Pe cy7 anti pd1

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).

The PBMCs were stained with PerCP-anti-CXCR5 (Biolegend, San Diego, USA), APC-anti-CD4, FITC-anti-ICOS, PEcy7-anti-PD-1, FITC-anti-CD19, PE-anti-IL-21 (ebioscience, San Diego, USA) after washing, as per manufacturer’s instructions. Isotype-matched control IgG was used in all procedures. For IL-21 staining, PBMCs were stimulated with Cell Stimulation Cocktail (ebioscience, San Diego, USA) at 37°C for 6 hours. Then the cells were harvested and stained with the above antibodies. The stained cells were then analyzed, using Becton Dickinson FACSCan and FlowJo software.