Anti tnf α fitc

Cryopreserved PBMCs from each participant at all time points were investigated simultaneously. After overnight rest of thawed PBMC, the cells were cultured in the presence of anti-CD107a-PE in complete RPMI containing 2 μg/mL of recombinant HBsAg (MyBioSource, USA) and 5 μg/mL of PHA (Sigma-Aldrich, USA) as the positive stimulation control or complete media alone as the unstimulated control at 37°C in a 5% CO₂ incubator for 16–18 hr.
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous whole blood by Ficoll-Hypaque density gradient centrifugation. In order to measure intracellular cytokines, PBMCs (2.5 × 10⁵) were seeded in 96-well plates in 100 µl of IMDM medium and stimulated with Leukocyte ActivationCocktail (Becton Dickinson GolgiPlug) for 4 h. After stimulation, the cells were collected and stained with the following monoclonal antibodies: anti-CD45-PerCP (BD phamingin, 2D1), anti-CD3-APC/H7 (BD phamingin, SK7), anti-CD4-V450 (BD phamingin, RPA-T4), and anti-CD8-PE/Cy7 (BD phamingin, SK1), followed by fixation and permeabilization, and staining with intracellular anti-IFN-γ-APC (BD phamingin, B27) antibody, anti-TNF-α-FITC (Biolegend, MAb11), and anti-IL-2-PE (Biolegend, MQ1-17H12). The cells were then analyzed with FACSCanto flow cytometer.

To assess in vivo macrophage numbers, digested cells and splenocytes were stained with fluorescence conjugated antibodies against MHC II, CD45, F4/80, and CD11b (eBioscience, San Diego, CA). LIVE/DEAD staining (Thermo Fisher Scientific) was used to assay for cell viability determination at 405 nm excitation. For intracellular cytokine staining, freshly isolated IELs and splenocytes were incubated with 50 ng/ml PMA (Sigma), 750 ng/ml ionomycin (Sigma, St. Louis, MO) and 5 μg/ml Brefeldin A (Biolegend, San Diego, CA) at 37°C for 4 h. Cells were first collected for surface staining with anti-CD8-BV650 (Biolegend, San Diego, CA), anti-CD62L-BV605 (Biolegend, San Diego, CA), anti-CD4-PE Cy7, and anti-CD44-APC (Thermo Fisher Scientific, Waltham, MA) antibodies, and then fixation and permeabilization were performed following the instructions from BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences, San Jose, CA). Intracellular staining for cytokines was detected with anti-IFN-γ-PE, anti-TNF-α-FITC, and anti-IL-17-Percp Cy5.5 antibodies (Biolegend, San Diego, CA). Data were collected using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo, LLC, Ashland, OR).

Mouse livers were perfused with 10 mL of PBS and homogenized in Hanks’ buffer. The homogenate was resuspended in 36.5% Percoll (Sigma-Aldrich), and lymphocytes were isolated by density gradient centrifugation. The lymphocytes were cultured in RPMI 1640 medium and stimulated with CD8+ T-cell epitope (L^d-HBV S28–39 epitope, IPQSLDSWWTSL, 10 μg/mL) as described previously (Schirmbeck et al., 2001 (link); Sette et al., 2001 (link)). The following antibodies were used for cell-surface staining and intracellular cytokine staining: BV421-anti-CD8, APC-Cy7-anti-CD4, PE-anti-CTLA4, and PE-Cy7-anti-PD1 (eBioscience, San Diego, CA, USA). For intracellular cytokine staining, APC-anti-IFN-γ, PerCP-Cy5.5-anti-IL-10, and FITC-anti-TNF-α (BioLegend, San Diego, USA) were used. Dead cells were excluded by staining with Fixable Viability Dye eFluor 506 (eBioscience). Samples were analyzed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo (version 10.0; TreeStar, Ashland, OR, USA).

Lymphocytes in the liver were isolated as described in a previous study (Ma et al., 2017 (link)). Briefly, the mouse liver was perfused with PBS and then digested with an enzyme solution containing 0.05% collagenase type IV (Sigma-Aldrich), 0.002% DNAase I (Sigma-Aldrich), and 10% fetal bovine serum for 30 min. Lymphocytes in the homogenate were isolated using Percoll (Sigma-Aldrich), following the manufacturer's instructions and cultured in RPMI 1640 medium in 96-well plates and stimulated with CD8+ T cell epitope (K^b-HBV Cor₉₃₋₁₀₀ epitope, MGLKFRQL, 10 μg/mL). For cell surface staining, the cells were stained with BV421-anti-CD8 (eBioscience, San Diego, CA, USA). For intracellular cytokine staining, the cells were fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (Invitrogen, Carlsbad, CA), and then stained with the following antibodies: APC-anti-IFN-γ, PE-anti-IL-2, and FITC-anti-TNF-α (Biolegend, San Diego, CA, USA). All the samples were stained with Fixable Viability Dye eFluor 506 (eBioscience) to exclude dead cells. The stained cells were analyzed using a BD FACSCanto II flow cytometer. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).