Bv421 anti cd8

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).