Anti tcrβ chain and anti trail cd253

All analyses were performed using a FACSCanto II Cytometer (BD Biosciences, Mountain View, CA, USA). To phenotype NK cell surface markers, liver and spleen leukocytes were stained with the following mAbs: anti-NK1.1 (PK136), anti-CD69 (H1.2F3), anti-CXCR3 (CD183), anti-CCR5 (CD195), anti-CCR7 (CD197), and anti-CD19 (1D3) (all obtained from BD Pharmingen, San Diego, CA, USA); anti-TCRβ chain and anti-TRAIL (CD253) (BioLegend, San Diego, CA, USA); anti-CX3CR1 and anti-CXCR6 (R&D Systems, Minneapolis, MN, USA); and anti-TRAIL (CD253) (eBioscience, San Diego, CA, USA). Chemokine production by lymphocytes was measured by staining for a combination of cell surface and cytoplasmic mAb staining and subsequent flow cytometric analysis. Briefly, 4 h after treatment with Protein Transport Inhibitor (containing brefeldin A; BD GolgiPlug, BD Biosciences), lymphocytes were stained with anti-NK1.1-FITC and anti-TCRβ-APC (BD Bioscience) or anti-F4/80-APC (eBioscience), permeabilized with fixation/permeabilization solution, and incubated with anti-CXCL9-PE (MIG-PE; eBioscience).

Flow cytometric analysis was performed using a FACS Canto II cytometer (BD Biosciences). Freshly isolated mononuclear cells were preincubated with anti‐CD16/32 (2.4G2) monoclonal antibody (mAb) to prevent nonspecific binding of the Fcγ II/III receptor. Subsequently, the cells were stained with appropriately diluted fluorescently labeled mAbs. For phenotyping of NK cell surface markers, liver and spleen leukocytes were stained with the following mAbs: anti‐NK1.1 (PK136), anti‐CD69 (H1.2F3), anti‐Dx5 (CD49b; all from BD Pharmingen) and anti‐TCRβ chain and anti‐TRAIL (CD253; BioLegend). Dead cells were excluded from the analysis through forward‐scatter and propidium iodide (PI; Sigma‐Aldrich) or 7‐amino‐actinomycin D (7‐AAD; BD Biosciences) staining.