Cells and cell lines. Four distinct sets of human cells were used in these studies: 1) PBMCs from

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1 Supplementary Methods Cells and cell lines. Four distinct sets of human cells were used in these studies: 1) PBMCs from hemophilic subjects who had participated in a gene transfer study of AAV-2-F.IX; these cells are denoted as subject E or subject G 3. 2) PBMCs from adults unaffected by hemophilia. These are summarized in Supplementary Table 2 online. Data collected on these subjects included both neutralizing antibody titer to AAV-2, and AAV-2 capsid-specific IFN-g ELISpot assays; 3) PBMCs from normal donors (ND) provided by the Center for AIDS Research of the University of Pennsylvania. Data collected from this third group of subjects included a variety of AAV capsid-specific T cell responses, and, in some cases, neutralizing antibody titer to AAV-2. 4) Splenocytes harvested from subjects undergoing splenectomy for a variety of indications at both an adult and a children s hospital. PBMC and splenocytes from all subjects in the study were collected as part of protocols approved by the Institutional Review Boards of the Children s Hospital of Philadelphia, the University of Pittsburgh Medical Center, or the Royal Prince Alfred Hospital. PBMC were isolated with Ficoll-Hypaque gradient centrifugation as per standard protocols and cryopreserved until assayed. Spleens were collected from the operating room or from the Collaborative Human Tissue Network on the day of splenectomy. Splenic tissue was homogenized, red cells lysed, and splenocytes cryopreserved until assayed. The JY cell line (B*0702 positive) was a kind gift from Dr. Robert Vonderheide (University of Pennsylvania). Viral vectors. Production of adenoviral vectors expressing AAV capsid was as previously described 4. Clinical grade recombinant AAV vectors used in human subjects were prepared 1

2 using Good Manufacturing Practices as previously described 5. Recombinant AAV-2 expressing human factor IX (AAV-2-F.IX) under the control of a liver-specific promoter was prepared as previously described 6. Empty capsids were purified by cesium chloride gradient centrifugation, as a contaminant band produced in purification of recombinant AAV vectors. In vitro expansion and cell culture. On d 0, 10 6 PBMC were stimulated with 10 mg/ml of the peptide indicated, or 10 mg/ml of AAV empty capsid particles, 0.5 mg/ml human b2- microglobulin (Sigma), and 10 ng/ml recombinant human IL-7 (Roche) irradiated autologous PBMC were added as feeder cells. Recombinant human IL-2 (Roche) was added to a final concentration of 20 IU/ml on d 1 and replenished every 2-3 d thereafter. IL-7 was replenished every 7 d. Cells were re-stimulated with peptide as above every 8-10 d. The following peptides were purchased from Genemed Synthesis, Inc.: B*0702-restricted peptides were HIV gag (IPRRIRQGL), EBV (RPPIFIRRL), and AAV-2 (VPQYGYLTL) and AAV-8 (IPQYGYLTL). HLA-A*0101-restricted peptides were Flu (CTELKLSDY) and AAV-2 (SADNNNSEY). The B*0702-restricted peptide is sometimes denoted peptide 74 or p74, and the A*0101-restricted peptide as peptide 99 or p99. The MHC:peptide pentamers for alleles A*0101 and B*0702 together allow monitoring of responses in up to 45% the Caucasian population 7. Splenocytes were expanded using 15-mers derived from the AAV VP1 capsid protein (735 amino acids, 145 peptides [Mimotopes]) individually. Cultures were maintained in AIM-V media supplemented with 3% human AB serum (Valley Biomedical) or RPMI supplemented with 10% human AB serum, L-glutamine, and antibiotics. 2

3 Staining and flow cytometry. In preparation for cell surface staining, cells were washed once in PBS-2% FBS. In cases where pentamer was used, pentamer staining was performed first for 20 min at 4 C. Antibody stains were also performed for 20 min at 4 C, followed by a wash in PBS-2% FBS and fixation in PBS-1% paraformaldehyde (Electron Microscopy Sciences). All pentamers and the antibody to CD8 were purchased from Proimmune. The following antibodies were purchased from BD Biosciences: CD3, CD4, CD14, CD20, IFN-g, CD80, CD86, HLA-DR, CD40, CCR7, CD27, CD45RA. For intracellular cytokine staining, cells were washed into fresh media and incubated at 37 o C with 10 mg/ml of the indicated peptide, 10 mg/ml of AAV empty capsid particles, or 50 ng/ml PMA (Sigma), 1 mg/ml ionomycin (Sigma), or medium alone for one h before adding monensin (GolgiStop, BD Biosciences) as per the manufacturer s directions. Incubation at 37 o C was continued for another four h, followed by cell surface staining, and fixation and permeabilization (BD Cytofix/Cytoperm), and intracellular staining. All samples were acquired on a Cyan flow cytometer (Dako Cytomation), and data were compensated and analyzed using FlowJo software (Treestar). Human dendritic cells. Adherent cells remaining after a two-h 37 o C incubation of fresh PBMC in plain AIM-V were treated with 800 U/ml recombinant human GM-CSF (Leukine, Berlex) and 500 U/ml recombinant human IL-4 (R&D Systems). After 6 d, the immature dendritic cells were harvested by washing in cold PBS and their phenotype was confirmed by flow cytometry after staining for HLA-DR, CD80, CD86, and CD83. Immature DC were then incubated for 48 h with an AAV-2-F.IX or an AAV-8-F.IX vector at an MOI of 500,000, or treated with a maturation cocktail (vide infra) for 24 h. The maturation cocktail consisted of 800 U/ml GM-CSF, 500 U/ml IL-4, 10 ng/ml TNF-a (R&D Systems), 150 ng/ml IL-6 (R&D Systems), 10 ng/ml IL-1b (R&D 3

