1. Volume 25, Issue 4, Pages 880-891 (April 2017)
The Balance between CD8+ T Cell-Mediated Clearance of AAV-Encoded Antigen in the Liver and Tolerance Is Dependent on the Vector Dose 
Sandeep R.P. Kumar, Brad E. Hoffman, Cox Terhorst, Ype P. de Jong, Roland W. Herzog 
Molecular Therapy 
Volume 25, Issue 4, Pages (April 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Molecular Therapy 2017 25, 880-891DOI: (10.1016/j.ymthe.2017.02.014)
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 1
CD8+ T Cell Response to the Transgene Product as a Function of Vector Dose
C57BL/6 WT mice were injected with different doses of AAV8-OVA, and mice were bled at indicated time points. Peripheral blood was analyzed for OVA-specific CD8+ T cell response using MHC-I H2-Kb-SIINFEKL tetramer. (A) Representative plots showing OVA-specific CD8+ T cell frequencies in mice receiving medium or high vector doses. (B) CD8+ T cell dose response to OVA after AAV8-OVA administration via tail vein. (C) Antibody response to vector-derived OVA. (D) Circulating levels of OVA in mouse plasma following tail vein injection. (E) OVA levels in liver lysates from mice injected with 1 × 109 vg at 4 and 12 weeks PI. Data points are presented as average ± SEM (n = 3–5/group). Statistical significance was determined using Student’s t test. *p ≤ 0.05; ***p ≤
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 2
OVA-Specific CD8+ T Cells Initially Express Multiple Markers of Exhaustion and Acquire Memory Phenotype over Time
C57BL/6 WT mice were injected with 1 × 109 vg of AAV8-OVA via tail vein. Mice were bled at indicated time points, and OVA-specific CD8+ T cells were characterized for the expression of various surface markers using flow cytometry. (A and B) Surface expression of different markers of exhaustion on OVA-specific H2-Kb-SIINFEKL tetramer+ CD8+ T cells. (C) Expression pattern of transcription factors T-bet and Eomes on OVA-specific CD8+ T cells. Data points are presented as average ± SEM (n = 5/group).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 3
OVA-Specific CD8+ T Cells Show Delayed Production of IFN-γ and TNF-α
(A) Representative plots showing ex vivo production of IFN-γ and TNF-α by CD8+ T cells from mice that develop an immune response to OVA. (B) Percentages of CD8+ (upper panels) and CD4+ (lower panels) T cells producing IFN-γ, TNF-α, and IFN-γ plus TNF-α following ex vivo re-stimulation with MHC class I- (SIINFEKL) and class II-specific (323–339) OVA peptide, respectively, at indicated time points. (C) Representative histograms from in vivo CD8+ T cell killing assay. A total of 2 million differentially CTV-labeled splenocytes (PBS- or SIINFEKL peptide-pulsed cells) were tail vein injected into either tetramer+ or naive mice. After 12 hr, mice were euthanized and splenocytes were analyzed for CTV-labeled cells. Numbers represent the percentage of adoptively transferred PBS- (brown) or SIINFEKL peptide (black)-pulsed CTV-labeled cells in tetramer+ or tetramer− mice, respectively. (D) Kinetics of memory phenotype on tetramer+ CD8+ T cells following tail vein injection of AAV8-OVA. Memory cells were characterized on the basis of surface expression of CD62L, CD44, and CCR7. Data points are presented as average ± SEM (n = 3–5/group). Statistical significance between different time points was determined using Student’s t test. *p ≤ 0.05; **p ≤ 0.01.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 4
Infiltration of CD8+ T Cells in the Livers of Mice as a Function of AAV8-OVA Vector Dose
OCT frozen liver tissues were sectioned and stained for CD8+ T cells. (A) Quantification of infiltrating CD8+ T cells in livers harvested at 4 weeks PI. (B) Quantification of infiltrating CD8+ T cells in livers harvested at 12 weeks PI. (C) Representative immuno-stained images of liver sections from mice injected either with PBS (naive) or with different doses of AAV8-OVA (green fluorescence shows CD8+ T cells; original magnification ×10). Five different focal areas per mouse were captured, and CD8+ T cells were manually counted. Statistical significance (comparison with naive mice) was determined using Student’s t test. **p ≤ 0.01; ***p ≤
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 5
Lack of PD-1 Pathway Results in Elimination of OVA Expression without Delay
Either C57BL/6 WT or PD-1−/− mice were intravenously injected with 1 × 109 vg of AAV8-OVA, and mice were bled at indicated time points. Peripheral blood and plasma were analyzed for OVA-specific CD8+ T cells and circulating OVA levels, respectively. (A) Frequency of OVA-specific CD8+ T cells in C57BL/6 WT and PD-1−/− mice. (B) Circulating OVA levels in plasma samples of C57BL/6 WT and PD-1−/− mice following intravenous injection of AAV8-OVA. (C) OVA-specific antibodies in plasma samples of C57BL/6 WT and PD-1−/− mice. (D) Quantification of infiltrating CD8+ T cells in liver sections of tetramer+ and tetramer− PD-1−/− mice harvested at 4 weeks PI. (E) Representative immuno-staining images showing infiltration of CD8+ T cells in liver sections of tetramer+ and tetramer− PD-1−/− mice harvested at 4 weeks PI. Data points are presented as average ± SEM (n = 3–6/group). Statistical significance was determined using Student’s t test. ***p ≤
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 6
Multiple Factors Contribute to a Lack of CD8+ T Cell Response to the Transgene Product at High Vector Dose
C57BL/6 WT, C57BL/6 FoxP3DTR, Fas-L−/−, and IL-10−/− mice were injected with 1 × 1010 vg of AAV8-OVA via tail vein. C57BL/6 FoxP3DTR received 1 μg of diphtheria toxin via intra-peritoneal route on days 7, 10, and 14 PI. Mice were bled at 2 and 4 weeks PI, and peripheral blood was analyzed for OVA-specific CD8+ T cells using MHC class I H2-Kb-SIINFEKL tetramer. (A) Representative plots showing OVA-specific CD8+ T cell in FoxP3DTR, Fas-L−/−, and IL-10−/− mice at 2 weeks PI. (B and C) Frequency of OVA-specific CD8+ T cells at 2 and 4 weeks PI, respectively. Data points are presented as average ± SEM (n = 3–5/group). Statistical significance was determined by Student’s t test. *p ≤ 0.05.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions