1. Volume 19, Issue 7, Pages 1263-1272 (July 2011)
Nonredundant Roles of IL-10 and TGF-β in Suppression of Immune Responses to Hepatic AAV-Factor IX Gene Transfer 
Brad E Hoffman, Ashley T Martino, Brandon K Sack, Ou Cao, Gongxian Liao, Cox Terhorst, Roland W Herzog 
Molecular Therapy 
Volume 19, Issue 7, Pages (July 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
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 < IL, interleukin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
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 < Original magnification: ×100 and ×200. Arrows, CD8+ cells within the parenchyma; arrowhead, CD8+ cell located in a blood vessel.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
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 < (d) Representative immunofluorescent labeling of liver sections obtained from mice treated as above at 2.5 weeks postgene transfer. Original magnification: ×100.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
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 <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
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 <
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
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.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions