Materials and Methods

2.1 Animals

Male C57BL/6 mice were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). PD-L1 KO mice were kindly provided by Dr.

Lieping Chen (59). All animals were housed in a specific pathogen-free environment in the animal facility of the Institute of Biomedical Sciences, Academia Sinica. All

experimental procedures complied with the regulations of the Academia Sinica Institutional Animal Care and Use Committee and the Council of Agriculture Guidebook for the Care and Use of Laboratory Animals.

2.2 Adeno-associated viral (AAV) vectors

The AAV2/8-5’HBV-SD and AAV2/8-3’HBV-SA which contain 5’-HBV and 3’-HBV genome, respectively, were generated and described previously (52). Briefly, plasmid pHBV1.3, containing the 1.3-times overlength HBV genome (genotype D), was split at the CAG/G site between nucleotides 2192 and 2193. A highly conserved

synthetic intronwas inserted at the split site. Plasmids pAAV5’-HBV-SD and

pAAV-3’-HBV-SA were generated by subcloning the two halves of HBV genome were subcloned into the pAAV-MCS vector (Stratagene, La Jolla, CA), which contains the inverted terminal repeat of AAV serotype 2 at both ends. AAV/Empty that contains no transgene served as a negative control. All AAV vectors were generated by the triple transfection method and purified by CsCl sedimentation (58). The physical vector genome (vg) of AAV was measured by quantitative PCR using SYBR Green reaction mix (Roche Diagnostics, Mannheim, Germany) (60).

2.3 AAV injection

Mice at 6-8 weeks of age were intravenously injected with the indicated titers (10¹⁰, 10¹¹, or 10¹² vg) of both AAV2/8-5'-HBV-SD and AAV2/8/-3'-HBV-SA (designated as AAV/HBV). Mice injected with AAV/Empty of the same titers were used as negative controls.

2.4 Immunization

In vivo electroporation following plasmid injection was performed as previously described (61). Briefly, mice were anesthetized with acepromazine maleate (Fermenta Animal Health Co., Kansas, Mo., USA). Fifty micrograms of plasmid DNA encoding CMV promoter-driven HBsAg, HBcAg, or OVA was injected into each bilateral quadriceps muscles using a disposable insulin syringe with a 27-gauge needle.

Immediately after injection, a pair of electrode needles was inserted into the muscle to a depth of 5 mm to encompass the DNA injection sites, and electric pulses were delivered using an electric pulse generator (Electro Square Porator ECM 830; BTX, San Diego, Calif., USA). The shape of the pulse was a square wave. The electrodes consisted of a pair of gold-plated stainless steel needles 5 mm in length and 0.8 mm in diameter with a fixed distance between them of 5 mm. Six pulses of 100 V each were administered to each injection site at a rate of 1 pulse/s, with each pulse being 50 miniseconds in duration. The procedure was repeated once two weeks after the first immunization. In experiments indicated, mice were i.v. injected with 1 x 10⁸ PFU of adenoviral vector expressing pre-S2 envelope protein one week after the second DNA immunization.

2.5 Isolation of splenocytes and intrahepatic lymphocytes

Splenocytes and intrahepatic lymphocytes were isolated on a Percoll density gradient

as described (62). Briefly, the liver was perfused with HBSS, removed and sliced, followed by incubation with collagenase IV (Sigma-Aldrich, St. Louis, MO) at 37°C for 30 min. Intrahepatic lymphocytes were separated from parenchymal cells by

centrifugation at 50×g for 5 min. Cells prepared from one mouse were suspended in 20 ml of 33% Percoll gradient solution (GE Healthcare, Piscataway, NJ) and centrifuged at 754×g for 18 min. After treated with red blood cell lysis buffer and washed with PBS, cells were used for IFN-γ ELISPOT and flow cytometric analysis.

