Martino et al.: Engineered AAV Vector Minimizes in vivo Targeting of

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1 Martino et al.: Engineered AAV Vector Minimizes in vivo Targeting of Transduced Hepatocytes by Capsid-specific CD8 + T Cells Supplementary Methods: In vitro CTL assay on murine hepatocytes: This assay was adapted from a protocol published by others. 1 H2.35 mouse hepatocytes (target: T) pulsed with 10 3, 10 4, 10 5, multiplicity of infection (MOI) of AAV2(Y-F)-hFIX or AAV2- hfix vector were co-cultured with cap-cd8 cells (effector: E) at 80:1, 40:1, 20:1, 10:1 and 5:1 E:T ratio as follows. H2.35 cells (2x10 4 ) were incubated with 10 μg/ml of DiOC18 viability stain in PBS for 45 minutes at 37 C. Subsequently, vector was added to the cells (in 96 well round bottom plates) in 100 µl of Dulbecco's Modified Eagle Medium (DMEM) and followed by 6-hr incubation. Additional 100 µl of DMEM (containing 10% FBS and 2% penicillin streptomycin) was added and incubated continued for another 12 hours. Pulsed target cells were rinsed 2X with PBS, and effector cells were added in 100 µl of RPMI-1640 media (containing 10% FBS and 1% penicillin and streptomycin) followed by incubation for 6 hours at 37 C. Cells were rinsed 2X with PBS and then incubated for 10 min with 20 µl of trypsin. Two µl of 7-AAD (diluted in 500 µl of PBS with 1% FBS) was added to detached target cells for 10 min at room temperature. Target cell death and percent hfix + cells were measured by flow cytometry. For control, effector cells were co-cultured with uninfected target cells to determine background target cell death (mock targets). Proteasome inhibition was performed by adding either 100 nm Bortezomib or 300 nm Mg-132 (Selleckchem, Houston, TX) to AAV vector infection media. 1

2 Liver target cells were gated by size using scatter plot, followed by histogram gating for DiOC18 + (3,3 -Dioctadecyloxacarbocyanine perchlorate, Invitrogen, viability dye fluorescing at 484 nm) cells and finally analyzed using dual stain using dot plot for DiOC18 vs 7-AAD (7- Amino Actinomycin D, Invitrogen, cell death stain fluorescing at 650 nm) stains. Unlabelled hepatocytes were assayed in parallel using identical co-cultures with T cells followed by intracellular staining for hfix with goat anti-hf.ix and anti-goat IgG-Alexa Fluor-488 (Invitrogen). All cells were counted with LSR II flow cytometer and analyzed with FCS 3.0 software (BD Biosciences, San Jose, CA). All target:effector ratios were assayed for killing and hf.ix expression in quadruplicate. ELISpot for IFN-γ secreting cells: Splenocytes were isolated 7 days after intramuscular (IM) injection of BALB/c mice with AAV vector (1x10 11 vg/mouse) and cultured for ELISpot analysis as published. 2 The stimulation media contained 5 μg/ml of the dominant CD8 + AAV2 capsid epitope (VPQYGYLTL, presented by H2-L d ), or other potential AAV2(Y-F) epitopes for the H-2 d haplotype (peptides were from AnaSpec, Fremont, CA). Positive control stimulation media contained 10 μg/ml of Staphylococcal Enterotoxin B (SEB) super antigen (Sigma, St Louis, MO). IFN-γ producing cells were counted with the CTL-ImmunoSpot S5 UV analyzer (Cellular Technology, Shaker Heights, OH). Intracellular imaging of human DCs and hepatocytes: Human dendritic cells (DCs) were prepared from fresh apheresis samples obtained from the Center for AIDS Research at the University of Pennsylvania. All samples were de-identified and 2

3 collected under protocols approved by the local Ethical Committees. DCs were prepared as previously described and stored in liquid nitrogen. 3 The human hepatocyte cell line HHL5 was previously described. 4 Image acquisition was performed with an Amnis ImageStreamX instrument (Amnis Corporation) at a 40x magnification. At least 5000 cells were acquired for each condition and data were analyzed, using specific algorithms for internalization and intracellular co-localization. DCs were incubated with virus in serum-free AIM-V (for DCs) at 37 C; all media used were from Invitrogen/Gibco. After one or four hours, cells were washed twice with PBS and stained. Intracellular staining was performed on 5x10 5 cells fixed with a fixation/permeabilization buffer (BD Bioscience) using the mouse anti-aav antibody A20 (Fitzgerald Industries International) at a dilution of 1:40 and a rabbit anti-cd71 antibody (Epitomics) at a dilution of 1:200. After 30 minutes at room temperature, cells were washed and secondary antibodies were added. Control stainings without primary antibody or with primary but without secondary antibody addition were performed for anti-aav and anti-cd71. At the end of the staining cells were fixed and analyzed. HHL5 human hepatocytes were incubated in serum free DMEM with the vectors (MOI = 1x10 5 ) in a 5%-CO 2 incubator at 37 o C for 1 to 24 hours. After the indicated times cells were washed with PBS and stained. Vectors used in the HHL5 experiments were conjugated with Alexa Fluor-488 using a commercially available kit (Invitrogen). Control stainings were performed with unlabeled vectors. Nuclear staining was performed with 1μM DRAQ5 (Biostatus Inc.). Internalization and nuclear colocalization analysis was performed using the IDEAS Software (Amnis Corporation). 3

