Patients and samples
Thirty-one treatment-naïve patients with chronic HBV infection were recruited and classified into immune tolerant carrier (IT; n = 13), hepatitis B e antigen (HBeAg)-positive CHB (n = 9), and inactive carrier (IC; n = 9) groups according to American Association for the Study of Liver Diseases guidelines . Ten healthy controls (HCs) were enrolled. We collected matched peripheral blood mononuclear cells (PBMCs) and intrahepatic mononuclear cells (HMCs) from 11 treatment-naïve patients with HBV-related HCC who underwent curative hepatectomy. In addition, matched PBMCs and intrasplenic mononuclear cells (SMCs) were obtained from another 11 patients who underwent splenectomy due to HBV-related liver cirrhosis-induced hypersplenism. All individuals were recruited at Nanfang Hospital (Guangzhou, China). Exclusion criteria for these studies were coinfection with hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, and human immunodeficiency virus (HIV). Patients with primary biliary cirrhosis and autoimmune diseases were also excluded. All individuals provided written informed consent, and the studies were approved by the Ethical Committee of Nanfang Hospital.
Mononuclear cells isolation
Twenty milliliters of heparinized blood was collected from patients with chronic HBV infection and HCs. The preparation of human HMCs was performed according to a previously described procedure . Briefly, the liver tissues were flushed using 4°C RPMI-1640 complete medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2% heat-inactivated fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 µg/mL streptomycin, 100 U/mL penicillin, and 2mM ethylenediaminetetraacetic acid (EDTA; Ambion; Applied Biosystems, San Mateo, CA, USA). Subsequently, PBMCs and HMCs were isolated by density-gradient centrifugation on Ficoll-Hypaque and were cryopreserved in liquid nitrogen. Human SMCs were isolated as previously described [11, 19]. Briefly, the splenic tissues (2 cm3) were placed into a 70 μm nylon mesh filter in a culture dish with the medium, and then the rounded side of a plunger from a 10 mL syringe was used to crush the tissues mechanically. The generated single-cell suspensions were used to obtain SMCs by Ficoll-Hypaque centrifugation. Cell viability, as assessed by trypan blue exclusion, was always higher than 90%.
PBMCs, HMCs, and SMCs were stained with the following monoclonal antibodies (mAbs) for 30 min at 4°C: CD14-PECy7, CD21-APC, FDC-FITC (cloned CNA.42), CD3-FITC, CD4-PECy7, CD8-APC, CXCR5-Brilliant Violet™ 421, CD19-APC, CD10-PE, CD38-FITC, and CD27-PerCPCy5.5. Dead cells were excluded using Live/Dead (Thermo Fisher Scientific; Waltham, MA, USA), cells were incubated with human BD Fc Block (5μL/million cell in 100 μL FACS buffer) for 10 min at room temperature to block the Fc-receptors. All samples were analyzed on a BD FACS CantoII flow cytometer (BD Biosciences). The data were analyzed with FlowJo software (Tree Star).
Enzyme-linked immunosorbent assay (ELISA)
The plasma levels of IL-6, IL-7, IL-15, BAFF (Invitrogen; Carlsbad, CA, USA), and C-X-C motif chemokine ligand 13 (CXCL13; R&D Systems; Minneapolis, MN, USA) and the concentrations of IL-6, IL-21, interferon (IFN)-γ (Invitrogen; Carlsbad, CA, USA), and CXCL13 in the culture supernatants were assessed by commercially available ELISA kits according to the manufacturers' instructions.
Isolation and culture of FDCs
Primary human FDCs were established as described previously . Briefly, human splenic tissues were placed in petri dishes and washed several times with PBS, followed by cut into small pieces. The spleen fragments were then digested with 0.5mM EDTA and 0.25% of trypsin for 20 min at 37°C and stopped the reaction with cold RPMI-1640 complete medium supplemented with 10% heat-inactivated FBS. The suspension was filtered through gauze and centrifuged. The supernatant was discarded, and the cell pellet was suspended in the medium and centrifuged on Ficoll-Hypaque. The interlayer cells were collected and incubated in culture flasks for 1 h at 37°C in RPMI-1640 complete medium with 10% heat-inactivated FBS to allow the adherence of macrophages and granulocytes. The supernatant cells were washed and incubated in a fibroblast medium, which consists of Opti-MEM reduced serum medium (Gibco; Thermo Fisher Scientific; Waltham, MA, USA) and a supplement containing 3% FBS, 100 U/mL penicillin, 50 μg/mL gentamicin, and 1 mmol/L glutamine. After overnight incubation, adherent cells attached to the flask, lymphocytes in the supernatant were discarded. The fibroblast medium was replaced and changed twice a week. FDCs matured after 2–4 weeks of culture.
Immunofluorescence staining and confocal microscopy
Cultured FDCs were transferred into the confocal petri dishes and washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were then treated with 0.5% Triton X-100 for 5 min and blocked with 1% bovine serum albumin (BSA; Fdbio Science; Hangzhou, China) for 30 min at room temperature. Subsequently, the cells were stained with mouse anti-human FDC monoclonal antibody (CNA.42, 1:300, eBioscience) overnight at 4°C. Following three washes with PBS, the cells were incubated with the goat anti-mouse antibody (1:400, Jackson ImmunoResearch) for 1 h at room temperature. Then confocal petri dishes were stained with DAPI (Abcam). Images were acquired using a confocal laser scanning microscope (Fluoview FV10i; Olympus, Tokyo, Japan).
Cell culture and supernatant analysis
Cultured FDCs were stimulated with IL-4, IL-10, IL-21, lipopolysaccharide (LPS), IFN-α-2α, lymphotoxin (LT)-α1β2, tumor necrosis factor (TNF)-α, CPG, and PMA for 3 days, respectively. The supernatants were collected and the levels of IL-6 and CXCL13 were assessed by ELISA. Autologous CD4+ T cells and CD19+ B cells were sorted from SMCs of patients with chronic HBV infection by FACS and cryopreserved in liquid nitrogen. Purified autologous CD4+ T cells were thawed and plated in a 96-well plate that pre-seeded with FDCs at an 80:1 (CD4+ T cells/FDCs) ratio, or with medium only as a control, and co-cultured in the presence of IL-2 (10ng/mL) and anti-CD3/CD28 (10μg/mL) for 3 days, the supernatants were collected and the levels of IL-21 and IFN-γ were assessed by ELISA.
Purified intrasplenic CD19+ B cells were thawed and labeled with carboxyfluorescein succinimidyl ester (CFSE; 1.5 mM; Molecular Probes; Eugene, OR) and suspended at 106 cells/mL. Labeled cells were plated in a 96-well plate that pre-seeded with FDCs at an 80:1 (CD19+ B cells/FDCs) ratio, or with medium only as a control, and co-cultured in the presence of CPG (10μg/mL) for 7 days. The proliferation rate of B cells was expressed as the percentage of cells that diluted CFSE intensity at least once at the time of harvest.
Data are expressed as either the median (range) or the mean ± SD. Statistical analyses were performed in GraphPad Prism v.8.0.1 (La Jolla, CA, USA). Mann-Whitney U test was used when two groups were compared. Correlations between variables were assessed with the Spearman rank-order correlation coefficient. All statistical analyses were based on two-tailed hypothesis tests with a significance level of p < 0.05.