malaria, as well as in various models of autoimmune disease and inflammation.32 Originally, IL-10 was considered as a Th2-type cytokine, inhibiting Th1 effector functions. But more recent reports suggest that the production of IL-10 is also associated with regulatory responses (such as Tr1, CD4+CD25+FoxP3+ Treg, or regulatory B cells), thereby inducing unresponsiveness.17 IL-10 deficient mice are highly susceptible to autoimmune disease and spontaneously develop enterocolitis/chronic inflammatory bowel disease (IBD) with age when not housed in specific pathogen free conditions. This occurs earlier in life in C3H/HeJ mice than in C57BL/6 mice. For these reasons, we used younger animals housed in specific pathogen free conditions.

Using mice with inducible knockout of FoxP3 expressing cells, it has been shown that CD4+CD25+FoxP3+ Treg are required to prevent catastrophic systemic autoimmune responses throughout the lifespan of a mouse.33,34 Using depletion with monoclonal anti-CD25, several labs have shown that Treg are required for tolerance to hepatic expressed transgene products,12,35,36 which is further supported by our experiments with α-GITR in C3H/HeJ mice. Interestingly, the Rudensky lab also showed that IL-10 expression by Treg is not required to prevent systemic autoimmunity. Rather, IL-10 is required for suppression of responses in specific locations, in particular the gut and other mucosal surfaces.33,34 Previously, we found that tolerogenic responses to hF.IX in C57BL/6 and immunogenic responses in BALB/c mice upon liver-directed adenoviral administration were independent of IL-10 expression.9 This discovery prompted us to speculate that IL-10 was not a critical factor in tolerance to hF.IX in hepatic gene transfer. Consistent with this interpretation, the data presented here indicate that expression of IL-10 is not a requirement for tolerance to AAV-delivered hF.IX in C57BL/6 mice. In addition, IL-10 deficiency was not associated with antibody formation against hF.IX in C57BL/6 or C3H/HeJ strains.

However, in C3H/HeJ mice, i.e., the strain with stronger T-cell responses against hF.IX, IL-10 deficiency resulted in robust CD8+ T-cell infiltrates in the liver and a loss of hepatic hF.IX expression. This occurred despite the fact that frequencies of splenic hF.IX-specific CD8+ T-cells were not elevated compared to wild-type mice. Interestingly, Breous et al. recently found that tolerance induction to human α-1 antitrypsin (hAAT) and β-galactosidase by hepatic AAV gene transfer correlated with induction of a hepatic population of IL-10+ cells that included CD4+CD25+FoxP3+ Treg and which was not seen in the spleen.5,6 Our previous studies demonstrated induction of splenic Treg by AAV2-hF.IX gene transfer to C3H/HeJ mice that, upon adoptive transfer, were able to suppress inflammatory CD8+ T-cell responses against hF.IX-transduced liver.7 Taking all of these observations into consideration, one can conclude that both Treg induction and hepatic IL-10 expression are required to control destructive CD8+ T-cell responses if the transgene product represents a strong antigen for T-cell activation in the specific mouse strain. A role for IL-10 in suppression of T-cell responses against viruses in the liver is further supported by clinical observations, such as recurrence of hepatitis C virus in patients with elevated systemic IL-10 levels, and increased viral burden in patients treated long-term with IL-10.37,38 Both Breous et al. and our studies suggest that IL-10 is less critical for Treg-mediated suppression of B-cell responses upon hepatic gene transfer.6

TGF-β is necessary for induction of a suppressive Treg response, thereby preventing B- and T-cell responses to the transgene product

TGF-β is a pleiotropic cytokine that has been shown to be involved in the maintenance of nTregs and the conversion of CD4+ T-cells into iTregs.16,39 This cytokine is also expressed by other regulatory and effector T-cells such as Tr1, Th2, and Th3, and promotes class switch to IgA in B cells of mucosa-associated lymphoid tissues. Of particular relevance to our study, TGF-β is required for suppression of CD8+ T-cells by Treg, resulting in a reversible state of reduced functionality.40 Suppression of the adaptive immune responses against hepatic AAV-hF.IX gene transfer, which normally occurs, was relegated in C3H/HeJ mice by administration of α-TGF-β. This antibody caused only minor changes in splenocyte populations in control mice. However, the introduction of exogenous antigens by hepatic gene transfer in mice with TGF-β blockade caused an immune response characterized by increased CD4+ and CD8+ T-cell frequencies and increased T-cell activation. Ultimately, antibodies against the transgene product were formed and transduced hepatocytes were targeted by CD8+ T-cells.