4 Capsid antigen presentation and CTL assay in human hepatocytes: Capsid antigen presentation assay was performed as described. 5 Briefly, the human hepatocyte cell line HHL5 was transduced with increasing MOIs of AAV vectors. Twenty four hours after transduction, a reporter T cell line was added at a reporter:target ratio of 10:1. In this assay, the intensity of the reporter gene signal correlates with the amount of capsid antigen being presented by MHC class I. 5 CTL assay was also performed as published. 6 Targets were transduced overnight with increasing MOIs vectors. Effector CD8 + T cells expanded in vitro from human PBMCs were co-cultured with target cells for four hours at an effector:target ratio of 10:1, and cytotoxicity was measured with a colorimetric assay. 3 Computer analyses of MHC I peptide presentation: Prediction of capsid-specific CD8 + T cell epitopes for H-2 d haplotype and for binding to L d and B*0702 molecules was performed with SYFPEITHI software (Biomedical Informatics, Heidelberg, Germany). For statistical analyses, differences between two experimental groups were analyzed with 2-tail student s t-test. The peptide/h2-l d complex was modeled using the crystal structure PDB code 3TJH, The peptide side chains were altered in Coot to generate peptide with the sequence VPQYGYLTL using the most frequently used solvent exposed rotamer conformations. 7 The peptide/hla-b*07:02:01 complex was modeled using the crystal structure of HLA-B8 from PDB code 3SPV (95.7 % identical to HLA-B*07:02:01) using the SWISS-MODEL program. 8 Atomic coordinates for the VPQYGYLTL peptide bound to L d were displayed in the antigen binding cleft of HLA-B*07:02:01 following superposition using Coot. Pymol was used to generate molecular graphic images. 4

5 Freezing of murine CD8 + T cells: All experiments in the manuscript were performed using freshly expanded CD8 + T cells. However, after in vitro expansion of cap-specific CD8 + T cells (rather than proceeding to purification), the cells can be frozen using the following protocol. Cells are suspended in1x10 7 cells/ml in freeze media (40% FBS, 50% Cell culture media, 10% DMSO) and added at ml to 2.0-ml cryotubes. Cells in freeze media should not be kept on ice for more than 15 minutes. Cryotubes with cells are frozen at -80 C overnight and then transferred to liquid nitrogen. To thaw the cells, 500 μl of cell culture media (RMPI in 10% FBS, pre-warmed to 37 C are added drop wise to frozen cells, cells are allowed to thaw at room temperature. Cells are spun down, freeze media are removed, and cells re-suspended to 1x10 7 viable cells/ml in stimulation media (RMPI in 10% FBS containing the peptide epitope). One ml of thawed cells are mixed with 1 ml of freshly isolated splenocytes from a naïve BALB/c mouse (5x10 6 cells/ml). Cells are stimulated for up to 3 days followed by purification of CD8 + T cells. References: 1. Hoppner M, Luhm J, Schlenke P, Koritke P, Frohn C. A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells. J Immunol Methods. 2002;267: Martino AT, Herzog RW, Anegon I, Adjali O. Measuring immune responses to recombinant AAV gene transfer. Methods Mol Biol. 2011;807: Mingozzi F, Maus MV, Hui DJ, et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med. 2007;13:

6 4. Clayton RF, Rinaldi A, Kandyba EE, et al. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int. 2005;25: Finn JD, Hui D, Downey HD, et al. Proteasome inhibitors decrease AAV2 capsid derived peptide epitope presentation on MHC class I following transduction. Mol Ther. 2010;18: Pien GC, Basner-Tschakarjan E, Hui DJ, et al. Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors. J Clin Invest. 2009;119: Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66: Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Res. 2009;37:D

7 Supplementary Figures: Fig. S1. AAV2(Y-F) mutations do not chance the dominant capsid-specific CD8 + T cells in BALB/c mice. Frequency of IFN-γ secreting cells upon in vitro stimulation (ELISpot) of splenocytes isolated from BALB/c mice (n=4 per vector) 10 days after IM injection of AAV2 or AAV2(Y-F). Cells were stimulated with peptide encoding the known dominant CD8 + T cell epitope E1 (VPQYGYLTL), or putative (AAV2(Y-F) epitopes E2 (RPKRLNFKL) or E3 (EPRPIGTRF). Data are average ±SD. ** P value < 0.01 by Student s t-test comparing E1 to E2, E3, or mock stimulation. 7

