Isolation and purification of human peripheral blood T lymphocytes
50 ml of peripheral blood was drawn from healthy volunteers (n=10, F/M is 4/6, age is 55.6±10.14 year) or patients diagnosed with HBV-related HCC (n=10, F/M is 3/7, age is 59.3±11.00 year). The general characteristics of voluneers and patients were shown in Table S1. The patients with HCV, HIV-coinfection, diabetes, chronic kidney diseases or autoimmune disorders were excluded from the study. PBMCs were harvested by density gradient centrifugation, washed 3 times with phosphate-buffered saline (PBS), and cultured in RPMI1640 medium at 37 °C in 5% CO2 for 2 h. After that, the medium was changed to remove macrophages. PBMCs were routinely cultured in RPMI1640 medium containing 10 IU/ml rhIL-2 for 4 weeks, incubated with fluorescently labeled antibody to label PBMCs, and then maintained at a low dose of rhIL-2. Total T lymphocyte numbers and T cell subpopulations were detected by Flow cytometry analysis. T lymphocytes were identified with surface markers, CD3, CD4, and CD8. CD3 represents the total number of T cells, and CD4 and CD8 represent T cell subsets. Purified T lymphocytes are collected according to the analysis results of flow cytometry.
HepG2.2.15 cells, which are capable of steady secreting HBsAg, HBeAg and intact Dane particles to the culture supernatant, were obtained from SHANGHAI CAFA BIOLOGICAL TECHNOLOGY CO., LTD and cultured in DMEM medium supplemented with 10% FBS.
For the co-culture experiments with T cells, HepG2.2.15 cells were seeded in 96 or 24-well flat-bottom plates at a density of 5×104 cells/ml. After co-cultured with T cells (5×105 cells/ml) for 24, 48 or 72h, T cells were removed, and HepG2.2.15 cells viability, apoptosis and migration ability was assessed.
Amygdalin was purchased from Macklin (Shanghai, China). T cells were treated with 5, 10, 15 or 20 µg/ml amygdalin for 24, 48 and 72 h or treated with 10µg/ml amygdalin for 48 h, T cells were collected for further experiments.
The cell proliferation was examined by MTT analysis. For T cell proliferation, T cells were stimulated with 5 μg/ml concanavalin A (Con A, Sigma-Aldrich, USA), 1 ×104 cells/well T cells were seeded into 96 well plates treated with or without amygdalin for 24, 48 and 72 h. For HepG2.2.15 cells proliferation, 5×103 cells/well HepG2.2.15 cells were co-cultured with 5 ×104 cells/well T cells into 96 well plates for 24, 48 and 72 h, then removed the T cells. 20 μl MTT (at a concentration of 5 mg/ml; Sigma-Aldrich) was added into the 96-well plates and incubated for 4 h in a humidified incubator. After that, the supernatant was discarded and 200 μl DMSO was added to dissolve the formazan. OD490 nm value was measured. The viability of the non-treated cells (control) was defined as 100%.
Total RNA was extracted from targeted cells using Trizol reagent (Invitrogen, CA, USA), and the expression of mRNA was determined using PCR-based analyses following the methods described previously . SYBR green PCR Master Mix (Qiagen, German) was used. GAPDH was employed as endogenous controls for mRNA expression determination, respectively. The data were processed using a 2-ΔΔCT method. The primer sequence were listed in Table S2.
The protein levels of p-STAT3, STAT3, p-JAK2, and JAK2 were detected by performing immunoblotting assays. Target cells were lysed in RIPA buffer with 1% PMSF; proteins were loaded onto an SDS-PAGE minigel and transferred onto a PVDF membrane. The blots were probed with anti-p-STAT3 (dilution 1:2000, ab76315, Abcam, Cambridge, MA, USA), anti-STAT3 (dilution 1:2000, ab119352, Abcam), anti-p-JAK2 (dilution 1:2000, ab32101, Abcam), anti-JAK2 (dilution 1:2000,ab108596, Abcam), anti-procaspase3 (dilution 1:1000, ab32150, Abcam), anti-cleaved caspase-3 (dilution 1:1000, ab32042, Abcam) and anti-GAPDH (dilution 1:5000, ab8245, Abcam) at 4°C overnight, subsequently incubated with the HRP-conjugated secondary antibody (1:5000, Santa Cruz, USA). ECL Substrates was used to visualize signals (Millipore, MA, USA). GAPDH was used as an endogenous protein for normalization.
Cytokines production determined by ELISA
T cells culture medium was collected for ELISA assay using ELISA kits specific for IFN-γ, TNF-α, IL-6, and IL-10 according to the manufacturer’s instructions (R&D systems, USA). First, 100μl of Assay Diluent was added to each well. 100 μl serially-diluted standard samples or supernatant samples were added into the microplate and were incubated at room temperature for 2 h. After aspirating each well and washing, 200 µl of Conjugate solution to each well and incubated at room temperature for 2 h. After aspirating each well and washing, each well was added with 200 μl substrate solution at dark for 30 min; Finally, each well was added with 50 μl stop solution and the optical density was assayed immediately at 540 nm with a spectrophotometer (Bio-Rad Laboratories).
Invasion and migration assays
HepG2.2.15 cells were co-cultured with T cells and plated on the top side of Transwell filter coated with (for invasion analysis) or without Matrigel (BD, New York, USA) (for migration analysis) in the top chamber. Cells were suspended in medium without serum and medium supplemented with serum were filled in the bottom chamber. The cells were incubated at 37 °C for 48 h. The noninvasive or non-migratoryion cells in the top chambers were removed with cotton swabs. The invaded cells or migrated cells on the lower membrane surface were fixed in 100% methanol for 10 min, air-dried, then stained staining with 0.1% crystal violet solution, and counted under a microscope.
Flow cytometer assay
After co-culture with NC-T cells, HBV-T cells or amygdalin treated HBV-T cells for 48 h, the HepG2.2.15 were harvested for apoptosis analysis. Quantification of apoptotic cells was performed with Annexin V-FITC apoptosis detection kit (Keygen, China). Briefly, the HepG2.2.15 cell samples were harvested with 0.25% trypsin without EDTA after 48 hours of infection and then washed twice with ice-cold PBS and re-suspended in 500 μl binding buffer. Then cells were incubated with 5 μl Annexin V-FITC specific antibodies and 5 μl propidium iodide (PI) then incubated for 15-20 minutes in the dark and detected by BD Accuri C6 flow cytometer (BD, USA) with an excitation wavelength of Ex = 488 nm and an emission wavelength of Em = 530 nm.
For T cell surface markers detection, Fixed T cells were permeabilized by adding 1 ml of cold Phosflow Perm Buffer III (BD Biosciences) and incubating for 30 min on ice. Samples were then washed and incubated with the mouse anti-human antibodies (BD Biosciences) anti-CD3-PE-Cy5, -CD4-FITC, -CD8-FITC, and p-STAT3-PE at room temperature for 1 h. Then, the cells were washed and collected for flow cytometry analysis on a BD Accuri C6 flow cytometer (BD, USA). Each experiment was repeated three times in triplicate.
Data were expressed as means ± SD of at least three independent experiments. A one-way ANOVA was used for the comparison of the differences among more three groups. The level of significance was based on the probability of *P < 0.05, **P < 0.01.