Pe conjugated anti pd l1

Cell suspensions were prepared from spleen, lymph nodes, VAT, or liver of ob/ob mice as previously described (Chang et al., 2015 (link); Xiang et al., 2016 (link)). DC single-cell suspensions were obtained from bone marrow following standard procedures. Cells were stained with antibodies against the following cell surface antigens: fluorescein isothiocyanate (FITC)-conjugated anti-CD86, allophycocyanin (APC)-conjugated anti-CD11c, phycoerythrin (PE)-conjugated anti-MHC-II and PE-conjugated anti-PD-L1 (eBioscience, USA); APC-conjugated anti-Foxp3, FITC-conjugated anti-CD4, PE-conjugated anti-CD25 (eBioscience, USA); APC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PE-conjugated anti-TCRβ (eBioscience, USA). Samples were detected on a BD LSRII flow cytometer or BD C6 (BD Pharmingen), and data were analyzed with Flowjo software, version 10.1.

For flow cytometry, the following antibodies for the analysis of human samples were used: FITC-conjugated anti-CD16, PE-conjugated anti-PD-L1, PerCP-conjugated anti- CD45, APC-conjugated anti-CD11b, APC-conjugated anti-CD14, FITC-conjugated anti-CD3, PE-conjugated anti-CD8, PerCP-conjugated anti-CD4 and APC-conjugated anti-PD-1. All human antibodies were purchased from BD Bioscience (San Jose, USA). Antibodies used in the mouse experiments were purchased from eBioscience (San Diego, CA) as follows: FITC-conjugated anti-Gr-1, PE-conjugated anti-PD-L1, PerCP-conjugated anti-CD11b, APC-conjugated anti-CD48, APC-conjugated anti-CD8, FITC-conjugated anti-CD3, PE-conjugated anti-PD-1, and PerCP-conjugated anti-CD4. Data were analysed with FlowJo5.6.7 (Tree star, Inc., Ashland, OR, USA).

Lymphomononuclear cells from the livers, spleens and peripheral blood were incubated with fluorochrome-labeled antibodies at 5×10⁵ / tube for 30 min at 4°C, and characterized using a FACS Canto II cytometer (BD Biosciences). For the intracellular cytokine staining, Caltag^TM, Fix&Perm^® reagents (Invitrogen, Carlsbad, CA, USA) were used following the manufacturer's instructions. The following antibodies were used: fluorescein isothiacyanate (FITC)-conjugated anti-CD4, FITC-conjugated anti-CXCR5, FITC-conjugated anti-CD19, Phycoerythrin (PE)-conjugated anti-PD-1, PE-conjugated anti-ICOS, PE-conjugated anti-PDL-1, PE-conjugated anti-ICOSL, PE-conjugated anti-IL-21R, allophycocyanin (APC)-conjugated anti-CD4 (eBioscience, USA), PE-conjugated anti-IL-21 (BD Biosciences, USA) and FITC-, APC- or PE-conjugated isotype antibodies (eBioscience, USA).
Lymphomononuclear cells from the livers and spleens were incubated with HBc-derived peptides HBc1-20 (1 mg / ml)at room temperature for 10 min. HBc-derived peptides HBc1-20 was purchased from Sangon Biotech (Sangon, Shanghai, China). The cells were then washed twice with PBS containing 1% BSA (BD Biosciences), and incubated with anti-CD4, anti-CXCR5 for 30 min at 4°C, and characterized using a FACS Canto II cytometer.