Patients and Samples
Tumor tissues and blood samples were collected from HCC patients including HBV- HCC and non-viral HCC who underwent surgical resection at the Fourth Hospital of Hebei Medical University between January 2016 and June 2021. The genomic DNA of tissues and serum were extracted by Wizard Genomic DNA extraction kit (Promega, Madison, WI, USA) according to the manufacturer's protocols. The HBV DNA concentration in HCC tissue was quantified as copies per microgram (mg) of genomic DNA with ABI 7300 TaqMan platform (Life Technologies, Carlsbad, CA, USA) by real-time PCR [24]. And the HBV DNA titers in serum was quantified as international units (IU) per milliliter (ml) of genomic DNA with ABI 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) by real-time PCR. Patients with the HBV DNA load in tissues greater or equal to 6×108 copies/mg were divided into High-copy HBV group [25], and the HBV DNA load in serum greater or equal to 1×105 IU/ml were divided into High-copy HBV group. The tumor tissues and blood samples from non-viral HCC were collected as control group. The blood samples of health volunteers were collected for the Lentiviral transduction assay. None of the patients had received antiviral therapy, chemotherapy or molecular targeted drugs prior to surgery. All procedures were supervised and approved by the hospital’s ethics committee. Written informed consent was obtained from the participants.
Illumina Infinium Human Methylation 450K BeadChip analysis and methylation assay
Epigenome-wide DNA methylation was analyzed by using the Illumina Infinium Human Methylation 450K BeadChip array (Illumina, Inc., San Diego, CA, USA). Briefly, the HCC tissues from High-copy HBV group and control group were collected and the genomic DNA of tissues was extracted by using DNeasy & Tissue Kit (QIAGEN, Hilden, Germany). A total of 1500 ng DNA was used for bisulfite conversion according to the manufacturer’s instructions of the EZ DNA Methylation-Gold Kit (Zymo research, Orange, CA, USA). Bisulfite-converted DNA was analyzed by using Infinium Human Methylation 450K BeadChips (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's protocol [26]. BeadChips were scanned using the Illumina Hi-Scan system. Methylation data was processed with Methylation Module (version 1.9.0) of Genome Studio software (Illumina; version 2011.1). DNA methylation measurements with P < 0.05 were considered statistically significant.
Cell culture
Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll-Paque (Sigma Chemical Co.St.Louis, MO, USA) density gradient centrifugation from peripheral blood of High-copy HBV group, control group and the healthy volunteers. CD8+ T cells were subsequently separated by CliniMACS CD8 (Miltenyi Biotec, Germany) according to the instruction. Then CD8+ T cells were activated by Phytohemagglutinin (5ug/ml) (Meilunbio, Dalian, China) and cultured in RPMI-1640 medium (GibcoTM Life Technologies, Grand Island, NY, USA) supplemented with IL-2 (200 IU/mL) (Meilunbio, Dalian, China), 1% Sodium Pyruvate (GibcoTM Life Technologies, Grand Island, NY, USA), 1% Glutamine (GibcoTM Life Technologies, Grand Island, NY, USA) and 10% human AB+ serum (Jacques Boy; StemcellTechnologies, Vancouver, CA) in a humidified incubator containing 5% CO2 at 37°C.
Generation of the lentiviral and construction of HBV infection in T cells
The plasmids pCS-CG and pCS-HBV1.3 which containing a 1.3-fold-overlength genome of HBV were kindly provided by Professor Xia Chuai from the Department of Microbiology, Hebei Medical University [27]. After packaging with lentivirus package systems, pCS-CG lentivirus and pCS-HBV1.3 lentivirus were transduced into CD8+ T cells from health volunteers. The HBV serological markers including hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the culture medium at 48, 72 and 120h were tested to confirm the successful transfection by the chemiluminescence method on the Cobas e 601analyzer (Roche Diagnostics GmbH, Mannheim, Germany) according to the instruction.
Detection the expression of PD-1 on CD8+ T cells by Flow cytometric analysis
The expression of PD-1 on CD8+ T cells was detected by Flow cytometric analysis. CD8+ T cells were harvested and washed twice with phosphate buffer (PBS) containing 2% fetal bovine serum (FBS) (GibcoTM Life Technologies, Grand Island, NY, USA). Then CD8+ T cells were incubated with a cocktail of antibodies CD8a-Pacific Blue (Biolegend, San Diego, CA) and PD1-FITC (Biolegend, San Diego, CA) at 4℃ for 10min in the dark. The mixture was subsequently washed once and analyzed by flow cytometer MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany).