4 Systems), and 1 mg/ml PGE2 (Cayman Chemicals). DCs were harvested and cell surface phenotypes were then re-assessed by staining and flow cytometry (as above). CTL assays. Cytotoxicity assays were performed as per standard protocols. Briefly, HLAmatched lymphoblastoid cells lines or JY cells were used as targets by loading them with 10 mg/ml of peptide overnight, and then labeling for one h with 500 mci of 51 Cr per million cells. Targets and effector cells were co-incubated for four h at 37 o C, and supernatants were counted with a gamma counter. The maximum release of targets was determined by treating targets with 1%-Triton X 100 (Bio-Rad). Percent of specific lysis was calculated with the formula (experimental release-spontaneous release) / (maximum release-spontaneous release). Neutralizing antibody assays to AAV. Neutralizing antibody titer to AAV-2 was determined as described previously 8. Briefly, half-log serial dilutions of serum samples in naïve mouse serum were mixed with vector genomes of AAV-2-LacZ vector in a 30 ml volume for 1 h at 37 C before addition of 15 ml of the mixture to a 96-well plate containing HEK293 cells per well in 200 ml of DMEM. Beta-galactosidase activity was measured the following day by a colorimetric assay. Percent activity was determined with the formula [test sample OD420 untransduced) / (maximum activity OD420 untransduced)] 100. The neutralization titer of a sample was determined as the highest dilution which results in 50% or greater inhibition of the beta-galactosidase activity. ELISpot assays for production of IFN-g by subject PBMCs. IFN-g ELISpot assays were performed as previously described 3. Peptide libraries consisting of 15-mers overlapping by 10 4

5 amino acids with the adjacent peptide were prepared for the AAV-2 VP-1 capsid sequence (735 amino acids, 145 peptides [GeneMed Synthesis, or Mimotopes]). For PBMC, peptides were screened in pools of 12 peptides each as previously described 3. For splenocytes, where numbers of cells were not limiting, each of the 145 peptides was screened individually. ELISpot assays were performed either at the Children s Hospital of Philadelphia, at Avigen, Inc., or under contract at the reference laboratory of the NIH Immune Tolerance Network (Cellular Technology, Ltd.). All assays were performed in triplicate. Positive controls included a 23 peptide pool 9 consisting of MHC class I-restricted peptides representing T cell epitopes of viruses common in the population (cytomegalovirus, Epstein-Barr virus, influenza), and phytohemagglutinin (PHA). Media alone served as negative control. Assays were scored positive if the number of spots was above 3 media control, and >10 spots per PBMC. Mouse experiments. Male mice transgenic for HLA-B*0702 and lacking mouse MHC H2K b and H2D b 10 were injected on d 0 as follows: a single injection of vector genomes of an AAV-2-F.IX vector into the lateral tail vein, a single injection into each anterior tibialis muscle to deliver a total of vector genomes of AAV-2-F.IX, or a single injection into each anterior tibialis muscle to deliver a total of AdHu5-AAV-2 Capsid vector particles 4. Mice were bled retroorbitally on d 7, 14, 21, 28 and 56 post-injection. 50 ml of blood was reserved for pentamer staining and plasma was extracted from the remainder of each sample for transgene expression analysis. For pentamer staining, 3 ml of HLA-B*0702-VPQYGYLTL pentamer was added directly to each whole blood sample. Following a 20 min incubation in the dark at room temperature, antibodies to mouse CD4, CD8, CD11b and CD19 were added and the samples were incubated for an additional 30 min in the dark at room temperature. After the 5

6 antibody incubation, cells were RBC lysed, washed in PBS, and fixed in paraformaldehyde for FACS analysis on a Cyan ADP cytometer. The percent of pentamer positive, CD8 + cells was determined after first performing a negative selection to remove CD4 +, CD11b + and CD19 + cells from the lymphocyte population. To verify successful vector injection, human Factor IX transgene expression levels were measured by sandwich ELISA as previously described 11. 6

7 Supplementary References 1. Nguyen, D.H., Hurtado-Ziola, N., Gagneux, P. & Varki, A. Loss of Siglec expression on T lymphocytes during human evolution. Proc. Natl. Acad. Sci. USA 103, (2006). 2. Bartlett, J.S., Wilcher, R. & Samulski, R.J. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J. Virol. 74, (2000). 3. Manno, C.S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat. Med. 12, (2006). 4. Sabatino, D.E. et al. Identification of mouse AAV capsid-specific CD8+ T cell epitopes. Mol. Ther. 12, (2005). 5. Manno, C.S. et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101, (2003). 6. Mingozzi, F. et al. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J. Clin. Invest. 111, (2003). 7. Mori, M., Beatty, P.G., Graves, M., Boucher, K.M. & Milford, E.L. HLA gene and haplotype frequencies in the North American population: the National Marrow Donor Program Donor Registry. Transplantation 64, (1997). 8. Kay, M.A. et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24, (2000). 9. Currier, J.R. et al. A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays. J. Immunol. Methods 260, (2002). 1

8 10. Rohrlich, P.S. et al. HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus. Int. Immunol. 15, (2003). 11. Herzog, R.W. et al. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc. Natl. Acad. Sci. USA 94, (1997). 2