Induction of immune regulatory mechanisms is a key component in establishment of tolerance by hepatic gene transfer or by alternative means, as these help tip the balance toward suppressive rather than effector responses.5,6,10,14,35,41 For example, induced Treg can control antibody formation against coagulation factors long-term, in part because they promote conversion of nonregulatory CD4+ T-cells to Treg, thereby amplifying the regulatory response.42 This process is TGF-β dependent.19 Here, we demonstrate that TGF-β blockage prevents the Treg response to hepatic AAV-hF.IX gene transfer, which is required to suppress B- and T-cell responses to hF.IX in C3H/HeJ mice. These results imply that TGF-β plays a critical role in the skewing of the host response toward the induction of antigen-specific tolerance by modulating the effector:Treg ratios. TGF-β blockade decreased the ratio of Treg to total CD4+ Teff, to activated CD4+ Teff, and to CD8+ T-cells, all of which were responses specific to gene transfer.

TGF-β is able to modulate peripheral immune tolerance by enhancing the survival of the nTregs and by promoting iTreg generation. It has been difficult to accurately distinguish these two populations until the recent development of a novel antibody design to target the Ikaros family transcription factor Helios in nTregs. As reported, the α-Helios antibody is capable of differentiating thymic-derived nTregs from iTregs based on the relative upregulation of Helios.23 The data present here show significantly more iTregs (FoxP3+Helios−) in C3H/HeJ that received only gene transfer, as opposed to those also receiving TGF-β blockade. This suggests that in this strain of mice, TGF-β plays a role in the generation of iTregs and induction of tolerance. In addition, our data also suggest an involvement of thymus-derived nTreg. Given that hF.IX is a systemic protein, transgene expression may well affect central T-cell tolerance to hF.IX, as suggested by our prior study on systemic delivery of ovalbumin by hepatic AAV gene transfer.12 However, further studies are needed to determine whether both subsets of Treg or only one of these (nTreg or iTreg) are required for tolerance.

Nonredundant immune regulatory roles of TGF-β and IL-10

In summary, IL-10 and TGF-β have nonredundant roles in establishing immune tolerance to hF.IX expressed from an AAV vector upon hepatic gene transfer to mice with strong immune responses to this therapeutic protein antigen. In the C3H/HeJ model, both cytokines were required to prevent CD8+ T-cell responses against transduced hepatocytes, while TGF-β was additionally involved in prevention of antibody formation against hF.IX. In C57BL/6 mice, a strain with typically weaker T responses to hF.IX, IL-10 was not required and the effects of TGF-β blockade were also minor. The dependence of antibody formation against hF.IX on T help may further explain why α-TGF-β administration was of lesser consequence in a strain with comparatively weak T-cell responses against hF.IX. However, antibody-mediated blockade is only transient and not nearly as effective or complete as a genetic approach. It is therefore likely that the experiment underestimated the importance of TGF-β signaling in tolerance in the C57BL/6 strain. Additional, more general differences between the two strains may also have had an effect.

In conclusion, we propose that TGF-β is critical to the general induction, amplification, and maintenance of Tregs, as well as to their functionality. Thus, TGF-β helps to tip the response of hepatic gene transfer toward regulation, thereby preventing systemic and hepatic immune responses. In contrast, the role of IL-10 is more specific in the suppression of CD8+ T-cell responses within the hepatic microenvironment.

Materials and Methods

Animal strains. Male inbred C57BL/6, C3H/HeJ, C3Bir.129P2(B6)-Il10tm1Cgn/Lt (C3H/HeJIL10−), and B10.129P2(B6)-Il10tm1Cgn/J (C57BL/6IL10−) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All studies were in accordance with protocols approved by Institutional Animal Care and Use Committees at the University of Florida, Gainesville.