2.6 IFN-γ and TNF-α ELISPOT

CD8 T cells were positively selected from intrahepatic and splenic lymphocytes using mouse CD8a MicroBeads and magnetic separator (Miltenyi Biotec, Bergisch-Gladbach, Germany). Mouse IFN-γ and TNF-α ELISPOT Ready-Set-Go kit (eBioscience, San Diego, CA) was used according to the manufacturer’s instruction. Briefly, CD8T cells (2.5 × 10⁴ to 5 × 10⁵ cells per well) were cocultured with EL4 cells, a murine thymoma cell line derived from C57BL/6 mice, pulsed with 10 µg/ml of peptides on

MultiScreen-IP plates (Millipore, Bedford MA) precoated with α-IFN-γ capture antibody. Three H-2K^b-restricted HBV epitopes, HBs190-197, HBs208-215, and HBc93-100

were used. The OVA257-264 peptide, an H2-K^b-restricted ovalbumin epitope and Con A (2 µg/ml) were used as negative and positive controls, respectively. Spots were

developed 18-24 h and detected sequentially with biotinylated antibody against IFN-γ, streptavidin-horseradish peroxidase, and AEC substrates. Spots were counted with AID ELISPOT Reader System with software 5.0 (AID GmbH, Ottobrunn, Germany).

2.7 Serological analysis

Sera samples were collected at different time points post AAV injection. Serological

markers for HBV (HBsAg and α-HBs antibodies) were quantified using an Elecsys Systems electrochemiluminescence kit and a Cobas e analyzer (Roche Diagnostics GmbH, Mannheim, Germany).

2.8 Flow cytometric analysis and fluorescence-activating cell sorting (FACS)

PD-1 expression on intrahepatic and splenic T lymphocytes was characterized by flow cytometric analysis. Cells were pre-incubated with α-CD16/32 monoclonal antibody (mAb, 2.4G2; ATCC, Manassas, VA) to block nonspecific binding of antibodies to Fc receptors. Cells were then incubated with the following mAbs for 30 min on ice: eFluor^® 450-conjugated rat α-mouse TCRβ (H57-597, eBioscience, San Diego, CA), allophycocyanin (APC)-eFluor^® 780-conjugated rat α-mouse CD8a

(53-6.7, eBioscience, San Diego, CA), phycoerythrin (PE)-conjugated hamster α-mouse PD-1 (J43, eBioscience, San Diego, CA), PE-conjugated rat α-mouse LAG-3 (C9B7W, eBioscience, San Diego, CA), fluorescein isothiocyanate (FITC)-conjugated rat

α-mouse 2B4 (eBio244F4, eBioscience, San Diego, CA),

Peridinin-Chlorophyll-Protein-Complex (PerCP)-conjugated rat α-mouse Tim-3 (215008, R&D Systems Inc., Minneapolis, MN), and Alexa Fluor 647-conjugated rat α-mouse CD11a (M17/4, Biolegend, San Diego, CA). After wash, cells were stained with fixable viability dye eFluor^® 506 for 30 min on ice. Cells were analyzed with FACS LSRII (BD Biosciences, San Jose, CA) and FlowJo V.7.2.5 software (Treestar, Ashland, OR). For intracellular cytokine staining, cells were harvested after culture for 6-12 hours in the presence of brefeldin A (Biolegend, San Diego, CA) in U-bottom 96-well plates. After stained with surface marker and viability dye, cells were fixed and permeablized with fixation/permeablilization kit (eBioscience, San Diego, CA) for 1

hour. The following mAbs were used for cytokine staining: PE-conjugated rat α-mouse IFN-γ (XMG1.2, BD Biosciences, San Jose, CA) and FITC-conjugated rat α-mouse TNF-α (MP6-XT22, eBioscience, San Diego, CA). For FACS, lymphocytes isolated from the liver and the spleen were stained with TCRβ, CD8a, and PD-1 as previous described after blocking. 7-amino-actinomycin D (7-AAD, eBioscience, San Diego, CA) was used to rule out dead cells.

2.9 In vivo blockade with mAb injection

Mice at 6-8 weeks of age were intraperitoneally injected with 200 µg of α-LAG-3 mAb (clone C9B7W, BioXCell, West Lebanon, NH) or Rat-IgG isotype control

antibody on day -7, -1, 4, 8, 15, 21, 25, 39, 47, and 56. On day 0, treated mice were i.v.

injected with 10¹¹ vg AAV/HBV.

2.10 Preparation of bone marrow-derived dendritic cells (BMDCs) and in

BMDCs were generated following the method reported by Lutz et al (63). In brief, bone marrow cells were isolated from femurs and tibiae, and cultured on Petri dish with RPMI containing 10% FBS, 50 µΜ b-ΜΕ, and 200 U/ml of GM-CSF. Fresh

GM-CSF-containing medium was replenished on day 3, 6, and 8. On day 10, suspended cells were collected and infected with 50 MOI of adenoviral vectors expressing either full-length HBV, pre-S2 envelope protein, HBcAg, by centrifugation at 2000×g, 37°C for 2 hours (64). After wash twice with PBS, BMDCs were cultured in RPMI

containing 100 U/ml of GM-CSF for 24 hours before subjected to further stimulation.

2.11 In vitro T-cell expansion

CD8 T cells were expanded in vitro with protocols modified from previous studies (23, 65). Briefly, Sorted CD8 T cells (2×10⁴ per well) were co-cultured in U-bottom 96-well plates with LPS-primed BMDCs (2×10⁴ per well) that were pulsed with overlapping peptide pools. Naïve splenocytes (2×10⁵ per well) that were previously irradiated with 45 Gy γ-radiation were added as feeder cells. The culture medium (RPMI with 10% FBS, 50 µM β-ME) contains interleukin-7 (IL-7, 5 ng/ml), IL-12 (100 pg/ml), IL-15 (1 ng/ml), and Dynabeads coated with α-CD3 plus α-CD28 mAbs (8×10⁴ per well). IL-2 (100 U/ml) was added on day 4 of culture. On day 10, 150 µl of culture medium was aspirated and peptide-pulsed irradiated naïve splenocytes were added along with 100U/ml of IL-2 for a second round of expansion. On day 20, all expanded cells were subjected to TNF-α ELISPOT.

2.12 T cell receptor (TCR) repertoire spectratype analysis

To analyze the TCR Vβ transcript size patterns, cDNA samples were amplified using a TCR Cβ (TCR β-chain constant region)-specific primer and a set of TCR Vβ (TCR β-chain variable region)-specific primers (66). Detail sequences are descried in Table 2.

Sorted cells were subjected to RNA extraction by RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse transcription by Transcriptor Reverse Transcriptase (Roche, Diagnostics, Mannheim, Germany). PCR was performed in a total volume of 12.5 µl containing 0.1 µl FastStart Taq DNA polymerase (5 U/µl) (Roche Diagnostics, Mannheim, Germany), 1.25 µl 10× reaction buffer, 1 µl dNTPs (2.5 mM), 0.75 µl MgCl2 (25 mM), 1 µl forward and reverse primers (5 mM) and 1.25 µl cDNA. After an initial denaturation step of 5 minutes at 95°C, the reactions were subjected to 45 cycles of polymerase chain reaction (PCR; 30 seconds at 95°C, 30 seconds at 60°C, 45 seconds

at 72°C) followed by a final elongation step for 10 minutes at 72°C. PCR products were then labeled with Cβ-carboxyfluorescein (Cβ-FAM) primer by 5 cycles of primer extension. The primer extension conditions include an initial denaturation of 2 min at 95°C and 5 PCR cycles of 2 min at 95°C, 2 min at 60°C and 20 min at 72°C. Labeled PCR products were mixed with the cocktail of 10 µl deionized formamide and

GeneScan-500 LIZ size standard (Applied Biosystems, Foster City, CA) and

cDNA-fragment length were analyzed with ABI 3700 Analyzer (Applied Biosystems, Foster City, CA) and PeakScanner Analysis Software 1.0 (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol.

2.13 Statistics

The unpaired Student t test was used for statistic analyses. Results were regarded as significant if the two-tailed p value was ≤ 0.05.