Cd226 dnam 1

Alive/dead stain was included to measure viability (Zombie Yellow, BioLegend). Cells were blocked with anti-FcγRIII (2.4G2) prior to surface staining. Cells were fixed with 1% paraformaldehyde for surface analysis or fixed with Cytofix/Cytoperm (BD Bioscience) for intracellular staining. For intranuclear staining, cells were fixed in Cytofix/Cytoperm and Cytoperm Plus buffer (BD) and DNase treated. For phospho mTOR staining, cells were fixed with 2% paraformaldehyde and permeabilized with methanol. For phospho AMPK staining, cells were fixed with Cytofix/Cytoperm (BD Bioscience), and stained overnight at 4C with anti-pAMPK. Data was acquired on a Cytek-modified FACScan (BD and Cytek) or LSRFortessa (BD); data was analyzed using FlowJo 7.6.5. Geometric mean fluorescence intensity and % proliferated were calculated using FlowJo proliferation analysis (see below). The following fluorochrome-conjugated antibodies were used: BrdU (BrdU kit, BD), CD107a (1D4B, BD), CD3ε (145-2C11, BD/BioLegend), CD11b (M1/70, BD), CD226/DNAM-1 (10E5, BioLegend), CD27 (LG.3A10, BD), CD45.1 (A20 Biolegend), anti-human IFN-γ (XMG1.2, BioLegend), Ly49C/I (5E6, BD), Ly49H (3D10, BD/BioLegend), NK1.1 (PK136, BD), NKG2A/C/E (20d5, BD), NKG2D (CX5, Biolegend), NKp46 (29A1.4, BD), pAMPKa, (40H9, Cell Signaling), and pMTOR (MRRBY, Invitrogen).

NK cells were pre-incubated with mAbs antibodies specific to CD226/DNAM-1 (clone: 11A8, BioLegend), CD11a (clone: TS1/22, Thermo Fisher Scientific) at a final concentration of 10 μg/mL or with the matched isotype control (BioLegend, mouse IgG1, κ isotype clone: MOPC-21) for 1 h prior to co-culture. mouse IgG1, κ anti-IFN-α antibody (clone: ST29, abcam) and anti-human IL-18 R α/IL-1 R5 Antibody (clone: 70625, R&D Systems) were used at a final concentration at 2.5 µg/ml. IL-12Rβ2 Ab (Cat #: AF1959-SP, R&D Systems) was used at a final concentration of 83.33 µg/mL. Goat polyclonal to human IL-15R (Cat #: PA5–46991, Thermo Fisher Scientific) and goat IgG isotype control antibody (Cat#: 31245, Thermo Fisher Scientific) were used at a final concentration of 4 µg/mL. mouse IgG1, κ anti-human CD119 IFN-γ receptor chain (clone: GIR-208, BD Pharmingen) was used at a final concentration of 50 µg/mL. Human IFN-γ monoclonal antibody (clone: NIB42, eBioscience) was used at a final concentration of 4 µg/mL. Purified NA/LE Rat anti-human IL-2 (clone MQ1–17H12, BD Pharmingen) and purified NA/LE Rat isotype control (clone: R35–95, BD Pharmingen) were used at a final concentration of 4 µg/mL. Recombinant human IL-2 protein (Cat# 202-IL-010, R&D Systems) was added at a final concentration of 10 U/mL.