Anti nkg2c

Manufactured by R&D Systems

Sourced in United States

Anti-NKG2C is a monoclonal antibody that recognizes the human NKG2C receptor. NKG2C is a type II transmembrane receptor expressed on the surface of natural killer (NK) cells and a subset of T cells. This antibody can be used for the detection and analysis of NKG2C-expressing cells.

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Close spelling variants of this product

Anti-NKG2C-PE (10 citations) NKG2C (5 citations) Anti-NKG2C (clone 134591) (2 citations) Anti-NKG2C-PE (clone 134591) (2 citations) Anti-NKG2C-Alexa Fluor 488 (2 citations)

9 protocols using anti nkg2c

NK Cell Immune Phenotyping Protocol

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Natural killer cells were analyzed after staining with an appropriate monoclonal antibody (mAb) cocktail, as previously described^{22 (link),23 (link)}: anti-CD45 (#J33), anti-CD3 (#UCHT1), anti-CD56 (#N901), anti-CD16 (#3G8), anti-CD159a/NKG2A (#Z199), CD335/NKp46 (#BAB281), anti-CD336/NKp44 (#Z231), anti-CD69 (#FN50), anti-Cx3CR1 (#2A9–1), and anti-CD127 (#R34.34) mAbs from Coulter; anti-CD62L (#DREG-56; BD Pharmingen), anti-CD337/NKp30 (#AF29–4D12), and anti-NKG2C (#134591) mAbs from R&D systems, and also a cocktail of anti-panKIR-L including anti-KIR2DL1 (#143211) and anti-KIR3DL1 (#177407); and anti-KIR2DL2/KIR2DL3 (#DX27; Miltenyi Biotech) mAbs. Populations of interest within the CD45⁺ lymphocytic population were gated on CD3⁻CD56⁺ NK cells. At least 20,000 CD45⁺ cells or 1000 CD3^-CD56⁺ NK cells were acquired on a Gallios flow cytometer (Beckman Coulter) and then analyzed with Flow Jo version 9 (TreeStar) (Supplementary Figure 1), as described.^{22 (link)}

Nel I., Lucar O., Petitdemange C., Béziat V., Lapalus M., Bédossa P., Debré P., Asselah T., Marcellin P, & Vieillard V. (2016). Accumulation of Intrahepatic TNF-α-Producing NKp44+ NK Cells Correlates With Liver Fibrosis and Viral Load in Chronic HCV Infection. Medicine, 95(19), e3678.

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Evaluating Vδ2 T Cells and PMN-MDSC

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The Vδ2 T cells and PMN-MDSC frequency and phenotype were evaluated utilizing the following monoclonal antibodies: anti-Vδ1 (Life technology), anti-NKG2A, anti-NKG2D (Beckman Coulter), anti-NKG2C (R&D system), anti-Vδ2, anti-CD3, anti-CD15, anti-CD33, anti-HLA-DR, cocktail of antibodies anti-CD3, -CD56, -CD19, anti-CD14, anti-CD11b (BD Biosciences). In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 1% paraformaldehyde and analyzed using a FACS Canto II (Becton Dickinson). For intracellular staining, membrane staining was performed as above described. After fixation cells were incubated with anti-IFNγ (BD Biosciences) for 30 min at room temperature. CD107a detection was accomplished by antibody staining during cell stimulation. After washing cells were analyzed using a FACS Canto II (Becton Dickinson). Apoptosis induction of Daudi cells were accomplished by evaluating Annexin V ligation to Daudi (Annexin V-FITC Apoptosis Detection Kit, eBiosciences) following the manufacturer’s instruction. Then cells were stained with anti-CD19, anti-Vδ2, anti-CD3, anti-CD15.

Sacchi A., Tumino N., Sabatini A., Cimini E., Casetti R., Bordoni V., Grassi G, & Agrati C. (2018). Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells. Frontiers in Immunology, 9, 1271.

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Immune Receptor Repertoire Analysis

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Peripheral blood mononuclear cells (PBMCs) were isolated from blood by density centrifugation and were cryopreserved with 5% DMSO. Samples were thawed and rested overnight prior to flow cytometry. Immune receptor repertoires were determined by flow cytometry on an LSRII (BD Biosciences). PBMCs were analyzed for NK cells (CD3^-CD56⁺), and adaptive (CD56^dimCD57⁺NKG2C⁺) NK cells using fluorescently conjugated antibodies: anti-CD3 (Invitrogen; MHCD0317), anti-CD56 (BD Biosciences; 335791), anti-NKG2C (R&D; FAB138P), anti-CD57 (Biolegend; 322316). Data analysis was performed using FlowJo 9.3.2 software (TreeStar).

Merino A.M., Mehta R.S., Luo X., Kim H., De For T., Janakiram M., Cooley S., Wangen R., Cichocki F., Weisdorf D.J., Miller J.S, & Bachanova V. (2020). Early Adaptive NK Cell Expansion is Associated with Decreased Relapse After Autologous Transplant for Multiple Myeloma. Transplantation and cellular therapy, 27(4), 310.e1-310.e6.

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Phenotyping of Human Lymphocyte Subsets

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Lymphocytes from blood/tissue samples were incubated for 30 minutes with the following mouse anti-human monoclonal antibodies: anti-CD45 (Biolegend, CA, USA); anti-CD3, anti-CD56, anti-perforine, anti-CD158a, anti-CD158b and anti-NKp44; anti-NKG2D, anti-NKp30, anti-CD226, and anti-NKp46 (BD Bioscience, San Jose, CA); anti-NKG2A and anti-NKG2C (R&D Systems, Inc., Minneapolis, MN); KIR2DL1/DS1 and KIR3DL1/DS1 (Beckman Coullter, Fullerton, CA). Mouse serum was used to block non-specific Fc receptor (FcR) binding, and isotype-matched IgGs were used as negative control antibodies. The samples were acquired using an fluorescence activating cell sorter (FACS) Calibur flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).

Xia Y., Zhang Q., Zhen Q., Zhao Y., Liu N., Li T., Hao Y., Zhang Y., Luo C, & Wu X. (2017). Negative regulation of tumor-infiltrating NK cell in clear cell renal cell carcinoma patients through the exosomal pathway. Oncotarget, 8(23), 37783-37795.

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Multiparameter Flow Cytometry Analysis of NK Cells

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FACS staining was performed using standard procedures. Staining was performed with the following conjugated antibodies: anti CD56, anti CD3, anti CD16 (all from Biolegend), anti 2B4, anti NKp46, anti CD69 (BD), anti NKp30, anti NKp44, anti NKG2D, anti DNAM1, anti NKG2C (R&D), anti CEACAM1 (R&D), anti TIGIT (eBioscience), anti NKG2A (R&D). Antibodies against chemokine receptors that were used included anti CCR1, anti CCR2, anti CCR3, anti CCR5, anti CCR7, anti CCR10, anti CXCR1, anti CXCR2, anti CXCR3, anti CXCR4, and anti CX3CR1. Other antibodies used were: anti CD57 and antibodies against the killer cell immunoglobulin-like receptors-KIR2DL1/DS1, anti KIR2DL2/DL3, and anti KIR3DL1/KIR3DS1. Unless otherwise noted, all antibodies in this study were purchased from Biolegend. In all FACS results shown in this paper, dead cells, neutrophils, and monocytes were excluded from analysis. We used the FCS Express version 4 program for FACS analysis. We performed correction for the MFI of different unrelated FACS staining, by dividing the individual staining MFI (of blood and SFs NK cells) by the background isotype control of the same staining (normalized MFI).

Yamin R., Berhani O., Peleg H., Aamar S., Stein N., Gamliel M., Hindi I., Scheiman-Elazary A, & Gur C. (2019). High percentages and activity of synovial fluid NK cells present in patients with advanced stage active Rheumatoid Arthritis. Scientific Reports, 9, 1351.

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Differentiation of iPSC-derived iNK Cells

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Human iPSC culture and differentiation to iCD34⁺ cells were performed as previously described (35 (link), 36 (link), 60 (link)). At the beginning of the iNK cell differentiation culture, iCD34 cells were plated on stroma cells in B0 media supplemented with cytokines that support NK cell differentiation from hematopoietic progenitors (61 (link)). After 21 days of culture, iNK cells were harvested and co-cultured with irradiated K562 cells transduced with membrane-bound IL-21 and 4–1BBL constructs (62 (link)) in supplemented B0 media for 2 – 3 weeks. K562 cells were propagated in RPMI 1640 media (Corning) containing 10% fetal bovine serum (FBS) (Hyclone). The following fluorescently-conjugated antibodies were used for phenotypic analysis of iNK cells: anti-KIR3DL1 (DX9), anti-KIR2DL1 (HP-MA4), anti-KIR3DL2/3 (DX27), anti-NKG2D (D11), anti-CD16 (3G8), anti-NKp44 (p44–8), anti-NKp46 (9E2), anti-NKp80 (5D12), anti-perforin (B-D48), anti-granzyme b (QA16A02), anti-2B4 (C1.7), anti-LFA-1 (HI111), anti-CD69 (FN50), anti-CD62L (DREG-56), anti-PD-1 (EH12.2H7), anti-LAG-3 (11C3C65), anti-TIGIT (A15153G) (all from Biolegend), anti-NKG2A (Z199; Beckman Coulter), anti-NKp30 (p30–15; BD Biosciences), and anti-NKG2C (134591; R&D Systems).

Cichocki F., Bjordahl R., Gaidarova S., Mahmood S., Abujarour R., Wang H., Tuininga K., Felices M., Davis Z.B., Bendzick L., Clarke R., Stokely L., Rogers P., Ge M., Robinson M., Rezner B., Robbins D.L., Lee T.T., Kaufman D.S., Blazar B.R., Valamehr B, & Miller J.S. (2020). iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy. Science translational medicine, 12(568), eaaz5618.

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PBMC Immunophenotyping Workflow

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PBMC were stained for extracellular markers (clones in parenthesis) with anti-CD2 (RPA-2.10, BioLegend, San Diego, CA, USA), anti-CD3 (BW264/56), anti-CD16 (3G8), anti-CD56 (REA196), anti-CD57 (TB03), anti-NKG2A (REA110), all from Miltenyi Biotec, Auburn, CA, USA and anti-NKG2C (134591, R&D Systems, Minneapolis, MN, USA). LIVE/DEAD Fixable Far Red (Invitrogen) was used to exclude dead cells. Fixation and permeabilization were performed using MACS Inside Stain Kit (Miltenyi Biotec) with polyclonal goat anti-human FcεRIγ (EMD Millipore, Etobicoke, ON, Canada), anti-IFN-γ (4S.B3), and anti-TNF-α (MAb11) from eBioscience, San Diego, CA, USA added for intracellular staining. The fluorescence minus one strategy was used to adjust multicolor compensation. Phenotypic analysis was performed using Kaluza Flow Cytometry Software 1.2 after data acquisition on a MoFlo Astrios EQ flow cytometer (both Beckman Coulter, Brea, CA, USA).

Comeau E.M., Holder K.A., Fudge N.J, & Grant M.D. (2019). Cytomegalovirus-Driven Adaption of Natural Killer Cells in NKG2Cnull Human Immunodeficiency Virus-Infected Individuals. Viruses, 11(3), 239.

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Cytotoxicity Assay for B-ALL, T-ALL, and K562 Cells

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Expression of CD107a and production of TNFα were measured as described previously.⁴¹ Briefly, PBMCs were incubated with B‐ALL, T‐ALL or K562 target cells at an effector‐to‐target ratio of 2:1 for 5 h. For some experiments, PBMCs were pre‐incubated with either 10 μg mL⁻¹ of isotype control or 10 μg mL⁻¹ of anti‐HLA‐A,B,C, anti‐HLA‐E (3D12), anti‐NKG2D (1D11), anti‐DNAM‐1 (11A8), anti‐NKp30 (P30‐15), anti‐NKp46 (9E2) and anti‐2B4 (C1.7) (all BioLegend), for 20 min at 37°C. Brefeldin A and monensin (both BD Biosciences) were added after 1 h. The following antibodies were used: anti‐NKG2C (134591; R&D Systems, Minneapolis, MN), anti‐KIR3DL1 (DX9; BioLegend), anti‐CD56 (clone B159), anti‐CD3 (SK7), anti‐CD57 (NK‐1), anti‐CD158a (HP‐3E4), anti‐CD158b (CH‐L), anti‐CD107a (H4A3), anti‐TNFα (MAb11) and fixable viability stain FV575 (all BD Biosciences). Cells were analysed on an BD LSR Fortessa and using FlowJo Version 10 software.

Foley B., Ta C., