1. Volume 22, Issue 1, Pages 28-41 (January 2014)
AAV8 Induces Tolerance in Murine Muscle as a Result of Poor APC Transduction, T Cell Exhaustion, and Minimal MHCI Upregulation on Target Cells 
Lauren E Mays, Lili Wang, Jianping Lin, Peter Bell, Alison Crawford, E John Wherry, James M Wilson 
Molecular Therapy 
Volume 22, Issue 1, Pages (January 2014)
DOI: /mt
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
AAV2/8 induces poor T cell activation and stable transgene expression in vivo. (a) C57BL/6 mice were injected intramuscularly with 1011 genome copies of AAV2/8 or AAV2/rh32.33 expressing CB.nLacZ. At day 21, whole blood was acquired for nLacZ-specific MHCI tetramer staining (right axis, gray bars), and mice were necropsied to harvest spleen for IFN-γ ELISPOT (left axis) and (b) muscle for X-gal histochemistry at day 28. (a) Splenocytes (left axis) were stimulated with the dominant H-2Kb CD8+ T cell epitopes for either the AAV8 or AAVrh32.33 capsid (white bars) or the β-gal transgene (black bars). PBS-injected mice showed no activation in response to the AAV8 or AAVrh32.33 capsid epitopes (stimulation against AAV8 capsid epitope is shown). AAVrh32.33-injected mice showed no activation in response to the AAV8 capsid; AAV8-injected mice showed no activation in response to the AAVrh32.33 capsid epitope (data not shown). Data represent a mean ± SD for four mice per group, where *P ≤ 0.05 compared with PBS or AAV2/8. Results were confirmed by three independent experiments. (b) Representative images from four mice per group are shown here under 10× magnification; scale bar represents 200 µm. AAV, adeno-associated virus; ELISPOT, enzyme-linked immunosorbent spot; IFN-γ, interferon-γ; MHCI, major histocompatibility complex class I; nLacZ, nuclear-targeted LacZ; PBS, phosphate-buffered saline.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
AAV2/8 induces transgene-specific tolerance to AAV2/rh32.33.nLacZ in the muscle. C57BL/6 mice were injected intramuscularly (i.m.) with PBS or 1011 genome copies (GC) of AAV2/8.CB.nLacZ at day 0 in the right hind leg. 14 days later, mice received an i.m. injection in the opposite leg, containing PBS or 1011 GC of AAV2/rh32.33 expressing either nLacZ (a and b, left panel) or eGFP (b, right panel). (a) At days 3, 7, 14, 21, and 28, whole blood was acquired for nLacZ-specific major histocompatibility complex class I tetramer staining (top panel). At day 28 postinjection, mice were necropsied to harvest muscle for X-gal histochemistry (bottom panel). (b) At the peak of the T cell response, day 28, splenocytes were harvested for interferon-γ (IFN-γ)-ELISPOT. Splenocytes from the left panel were stimulated independently with the dominant H-2Kb CD8+ T cell epitopes for both the AAV8 capsid (data not shown), the AAVrh32.33 capsid (white bars), and the β-gal transgene (black bars). Capsid-specific IFN-γ–producing T cell responses toward the AAV8 capsid were negative for all groups (data not shown). Splenocytes from the right panel were stimulated with the dominant, GFP-specific H-2Kb CD8+ T cell epitope to assess transgene-specific T cell responses to GFP (black bars). Data represent a mean ± SD for four mice per group, where *P ≤ 0.05 compared with (a) PBS or AAV2/8 or (b) the indicated comparisons. Results were confirmed by three independent experiments. (b) Representative tissue sections from four mice per group are shown here under 10× magnification; scale bar represents 250 µm. AAV, adeno-associated virus; nLacZ, nuclear-targeted LacZ; ns, nonsignificant; PBS, phosphate-buffered saline.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
A suppressive factor is not sufficient to account for AAV2/8-induced tolerance. (a) Following intramuscular (i.m.) injection of PBS or 1011 genome copies (GC) of either AAV2/8 or AAV2/rh32.33 expressing CB.nLacZ, splenocytes were harvested at day 21 and stained for CD4, CD25, and Foxp3 by intracellular Foxp3 staining. Data represent the percentage of CD4+ T cells expressing CD25+ and Foxp3+ within the lymphocyte population, as determined by flow cytometry and FlowJo analysis. Representative dot plots were taken from the median of each group. (b) C57BL/6 mice were administered either PBS or the anti-CD25 depleting antibody, PC-61, at days −1 and every 2 weeks thereafter. At day 0, mice were injected i.m. with either PBS or 1011 GC of AAV2/8.CB.nLacZ in the right hind leg. Fourteen days later in the opposite leg, mice received either PBS or 1011 GC of AAV2/rh32.33.CB.nLacZ. At day 21, whole blood was acquired for nLacZ-specific MHCI tetramer staining (top panel) and mice were necropsied to harvest muscle for X-gal histochemistry at day 28 (bottom panel). (c) C57BL/6 mice were injected i.m. with 1011 GC of AAV2/8.CB.nLacZ. At day 21, total splenocytes or sorted CD4+ T cells, by magnetic-activated cell sorting, were isolated and adoptively transferred to naive mice via tail vein injection. The following day, mice were immunized i.m. with 1011 GC AAV2/rh32.33 to determine if a suppressive factor present in the splenocyte or CD4 cell population could blunt the normally strong T cell response seen with AAV2/rh Twenty-one days after adoptive transfer, whole blood was isolated for nLacZ-specific MHCI tetramer stain (top panel). At day 28 post-transfer, muscle was harvested for X-gal histochemistry (bottom panel). (b) Data represent means ± SD for four mice per group, where **P ≤ 0.01 for the indicated comparisons. Results were confirmed by two independent experiments. (c) Data represent means ± SD for a cumulative n = 8, generated from two independent experiments each with an n of 4. Representative muscle sections from four mice per group are shown here under 10× magnification; scale bars represent 250 µm. AAV, adeno-associated virus; nLacZ, nuclear-targeted LacZ; PBS, phosphate-buffered saline.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