Assays for human F.IX antigen and anti-hF.IX. Levels of hF.IX in plasma samples were determined by ELISA. IgG1 and IgG2a antibody titers to hF.IX were measured by immunoglobulin subclass-specific immuno-capture assay as previously described.43

AAV2 vectors. AAV-ApoE/hAAT-hF.IX carries the hepatocyte-specific expression cassette for hF.IX.44 This cassette includes an apolipoprotein E (ApoE) enhancer/hepatocyte control region, a human a1-antitrypsin promoter, hF.IX cDNA, a 1.4-kb portion of intron 1 of the F9 gene, and the bovine growth hormone poly(A) signal. Expression cassettes are flanked by AAV2 inverted terminal repeats. AAV vector serotype 2 was produced by triple transfection of HEK-293 cells, purified by CsCl gradient centrifugation, filter sterilized, and quantified by slot-blot hybridization as described.45 Unless otherwise specified, mice were injected with 1 × 1011 vg (4 × 1012 vg/kg) via the splenic capsule.

IFN-γ ELISpot assays. ELISpot kits for mouse IFN-γ (R&D Systems, Minneapolis, MN) were used as per the manufacturer's recommendations. Briefly, splenocytes from individual animals were applied in quadruplicate at 106 cells/well and either 10 µg/ml hF.IX-derived peptide containing an immunodominant CD8+ T-cell epitope (p74, SGGPHVTEVEGTSFL) (as described in ref. 3) (Anaspec, San Jose, CA), phosphate-buffered saline (PBS) (mock), or Staphylococcal enterotoxin B. Plates were incubated in 10% CO2 at 37 °C for 18 hours. Plates were washed and incubated with cytokine-specific biotinylated detection antibodies. Spots were developed using ELISpot Blue Color Module (R&D Systems) and counted with the ImmunoSpot Analyzer (Cellular Technology, Shaker Heights, OH). Results were calculated as spot-forming units per 106 total cells and compared to mock-stimulated cultures.

Immunofluorescent staining. At least two liver lobes were collected from each animal and snap frozen in optical cutting temperature compound (Sakura Finetek USA, Torrance, CA) with liquid N2. Multiple cryosections (10 µm thick) were fixed in acetone for 5 minutes, air-dried, and then rehydrated in PBS. Sections were blocked with 2% donkey serum in PBS for 30 minutes. Goat α-hF.IX (1:400; Affinity Biologicals, Ancaster, ON, Canada) and rat α-CD8a (clone: 53.6.7), were applied in 2% donkey serum for 30 minutes. After a washing, tissue sections were incubated with the secondary antibodies donkey α-rat IgG-Alex Fluor-488 and donkey α-goat IgG-Alex Fluor-568 (1:100 dilution; Invitrogen, Carlsbad, CA). After being washed, slides were mounted with or without 4′,6-diamidino-2-phenylindole. Images were captured using a Nikon Eclipse 80i fluorescence microscope and Retiga 2000R digital camera (QImaging, Surrey, BC, Canada) and analyzed with Nikon Elements software.

TGF-β antibody and blockade. Anti-TGF-β antibody was produced by the University of Florida ICBR Hybridoma Research Laboratory from the hybridoma HB-9849 (ATCC, Manassas, VA). Mice were injected intraperitoneal with 25 mg/kg α-TGF-β (clone: 1D11.16.8) in a total of 200 µl PBS. Injections were performed 1 hour prior to splenic injection of vector and every 3rd day for 2 weeks.

Anti-GITR treatment. Monoclonal rat α-GITR (clone: DTA-1) was kindly provided by S Sakaguchi. C3H/HeJ mice received intraperitoneal injections of α-GITR every 2 weeks for a total of four injections. Injections began on the day of vector administration or 6 weeks later. Antibody or rat IgG2b isotype control were given at a dose of 10 mg/kg.

Flow cytometry. Cell suspensions from individual mice were prepared and labeled with fluorescently conjugated antibodies as previously described.15 Antibodies to murine CD4 (RM4-5), CD25 (PC61), CD3 (145-2C11) were from BD Biosciences (San Jose, CA). Anti-Mouse/Rat Foxp3 Alexa Fluor 647 (FJK-16s) was purchased from eBioscience (San Diego, CA). Anti-Helios antibody was purchased from Biolegend (San Diego, CA). Controls for all stains included isotype controls, single positive, and unstained cells. Flow cytometry was performed with the LSR II system (BD Biosciences), and data were analyzed with Diva and FCSExpress software.

Statistical analysis. Statistical analysis of the data presented was performed using GraphPad Prism Ver 5.02, (GraphPad Software, San Diego, CA). Values of *P < 0.1, **P < 0.05, ***P < 0.01, ****P < 0.001 were considered significant.

