Cd161 fitc

Flow cytometry was performed on cryopreserved cells. As per the gating strategy in figure 1A, NK cells were identified using a combination of Fixable blue dead cell stain (Life technologies), CD3-Pacific Blue, CD56-PE-Cy7 and CD16- APC-Cy7 (BD Biosciences). NK cell receptor expression was assessed using combinations of CD158a-PerCP-Cy5.5 (eBioscience), CD158b-FITC, KIR3DL1-Alexafluor700 (both Biolegend), KIR2DL3-PE, KIR2DL1-APC (both R&D Systems), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). For intracellular staining, cells were fixed and permeabilized (PermA/B solution, Caltag) according to the manufacturers' instructions, prior to incubation with Perforin-PerCP-Cy5.5 (eBioscience). At least 1500 NK cells were acquired for all samples on either a five laser BD LSRFortessa or a four laser BD LSRII system, equipped with FACSDiva Version 8.8.3 (BD biosciences). Rainbow beads ensured a consistent, comparable level of fluorescence across all samples on different days of acquisition. Gates were set using fluorescence minus one or unstimulated samples where appropriate. The data were analysed using FlowJo version 9.5.3 (Treestar, OR, USA).

Cryopreserved peripheral blood mononuclear cells (PBMC) were stained with combinations of antibodies that comprehensively interrogate the breadth of NK-cell receptors including the killer immunoglobulin receptors (KIRs), the c-type lectin receptors (NKG2), and the natural cytotoxicity receptors (NCRs). These included CD158a-PerCP-Cy5.5 (eBioscience), KIR3DL1-Alexafluor700 (Biolegend), NKG2A-PE (Beckman Coulter), NKG2D-APC, CD94-FITC, 2B4-FITC, NKp46-PE, NKp30-APC, CD158b-FITC (all BD Biosciences), TRAIL-PE (Biolegend), CD161-FITC (Miltenyi) and CD160-Alexafluor647 (Biolegend). Dead cells were excluded using the Fixable Blue Dead Cell Stain (Life Technologies) prior to surface staining and fixation. For perforin staining, samples were then permeabilized (Perm A/B, Caltag) and stained with anti-Perforin-PerCP-Cy5.5 (eBioscience). NK cells were identified as CD3 negative lymphocytes expressing CD56 and/or CD16. At least 1500 NK cells were acquired per sample. Fluorescence minus one was used to set gates. The data were analyzed with Flowjo v9.5.4 (Treestar).

Aliquots of 1 × 10⁶ isolated liver-derived cells and PBMC were stained for surface markers with pre-diluted fluorochrome-conjugated anti-human monoclonal antibodies: BD Multitest™ CD16-PE (IgG1, clone B73.1) and CD56-PE (IgG1, clone NCAM 16.2); CD3/CD16+CD56/CD45/CD19 reagent (CD3-FITC (IgG2a, clone SK7); CD45-PerCP (IgG1, clone 2D1); CD335/NKp46-PE-Cy7 (IgG1, clone 9E2/Nkp46); CD19-APC (IgG1, clone SJ25C1)) and CD3-FITC (IgG2a, clone SK7); CD4-PE-Cy7 (IgG1, clone L200); CD56-AlexaFluor-700 (IgG1, clone B159); CD8-APC-Cy7 (IgG1, clone SK1); antiCD337/NKp30-AlexaFluor-647 (IgG1, clone p30-15); and CD314/NKG2D-PerCP-Cy5.5 (IgG1, clone 1D11). These antibodies were purchased from BD Biosciences (Europe, dilution 1:10). CD161-FITC (IgG2a, clone 191B8); CD3-PerCP (IgG2a, clone BW264/56); CD16-APC (IgM, clone VEP13); and CD336/NKp44-PE (IgG1, clone 2.29) from Miltenyi Biotec (Bergisch Gladbach, Germany, dilution 1:20). TCRVa7.2-PE (IgG1, clone 3C10) was obtained from Biolegend (Campoverde s.r.l., Milan, Italy, dilution 1:25). At room temperature in the dark, LP samples were incubated for 30 min, and washed once with PBS/2% FBS. They were analyzed by flow cytometry with a FACSAria II cytometer (BD Biosciences, Eysins, Switzerland).