Tissue suspension was incubated (37°C, 30 minutes), ground through a 100-μm strainer (BD Biosciences, Franklin Lakes, NJ), and eventually centrifuged (50g, 5 minutes). The supernatant was transferred onto a two-step density gradient (Optiprep; Sigma-Aldrich) and centrifuged (800g, 20 minutes). Cells from the gradient interface were transferred onto flat-bottom 96-well Selleck MK-2206 plates (5 × 105 cells/well) for 30 minutes at

37°C and nonadhesive cells were removed. Expression was analyzed in TAM seeded on 24-well plates (1 × 106 cells/well). Murine liver associated lymphocytes were isolated as described.15 Sorafenib (sc-220125; Santa Cruz Biotech., Santa Cruz, CA) or dimethyl sulfoxide (DMSO) carrier (mock) was added to cell cultures (0.07-2.5 μg/mL [0.02-10 μM]) for 3 hours. Treatment was followed by a medium exchange and NK cell coculture for 24 hours. Lipopolysaccharide (LPS) (1 ng/mL), Celastrol, TPCA-1 (all Sigma-Aldrich), or Q-VD-OPh (R&D Systems, Minneapolis, MN) were applied as indicated. Blocking experiments were performed with IL12 (BD Biosciences), IL15 (R&D Systems) and IL18 (MBL, Woburn, MA) specific antibodies (5 μg/mL) for 24 hours or with IgG1 control antibodies Crizotinib in vivo (Abcam,

Cambridge, UK). Flow cytometry was performed with a Canto-II reader (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR). Cells were permeabilized in Cytofix/Cytoperm reagent (BD Biosciences) and live-dead discriminated by EMA or propidium-iodide. Fluorochrome-conjugated antihuman antibodies: CD3-FITC, CD16-PerCP, CD69-FITC (all eBioscience, San Diego, CA), CD56-APC (R&D Systems), interferon-gamma (IFN-γ)-FITC (BD Biosciences), CD107a-PE-Cy5.5 (Biolegend, San Diego, CA), or antimurine antibodies: CD3-PacB, IFN-γ-PE, CD69-FITC, CD107a-APC, NK1.1-PerCp/Cy5.5, NK1.1-APC (all eBioscience) as well as appropriate isotype controls (BD Biosciences) were subsequently

applied. NK cells designated for CD107a degranulation assays and IFN-γ stains were stimulated with K562 (effector to target ratio [E:T] = 1) or ionomycin/phorbol myristate acetate (PMA) (500/5 G protein-coupled receptor kinase ng/mL) for 5 hours; Brefeldin-A (5 μg/mL) was added after 1 hour of stimulation (all Sigma-Aldrich). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed as described.15 Primers specific for CD14, CD68, albumin, α-fetoprotein (AFP), liver/lymph node-specific ICAM-3-grabbing non-integrin (L-SIGN), interleukin-6 (IL6), IL10, interleukin-12 p40 (IL12), IL18, tumor necrosis factor-α (TNF-α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used (Supporting Information). Messenger RNA (mRNA) expression was normalized to GAPDH. Cytokines were quantified by ELISA for IL6, IL10, TNF-α (all BD Biosciences), IFN-γ, IL12 (p40/70) (all Biolegend), and IL18 (Bender Med Systems, Burlingame, CA).