AAV2/8-induced tolerance can be broken in the presence of Toll-like receptor (TLR)-mediated inflammation. C57BL/6 mice were injected intramuscularly with either PBS or 1011 genome copies (GC) of AAV2/8.CB.nLacZ in the right hind leg at day 0. Fourteen days later in the opposite leg, mice received 1011 GC of AAV2/rh32.33.CB.nLacZ either alone or in combination with four consecutive injections of the ligands for TLR3 and TLR4, respectively. At days 14, 21, and 28, whole blood was acquired for nLacZ-specific major histocompatibility complex class I tetramer staining (top panel) and mice were necropsied to harvest muscle for X-gal histochemistry (bottom panel). Data represent means ± SD for four mice per group, where *P ≤ 0.05 compared with the “AAV8 + AAVrh32.33” group. Results were confirmed by two independent experiments. Representative muscle sections from four mice per group are shown here under 10× magnification; scale bar represents 200 µm. AAV, adeno-associated virus; LPS, lipopolysaccharide; PBS, phosphate-buffered saline.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
AAV2/8 primed T cells exhibit markers of exhaustion. C57BL/6 mice were injected intramuscularly with 1011 genome copies of either AAV2/8 or AAV2/rh32.33 expressing nuclear-targeted LacZ (nLacZ). Splenocytes were harvested for flow cytometry, 14, 21, and 28 days postinjection. Data represent MFI of (a) LAG3, (b) 2B4 (B), (c) PD1 and nLacZ-tetramer double positive T cells. Data represent means ± SD for four mice per group, where *P ≤ 0.05 for the indicated comparisons. AAV, adeno-associated virus; MFI, mean fluorescent intensity.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
AAV2/8 shows reduced MHCI expression on the surface of myocytes in comparison to AAV2/rh C57BL/6 mice were injected intramuscularly with 1011 genome copies of either AAV2/8 or AAV2/rh32.33 expressing nLacZ. Mice were killed and muscles were isolated, 14, 21, and 28 days postinjection. (a) Muscle sections were blocked with avidin and biotin, treated with mouse-specific blocking solution, and stained with monoclonal antibody (anti-MHCI) diluted 1:50 for 1 hour followed by biotinylated anti-mouse antibodies and rhodamin-avidin; scale bar represents 500 µm. Scale bars for the image with DAPI costaining represent 25 µm. For controls, primary antibodies were replaced by normal sera from C57BL/6 mice (1:1000), or BALB/C muscle sections (haplotype d) were stained with the haplotype b-specific antibody (data not shown). (b) To quantify MHCI expression, a low magnification image (4× objective) was taken from each muscle section stained for H-2K/H-2D. The brightness values of the images (i.e., the sum of all pixel values of each image) were determined with ImageJ software and averaged for each group. Experiments were performed with an n of four mice per group (*P ≤ 0.05) for the indicated comparisons. AAV, adeno-associated virus; MHCI, major histocompatibility complex class I.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
AAV2/8 shows reduced in vitro transduction and in vivo activation of antigen-presenting cells (APCs). (a) AAV2/8 or AAV2/rh32.33 expressing enhanced GFP (eGFP) was used to transduce JAWSII murine dendritic cells (DCs) (top two panels) at a multiplicity of infect (MOI) of 1 × 107 genome copies (GC)/cell. PBS and Ad5.eGFP were used as negative and positive controls, respectively. AAV2/8 or AAV2/rh32.33 vectors were used to cotransduce U937 human monocyte derived DCs (bottom panel) at an MOI of 1 × 106 with wtAd5 (MOI: 1 × 104) and anti-Ad5 antibody (1/8,000). Images were taken 4 days post-transduction. Scale bar is estimated to represent 50 µm. (b) RAW murine macrophage cells were transfected with vector expressing firefly luciferase. (c) Transduced U937 cells were then run on a flow cytometer and gated for GFP fluorescence (FL1) to compare mean fluorescence intensity between cells transduced with AAV2/8 versus AAV2/rh32.33 (representative image shown). (d–f) C57BL/6 mice were injected intramuscularly with 1011 GC of AAV2/8 or AAV2/rh32.33 expressing nLacZ. At 12 hours postinjection, spleen, popliteal, and inguinal lymph nodes were harvested and stained for antibodies against the DC marker CD11c, and the APC activation markers, CD80/86, CD40, and MHCII. (d) The percentage of CD11c+ DCs in the total cell population within each tissue 12 hours postinjection. (e) The percentage of expression of DC activation markers within the popliteal lymph node 12 hours postinjection. (f) Representative histograms demonstrating differential expression of surface molecules typically upregulated on activated DCs; taken from the popliteal lymph node at 12 hours. An intraperitoneal injection of LPS, a known activator of innate immunity, was used as a positive control. Experiments were performed in triplicate (in vitro) or with an n of four (in vivo) and repeated in at least two independent studies. Data are shown as mean ± SD; *P ≤ 0.05 for the indicated comparisons as shown in b, or when compared with PBS or AAV2/8 groups as shown in d,e. AAV, adeno-associated virus; GFP, green fluorescent protein; LPS, lipopolysaccharide; MHCII, major histocompatibility complex class II; PBS, phosphate-buffered saline.
Molecular Therapy  , 28-41DOI: ( /mt )
Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions