Nkg2c 134591

Human PBMC were stained using directly conjugated mAb against human CD3 (BW264/56), CD56 (REA196), CD57 (TB03), from Miltenyi Biotec (San Diego, CA, USA), Tim-3 (F38-2E2) from BioLegend and NKG2C (134591) from R&D Systems. Cells were stimulated with 1 μg anti-CD16 mAb (3G8; BioLegend) per 10⁶ PBMC and prepared for intracellular staining by adding brefeldin A (Sigma-Aldrich) 1 h after the start of incubation to a final concentration of 10 μg/mL and continuing the incubation for an additional 4 h. NK cell degranulation was detected by introducing directly conjugated anti-CD107a mAb (H4A3; BioLegend) at a 0.25 μg per 10⁶ PBMC at the time of brefeldin A addition. Cells were fixed and permeabilized after 5 h incubation using the Inside Stain Kit (Miltenyi Biotec) as per manufacturer's instructions and then stained with directly conjugated polyclonal Ab against human FcRγ from MilliporeSigma (Burlington, MA, USA) and anti-human IFN-γ mAb (4S.B3) from eBioscience (San Diego, CA, USA). Non-viable cells were excluded by fixable live/dead stain (Invitrogen) as per manufacturer's instructions. Data were acquired using a MoFlo Astrios EQ flow cytometer and data analyses and illustration performed using Kaluza software (both Beckman Coulter, Brea, CA, USA).

NK cell phenotype was analyzed with fluorochrome-conjugated antibodies against CD2 (RPA-2.10), CD11a (HI111), CD11b (ICRF44), CD27 (M-T271), CD44 (G44-26), CD57 (NK-1), CD62L (DREG-56), CD107a (H4A3), DNAM1 (DX11), KI-67 (B56), NKp44 (p44-8.1) NKp46 (9E2), NKp30 (p30-15), NKG2D (1D11), Siglec 9 (E10-286) (BD Biosciences, Franklin Lakes, NJ, USA); NKG2C (134591) (R&D Systems, Abingdon, UK); CD2 (TS1/8), CD56 (HCD56), NKp80 (5d12), CD160 (BY55), CD161 (HP-3G10), CD319 (CRACC) (162.1), and CD352 (NTBA) (NT-7) (Biolegend, San Diego, CA, USA); CD244 (2B4) (C1.7) (Beckman Coulter, Brea, CA, USA); NKG2A (REA110), Siglec 7 (REA214) (Miltenyi) and handled according to the manufacturers’ protocol. For intracellular staining of Granzyme A (CB9) (Biolegend), Granzyme B (GB11), and Perforin (dG9) (BD Biosciences), cells were washed with PBS followed by fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) and incubated for 20 min. Following fixation and permeabilization, cells were washed and stained in Permwash according to manufacturer’s protocol. All cells were acquired by a BD LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

NK cells were stained with antibodies recognizing CD56 (NCAM16.2), CD3 (UCHT1), CD16 (3G8), CD38 (HIT2), CD34 (8G12), NKp44 (P44-8.1), KIR3DL1 (HP-3E4), KIR2DL1 (HP-3E4), CD107a (H4A3), TNF-α (6401.1111), and IFN-γ (4S.B3) from BD Biosciences (Franklin Lakes, New Jersey, USA), DNAM-1 (11A8), 2B4 (2–69), NKG2D (1D11), NKp30 (p30-15), NKp46 (9E2), CD62L (DREG-56), CXCR4 (12G5), Lir-1 (GHI/75), TRAIL (RIK-2), and FLAG tag (L5) from BioLegend (San Diego, California, USA), NKG2A (Z199) from Beckman Coulter (Brea, California, USA), NKG2C (134591) from R&D Systems (Minneapolis, Minnesota, USA) along with annexin V (BioLegend), and/or a Live/Dead fixable aqua dead cell stain kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Flow cytometry was performed using a LSRFortessa instrument (BD Biosciences), and data were analyzed with FlowJo V.10.1 software (BD Biosciences).