Cytokine measurement
The concentrations of cytokine including interleukin-1β (IL-1β), interferon‐α2 (IFN‐α2), IFN‐γ, tumor necrosis factor‐α (TNF‐α), monocyte chemotactic protein-1 (MCP‐1), (IL‐6), (IL‐8), (IL‐10), IL‐12p70, IL‐17A, IL‐18, IL‐23, and IL-33, were detected by using BioLegend LEGENDplex human inflammation panel (Biolegend, San Diego, CA). The serum from high-copy HBV group and control group, the culture medium from pCS-CG lentivirus group and pCS-HBV1.3 lentivirus group were collected. serum or culture medium of 25 µL was diluted 2-fold with Assay Buffer and incubated with 25µL of microbeads and detection antibody each at 25℃ for 2 hours in the dark. The 25µL Streptavidin-phycoerythrin (SA-PE) was added into each medium subsequently, shaking at approximate 500 rpm for 30 minutes at room temperature in the dark. The flow cytometer MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to quantify the PE fluorescence signal of the analyte-specific bead region, and BioLegend’s LEGENDplexTM Data Analysis Software (Biolegend, San Diego, CA) was used to generate a standard curve and determine the concentration of the specific analyte. The absolute concentrations in the serum were calculated to known standards.
Western blot analysis
The protein of HCC tissues collected from High-copy HBV group and control group were extracted by Total Protein Extraction Kit (Invent Biotechnologies, USA) according to the manufacturer's instructions. And the cell protein of CD8+ T cells collected from High-copy HBV group and control group were extracted by radio immunoprecipitation assay (RIPA) lysis buffer containing 1% protease inhibitor (Roche, Basel, Switzerland). Protein extract was subjected to 10% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) followed by transfer onto a polyvinylidene difluoride (PVDF) membranes (Roche, Basel, CH). After blocking with 5% skim milk, the membrane was incubated with primary antibody against human PD-1/CD279 (PROTEINTECH, Chicago, USA) or β-actin (Santa Cruz, CA, USA) overnight at 4°C, followed by incubation with secondary HRP-conjugated anti-rabbit IgG antibody (Thermo Fisher, New York, USA). The relative intensities of protein bands were visualized with an enhanced chemiluminescence reagent (Thermo Fisher, New York, USA) using the FluorChem® HD2 protein imprinting imaging system (Alpha InnoTec, San Leandro, CA).
Immunofluorescence staining and imaging
The paraffin tissue sections (4 µm thick) were baked at 60°C for 1 hour and dewaxing with xylene. The tissue sections collected from High-copy HBV group and control group were subsequently rehydrated and subjected to microwave antigen retrieval with citrate buffer (pH, 6.0) for 30 min. After blocking with 2% normal goat serum (Invitrogen, California, USA), sections were incubated with primary antibodies including Mouse Monoclonal anti-CD8 (PROTEINTECH, Chicago, USA), Rabbit Polyclonal anti-PD1/CD279 (PROTEINTECH, Chicago, USA), CD4 Monoclonal Antibody (ImmunoWay, TX, USA), FOXP3 Polyclonal Antibody (ImmunoWay, TX, USA), CD68 Polyclonal Antibody (ImmunoWay, TX, USA) and CD163(ABT-CD163) mouse mAb (ImmunoWay, TX, USA) overnight at 4°C. Subsequent to washing with PBS (pH, 7.2) three times, the slides were incubated with a cocktail of Goat Anti Mouse IgG (H&L) - Alexa Fluor 488 (ImmunoWay, TX, USA) and Goat Anti Rabbit IgG (H&L) - Alexa Fluor 568 (ImmunoWay, TX, USA) for 1 hours in the dark. After washing with PBS three times, the slides then stained with 4,6-diamidino-2-phenylin-dole (DAPI) (Solarbio, Beijing, China) and mounted with Prolong Gold mounting reagent (Life Technologies, Alaska, USA). Then the slides were visualized using the Leica DMi8-M microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Quantitative methylation analysis
The DNA of CD8+ T cells and HCC tissues were extracted by using DNeasy & Tissue Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Then, the extracted DNA was subjected to bisulfite conversion and purification by EZ DNA Methylation-Gold Kit (Zymo research, Orange, CA, USA). Quantitative methylation analysis of PD-1 promoter was performed by using the Sequenom MassARRAY platform (CapitalBio, Beijing,China), which was composed of matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry in combined with RNA base-specific cleavage. PCR primers were designed using Methprimer (http://www.urogene.org/methprimer/), which were listed in TableS1. For each reverse primer, an additional T7 promoter tag for in vivo transcription was added, whereas a 10-mer tag on the forward primer was used to adjust melting temperature differences. Mass spectra were obtained via MassARRAY Compact MALDI-TOF (Sequenom) and spectra’s methylation ratios were generated using the Epityper software version 1.0 (Sequenom).
Statistical Analysis
We used descriptive statistics (means and standard deviations or median and ranges) to summarize the data. Statistical analyses were performed by χ2 test or Student’s t test. P < 0.05 was considered statistically significant difference. Data was obtained from at least three independent experiments with a similar pattern. All statistical analyses were carried out using SPSS statistical software, version 19.0 (IBM Corporation, Armonk, NY).