SUPPLEMENTARY MATERIAL Figure S1. Administration of α-GITR abrogates induction of immune tolerance to hF.IX in C3H/HeJ mice. (a-c) C3H/HeJWT mice (n=5 per group) were injected with a α-GITR antibody (clone: DTA-1) at the time of hepatic AAV2-hF.IX gene transfer and once every 2 weeks for a total of 4 injections followed by challenge with rhF.IX/CFA at 6 weeks. ELISA analysis of plasma samples for (b) hF.IX and (c) anti-hF.IX IgG1 obtained before the protein challenge and those obtained 2 weeks later. (d-f) Administration of α-GITR starting 6 weeks after AAV-hF.IX. Comparative ELISA analysis of plasma samples for (e) hF.IX and (f) anti-hF.IX IgG1 obtained at 12 weeks (before the protein challenge) and those obtained 2 weeks later (at 14 weeks). Data are mean ±SEM. One-way ANOVA with Bonferroni's post test was used for statistical analysis *P<0.05.

Acknowledgments

This work was supported by NIH grants R01 AI51390 (R.W.H.) and P01 HL078810 (R.W.H., C.T.). B.K.S. was supported by T32AI007110-27. The authors thank Dr George Perrin for help with the manuscript and the University of Florida ICBR Hybridoma Core Facility for production of clone 1D11. R.W.H. has been receiving royalty payments from Genzyme for license of AAV-FIX technology.

Supplementary Material

Figure S1.

Administration of α-GITR abrogates induction of immune tolerance to hF.IX in C3H/HeJ mice. (a-c) C3H/HeJWT mice (n=5 per group) were injected with a α-GITR antibody (clone: DTA-1) at the time of hepatic AAV2-hF.IX gene transfer and once every 2 weeks for a total of 4 injections followed by challenge with rhF.IX/CFA at 6 weeks. ELISA analysis of plasma samples for (b) hF.IX and (c) anti-hF.IX IgG1 obtained before the protein challenge and those obtained 2 weeks later. (d-f) Administration of α-GITR starting 6 weeks after AAV-hF.IX. Comparative ELISA analysis of plasma samples for (e) hF.IX and (f) anti-hF.IX IgG1 obtained at 12 weeks (before the protein challenge) and those obtained 2 weeks later (at 14 weeks). Data are mean ±SEM. One-way ANOVA with Bonferroni's post test was used for statistical analysis *P<0.05.

Click here for additional data file. (280.8KB, tiff)

REFERENCES

  1. Gao G, Wang Q, Calcedo R, Mays L, Bell P, Wang L.et al. (2009Adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses Hum Gene Ther 20930–942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Niemeyer GP, Herzog RW, Mount J, Arruda VR, Tillson DM, Hathcock J.et al. (2009Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy Blood 113797–806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cao O, Hoffman BE, Moghimi B, Nayak S, Cooper M, Zhou S.et al. (2009Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B Mol Ther 171733–1742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Wang L., and, Herzog RW. AAV-mediated gene transfer for treatment of hemophilia. Curr Gene Ther. 2005;5:349–360. doi: 10.2174/1566523054065048. [DOI] [PubMed] [Google Scholar]
  5. Breous E, Somanathan S, Vandenberghe LH., and, Wilson JM. Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver. Hepatology. 2009;50:612–621. doi: 10.1002/hep.23043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Breous E, Somanathan S., and, Wilson JM. BALB/c mice show impaired hepatic tolerogenic response following AAV gene transfer to the liver. Mol Ther. 2010;18:766–774. doi: 10.1038/mt.2009.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dobrzynski E, Fitzgerald JC, Cao O, Mingozzi F, Wang L., and, Herzog RW. Prevention of cytotoxic T lymphocyte responses to factor IX-expressing hepatocytes by gene transfer-induced regulatory T cells. Proc Natl Acad Sci USA. 2006;103:4592–4597. doi: 10.1073/pnas.0508685103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mingozzi F, Liu YL, Dobrzynski E, Kaufhold A, Liu JH, Wang Y.et al. (2003Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer J Clin Invest 1111347–1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fields PA, Armstrong E, Hagstrom JN, Arruda VR, Murphy ML, Farrell JP.et al. (2001Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice Gene Ther 8354–361. [DOI] [PubMed] [Google Scholar]
  10. Nayak S, Cao O, Hoffman BE, Cooper M, Zhou S, Atkinson MA.et al. (2009Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T-cells J Thromb Haemost 71523–1532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoffman BE, Dobrzynski E, Wang L, Hirao L, Mingozzi F, Cao O.et al. (2007Muscle as a target for supplementary factor IX gene transfer Hum Gene Ther 18603–613. [DOI] [PubMed] [Google Scholar]
  12. Cao O, Dobrzynski E, Wang L, Nayak S, Mingle B, Terhorst C.et al. (2007Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer Blood 1101132–1140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Prochazkova J, Fric J, Pokorna K, Neuwirth A, Krulova M, Zajicova A.et al. (2009Distinct regulatory roles of transforming growth factor-beta and interleukin-4 in the development and maintenance of natural and induced CD4+ CD25+ Foxp3+ regulatory T cells Immunology 1281 Supple670–e678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Martino AT, Nayak S, Hoffman BE, Cooper M, Liao G, Markusic DM.et al. (2009Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity PLoS ONE 4e6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cooper M, Nayak S, Hoffman BE, Terhorst C, Cao O., and, Herzog RW. Improved induction of immune tolerance to factor IX by hepatic AAV-8 gene transfer. Hum Gene Ther. 2009;20:767–776. doi: 10.1089/hum.2008.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sanjabi S, Zenewicz LA, Kamanaka M., and, Flavell RA. Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol. 2009;9:447–453. doi: 10.1016/j.coph.2009.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Saraiva M., and, O'Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10:170–181. doi: 10.1038/nri2711. [DOI] [PubMed] [Google Scholar]
  18. Wang L, Cao O, Swalm B, Dobrzynski E, Mingozzi F., and, Herzog RW. Major role of local immune responses in antibody formation to factor IX in AAV gene transfer. Gene Ther. 2005;12:1453–1464. doi: 10.1038/sj.gt.3302539. [DOI] [PubMed] [Google Scholar]
  19. Presser K, Schwinge D, Wegmann M, Huber S, Schmitt S, Quaas A.et al. (2008Coexpression of TGF-beta1 and IL-10 enables regulatory T cells to completely suppress airway hyperreactivity J Immunol 1817751–7758. [DOI] [PubMed] [Google Scholar]
  20. Takaku S, Terabe M, Ambrosino E, Peng J, Lonning S, McPherson JM.et al. (2010Blockade of TGF-beta enhances tumor vaccine efficacy mediated by CD8(+) T cells Int J Cancer 1261666–1674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ahmadzadeh M., and, Rosenberg SA. TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells. J Immunol. 2005;174:5215–5223. doi: 10.4049/jimmunol.174.9.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Haribhai D, Lin W, Edwards B, Ziegelbauer J, Salzman NH, Carlson MR.et al. (2009A central role for induced regulatory T cells in tolerance induction in experimental colitis J Immunol 1823461–3468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y.et al. (2010Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells J Immunol 1843433–3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shimizu J, Yamazaki S, Takahashi T, Ishida Y., and, Sakaguchi S. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002;3:135–142. doi: 10.1038/ni759. [DOI] [PubMed] [Google Scholar]
  25. Cohen AD, Schaer DA, Liu C, Li Y, Hirschhorn-Cymmerman D, Kim SC.et al. (2010Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5e10436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liao G, Nayak S, Regueiro JR, Berger SB, Detre C, Romero X.et al. (2010GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T-cells Int Immunol 22259–270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LoDuca PA, Hoffman BE., and, Herzog RW. Hepatic gene transfer as a means of tolerance induction to transgene products. Curr Gene Ther. 2009;9:104–114. doi: 10.2174/156652309787909490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fields PA, Kowalczyk DW, Arruda VR, Armstrong E, McCleland ML, Hagstrom JN.et al. (2000Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX Mol Ther 1225–235. [DOI] [PubMed] [Google Scholar]
  29. Verma D, Moghimi B, LoDuca PA, Singh HD, Hoffman BE, Herzog RW.et al. (2010Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice Proc Natl Acad Sci USA 1077101–7106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Xu L, Gao C, Sands MS, Cai SR, Nichols TC, Bellinger DA.et al. (2003Neonatal or hepatocyte growth factor-potentiated adult gene therapy with a retroviral vector results in therapeutic levels of canine factor IX for hemophilia B Blood 1013924–3932. [DOI] [PubMed] [Google Scholar]
  31. Herbert DR, Orekov T, Perkins C., and, Finkelman FD. IL-10 and TGF-beta redundantly protect against severe liver injury and mortality during acute schistosomiasis. J Immunol. 2008;181:7214–7220. doi: 10.4049/jimmunol.181.10.7214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Dardalhon V, Awasthi A, Kwon H, Galileos G, Gao W, Sobel RA.et al. (2008IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells Nat Immunol 91347–1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maynard CL, Harrington LE, Janowski KM, Oliver JR, Zindl CL, Rudensky AY.et al. (2007Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3- precursor cells in the absence of interleukin 10 Nat Immunol 8931–941. [DOI] [PubMed] [Google Scholar]
  34. Williams LM., and, Rudensky AY. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat Immunol. 2007;8:277–284. doi: 10.1038/ni1437. [DOI] [PubMed] [Google Scholar]
  35. Annoni A, Brown BD, Cantore A, Sergi LS, Naldini L., and, Roncarolo MG. In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance. Blood. 2009;114:5152–5161. doi: 10.1182/blood-2009-04-214569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sun B, Kulis MD, Young SP, Hobeika AC, Li S, Bird A.et al. (2010Immunomodulatory gene therapy prevents antibody formation and lethal hypersensitivity reactions in murine pompe disease Mol Ther 18353–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nelson DR, Tu Z, Soldevila-Pico C, Abdelmalek M, Zhu H, Xu YL.et al. (2003Long-term interleukin 10 therapy in chronic hepatitis C patients has a proviral and anti-inflammatory effect Hepatology 38859–868. [DOI] [PubMed] [Google Scholar]
  38. Schnell MA, Zhang Y, Tazelaar J, Gao GP, Yu QC, Qian R.et al. (2001Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors Mol Ther 35 Pt 1708–722. [DOI] [PubMed] [Google Scholar]
  39. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR., and, Neurath MF. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol. 2004;172:5149–5153. doi: 10.4049/jimmunol.172.9.5149. [DOI] [PubMed] [Google Scholar]
  40. Mempel TR, Pittet MJ, Khazaie K, Weninger W, Weissleder R, von Boehmer H.et al. (2006Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation Immunity 25129–141. [DOI] [PubMed] [Google Scholar]
  41. Dobrzynski E, Mingozzi F, Liu YL, Bendo E, Cao O, Wang L.et al. (2004Induction of antigen-specific CD4+ T-cell anergy and deletion by in vivo viral gene transfer Blood 104969–977. [DOI] [PubMed] [Google Scholar]
  42. Miao CH, Harmeling BR, Ziegler SF, Yen BC, Torgerson T, Chen L.et al. (2009CD4+FOXP3+ regulatory T cells confer long-term regulation of factor VIII-specific immune responses in plasmid-mediated gene therapy-treated hemophilia mice Blood 1144034–4044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Fields PA, Kowalczyk DW, Arruda VR, Armstrong E, McCleland ML, Hagstrom JN.et al. (2000Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX Mol Ther 1225–235. [DOI] [PubMed] [Google Scholar]
  44. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ.et al. (2006Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response Nat Med 12342–347. [DOI] [PubMed] [Google Scholar]
  45. Liu YL, Wagner K, Robinson N, Sabatino D, Margaritis P, Xiao W.et al. (2003Optimized production of high-titer recombinant adeno-associated virus in roller bottles Biotechniques 34184–189. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Administration of α-GITR abrogates induction of immune tolerance to hF.IX in C3H/HeJ mice. (a-c) C3H/HeJWT mice (n=5 per group) were injected with a α-GITR antibody (clone: DTA-1) at the time of hepatic AAV2-hF.IX gene transfer and once every 2 weeks for a total of 4 injections followed by challenge with rhF.IX/CFA at 6 weeks. ELISA analysis of plasma samples for (b) hF.IX and (c) anti-hF.IX IgG1 obtained before the protein challenge and those obtained 2 weeks later. (d-f) Administration of α-GITR starting 6 weeks after AAV-hF.IX. Comparative ELISA analysis of plasma samples for (e) hF.IX and (f) anti-hF.IX IgG1 obtained at 12 weeks (before the protein challenge) and those obtained 2 weeks later (at 14 weeks). Data are mean ±SEM. One-way ANOVA with Bonferroni's post test was used for statistical analysis *P<0.05.

Click here for additional data file. (280.8KB, tiff)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES