Anti nkg2c pe

PBMCs were stained in 96-well U-bottom plates as described previously (6 (link)). In brief, cells were stained with fluorophore-labeled Abs to cell-surface markers, fixed, permeabilized (Cytofix/Cytoperm; BD Biosciences), and stained for intracellular molecules. The following mAbs were used: anti–CD3-V500, anti–CD56-PECy7, anti–IFN-γ–allophycocyanin, anti–CD107a-A488, anti–CD16-allophycocyanin-H7, anti–CD25-allophycocyanin-H7 (all BD Biosciences), anti–CD57-e450, anti–CD25-PErCPCy5.5, anti–CD16-allophycocyanin, anti–CD25-PE, anti–IL-18Rα–PE, anti–IL-18Rα–FITC, anti–IFN-γ–allophycocyanin-e780, anti–CD16–allophycocyanin-e780 (all e-Biosciences), anti–NKG2C-allophycocyanin, anti–NKG2C-PE (both R&D Systems), and anti–NKG2A-FITC (Miltenyi). IL-12Rβ2 Ab was conjugated using EasyLink PE-Cy5 (Abcam). Cells were acquired on an LSRII flow cytometer (BD Biosciences) using FACSDiva software. Data analysis was performed using FlowJo V10 (Tree Star). FACS gates set on unstimulated cells (medium alone or isotype controls) were applied in standard format across all samples and all conditions.

PBMCs from 42 MS patients (22 RRMS, 8 SPMS and 12 PPMS) and 17 controls matched for HCMV serostatus were incubated overnight at 37°C with recombinant IL-2 (200 U/ml). The response of NK cells to the HLA class I-defective 721.221 B-lymphoblastoid cell line with or without rituximab (50 ng/ml) was assessed following a 4-h incubation (E/T = 1/1). A complementary approach was performed using EBV(+) AKBM cells as targets following induction of the lytic cycle in the presence of EBV(+) or EBV(–) sera, as previously described (32 (link), 33 (link)). Surface expression of CD107 as a marker of degranulation and intracellular TNFα production was analyzed by flow cytometry as previously reported (34 (link)), using the anti-CD107-APC (BD Pharmigen) monoclonal antibody during incubation together with monensin (GolgiStop® BD) and brefeldin (GolgiPlug® BD). Cultures were then stained with anti-CD3-PerCP (BD Pharmigen), anti-CD56-APC-Cy7 (Biolegend), and anti-NKG2C-PE (R&D System), permeabilized, fixed and stained intracellularly with anti-TNFα-CFBlue (labeled by Immunostep), anti-FcRγ-FITC (Millipore), and anti-PLZF-PE CF594 (BD Biosciences). Data acquisition was performed with an LSRFortessa cytometer (BD Biosciences).

NK-cell subsets were analyzed by flow cytometry within the CD45⁺ lymphocyte population (anti-CD45-Chrome Orange; #J33), gated on the CD3⁻ (anti-CD3-ECD; #UCHT1) and CD56⁺ (anti-CD56-PC7; #N901) population, with an appropriate cocktail of 11 antibodies, including anti-CD159a/NKG2A-APC (#Z199); anti-CD335/NKp46-APC (#BAB281); anti-NKG2D-APC; (#ON72); anti-HLA-DR-PE (#Immu357); anti-CD57-FITC (#S-HCL-1) from Coulter, DNAM-1-FITC (#DX11) from Becton Dickinson, anti-CD337/NKp30-PE (#AF29-4D12), and anti-KIR2DL2/KIR2DL3-APC (#DX27) from Miltenyi Biotech; anti-NKG2C-PE (#134591); anti-KIR2DL1-FITC (#143211); and anti-KIR3DL1-APC (#DX9) from R&D systems.
At least 10,000 CD45⁺CD3⁻CD56⁺ cells were analyzed on a Gallios cytometer (Beckman Coulter). Expression of each marker was measured as a percentage of the total CD3⁻CD56⁺ NK cells, as described earlier [36 (link)]. A hierarchical clustering was applied for the 11 NK cell-specific phenotypic markers, with the results displayed using the GENESIS program (software available at www.genome.tugraz.at), as described previously [37 (link), 38 (link)].

Four-color multiparameter flow cytometry was performed using a BD FACSCalibur instrument (BD Biosciences, San Jose, CA) compensated with single fluorochromes and analysed using CellQuest software (BD Biosciences). Flurochrome-labeled (FITC/PE/PerCP/APC) monoclonal antibodies (MAbs) specific for CD3, CD4, CD8, CD56, CD161, CD94, CD16, CD158A, CD158B, CD158E (NKB1), and NKG2D were purchased from BD Biosciences. Anti-NKG2C-PE and TRAIL-PE MAbs were supplied by R&D systems (Minneapolis, MN). Anti-NKG2A-PE, NKp30-PE, NKp44-PE, and NKp46-PE were obtained from Immunotech (Beckman Coulter, Fullerton, CA). PBMCs (2.5×10⁵), were stained for cell surface antigen expression at 4°C in the dark for 30 minutes. Then, washed twice in 2 ml phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.01% sodium azide (Facs Wash) and subsequently fixed in 200 µl of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Isotype-matched fluorescently-labeled control antibodies were used to determine background levels of staining. Lymphocytes were identified by characteristic forward scatter (fsc) and side scatter (ssc) parameters and populations of interest were gated on positive staining for CD56 and negative staining for CD3 within the lymphocyte population (Figure 1A). Results are expressed as % positive of gated populations or as median fluorescent intensity (MFI) where appropriate.

Peripheral blood-derived and in vitro cultured mononuclear cell populations were identified by a combination of physical parameters and immunostaining with saturating concentrations of the following fluorochrome-conjugated mAbs: anti-CD3 PerCP (clone: SK7, cat #: 347344; BD Biosciences) or PerCP-Vio700 (clone: REA613, cat #: 130-109-465; Miltenyi Biotec), anti-CD56 APC (clone: B159, cat #: 555518; BD Biosciences) or APC-Vio770 (clone: REA 196, cat #: 130-100-694; Miltenyi Biotec), CD16 PE (clone: B73.1, cat #: 347617; BD Biosciences) or PE-Vio770 (clone: REA423, cat #: 130-106-706; Miltenyi Biotec), anti-FcεRIγ subunit FITC (polyclonal antibody, cat #: FCABS400F; Merck), anti-NKp46 APC (clone: REA808, cat #: 130-112-122; Miltenyi Biotec), anti-NKG2C PE (clone: 134591, cat #: FAB138P; R&D Systems), anti-PLZF PE (clone: Mags.21F7, cat #: 12-9320-82; ThermoFisher Scientific), anti-PD-1 PE (clone: EH12.2H7, cat #: 329906; BioLegend), and anti-PD-L1 APC (clone: 29E.2A3, cat #: 329708; BioLegend). Samples were stained for surface antigens for 30 min at 4°C, washed with PBS (Euroclone) containing 2% FCS and 2 mM EDTA (used for all washing steps), fixed with 2% paraformaldehyde for 20 min at room temperature (RT), washed, permeabilized with washing solution supplemented with 0.05% Triton-X 100 for 30 min at RT, and stained for intracellular antigens for 30 min at 4°C.