Masson’s trichrome and Sirius Red stain was also used for visualizing fibrosis on liver tissue sections. Liver infiltrating mononuclear cells (MNCs) were isolated as described.10 The cells were resuspended in staining buffer (0.2% bovine serum albumin [BSA], 0.04% ethylenediaminetetraacetic acid [EDTA] and 0.05% sodium azide in phosphate-buffered saline [PBS]), divided into 25-μL aliquots, and incubated with antihuman FcR blocking reagent (eBioscience) for 15 minutes at 4°C. Cells were then washed and

stained for 30 minutes at 4°C with cocktails containing combinations of fluorochrome-conjugated anti-PD-1 antibody inhibitor monoclonal antibodies for different cell surface markers including CD8a, CD4, NK1.1 (Biolegend, San Diego, CA), CD19 and TCRβ (eBioscience, San Diego, CA). IgG isotype antibodies BVD-523 solubility dmso with matching conjugates were used as negative controls. The cells were then washed once with PBS containing 0.2% BSA. For intracellular cytokine staining, cells were resuspended in 10% FBS RPMI and stimulated with Leukocyte Activation Cocktail in the presence of BD GolgiPlug (BD Pharmingen, San Diego, CA) at 37°C for 4 hours. The cells were stained for surface CD4, NK1.1, and TCRβ, fixed, and permeabilized with BD

Cytofix/Cytoperm Solution (BD Biosciences, San Diego, CA), then stained for intracellular interferon γ (IFN-γ), IL-4, and IL-17 (BioLegend). Normal IgG isotype controls were used in parallel. A FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) upgraded for the detection of five colors by Cytek Development (Fremont, CA) was used to acquire data, which were analyzed with Cellquest PRO software (BD Immunocytometry Systems). For analysis of secreted cytokines, 2.0 × 105 hepatic MNCs were cultured in 96-well round-bottom plates in 200 μL of RPMI ADP ribosylation factor supplemented with 10% heat-inactivated FBS (GIBCO-Invitrogen, Grand Island, NY), 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.5 μg/mL each of anti-CD3 (BioLegend) and anti-CD28 (BioLegend). The cells were incubated

for 72 hours at 37°C in a humidified 5% CO2 incubator, then centrifuged. The supernatant was collected and analyzed for concentration of cytokines. IL-22 level was measured using an ELISA kit (BioLegend). The levels of IFN-γ, tumor necrosis factor α (TNF-α), IL-6, and IL-17 were measured simultaneously with a mouse cytometric bead array (CBA) kit (BD Biosciences), using a FACScan flow cytometer with CBA software (BD Biosciences). Hepatic hydroxyproline content was quantified using a hydroxyproline assay kit (BioVision, Mountain View, CA). Briefly, frozen liver tissue from 24-week-old mice, in groups of 5-8 mice, were weighed and homogenized in distilled H2O and hydrolyzed.