8 Fig. S2. AAV capsid-specific CD8 + T cells adoptively transferred at later time points (day 7 after gene transfer or later) do not effect hf.ix levels. AAV2 gene transfer and capsidspecific T cell transfer was carried out as in Fig. 3, except that T cell administration was performed either 7 days or 14 days after vector administration. Systemic hf.ix levels were compared to mice that received vector but no T cells (n=4 per experimental group). Fig. S3. Adoptively transferred AAV capsid-specific CD8 + T cells do not effect hf.ix levels in adenoviral vector-transduced mice. Viral gene transfer and capsid-specific T cell transfer was carried out as in Fig. 3, except that Ad-hF.IX vector injected instead of AAV-hF.IX. Systemic hf.ix levels were compared to mice that received AAV capsid-specific or control CD8 + T cells (n=4 per experimental group). 8

9 Fig. S4. Outline of in vitro killing assay. Diagram of how cell death was measured in murine H2.35 liver target cells pulsed with AAV vector and co-cultured with either control or capsidspecific CD8 + T cells. Target cells were pre-stained with DioC 18 (intracellular staining) and later stained with 7AAD for cell death. Cells were first gated by scatter pattern to distinguish effector T cells from liver target cells. A DioC 18 gate was used to unequivocally identify H2.35 target cells, and finally the percentage of 7AAD and DioC 18 co-stained target cells was determined. 9

10 Fig. S5. In vitro killing of murine hepatocytes by capsid-specific CD8 + T cells and survival of hf.ix-expressing murine hepatocytes after co-culture with capsid-specific CD8 + T cells (adjusted for similar transduction efficiency). H2.35 cells were infected with a multiplicity of infection (MOI) of 10 4 for AAV2 or MOI of 10 3 for AAV2(Y-F), which results in similar transduction efficiencies. A. Percent death of target cells (adult BALB/c hepatocyte cell line H2.35), pulsed with either AAV2 or AAV2(Y-F), as a function of effector to target ratio. B. Percent surviving hf.ix + H2.35 hepatocytes (as determined by flow cytometry after completion of in vitro killing assay) pulsed with either AAV2 or AAV2(Y-F), as a function of effector to target ratio. In panel D) an MOI of 10 4 for AAV2 is compared to MOI of 10 3 for AAV2(Y-F), which results in similar transduction efficiencies. Statistically significant differences between AAV2 and AAV2(Y-F) for a specific traget:effector ratio are indicated with * for P<0.05, unpaired, two-tailed t test. 10

11 Fig. S6. Binding and entry of AAV vectors to human dendritic cells. Mature human DCs cells were treated at a multiplicity of infection (MOI) of 10 5 for one hour. After staining cells were acquired on ImageStream imaging flow cytometer. Analysis of acquired data was performed using algorithms specific for internalization (panels A and B) and endosomal colocalization (panels C, D). From top to bottom: A. Percent of cells showing positive staining for the AAV2 or AAV2(Y-F) capsid. B. Percent of cells with positive staining for the AAV capsid inside the cell after exclusion of viral particles bound to the cell surface (internalization). C. Percent of cells with positive staining for the AAV capsid and for the early endosomal marker 11

12 CD71. D. Histogram plots depicting the percent of double positive cells in which the signals of capsid and early endosome is co-localized. Shown are results from one representative experiment. All experiments were repeated and analyzed at least twice. Fig. S7. Capsid-specific CD8 + T cells infiltrate livers of AAV8 transduced mice. Images show Cap-CD8 cells (green cell, highlighted with arrows) 6 weeks after AAV8 gene transfer in mice that had received the cells 1 day, 7 days, or 14 days after vector administration. Tope left: liver of a mouse that had not received cells. Red stain: hf.ix-expressing hepatocytes. 12

13 Fig. S8. Mild increase in systemic liver enzyme levels 7 after administration of AAV capsid-specific CD8+ T cells (Cap-CD8) to mice that had received hepatic AAV8-hF.IX gene transfer. Gene transfer (1x10 11 vg/mouse) had been performed 1 day after CD8 cell administration. Control animals received vector plus control cells or vector only (n=4 per experimental group). 13

14 Fig S9: Human F.IX expression in immune competent mice as a function of time after hepatic gene transfer. AAV2 or AAV2(Y-F) vector was administered via tail vein. A. Transduction of C57BL/6 mice with 5x10 10 vg/mouse (n=5 per experimental group). Vector was given alone or mixed with 25 mg of the proteasome inhibitor bortezomib. B. Transduction of BALB/c mice at the indicated vector doses. 14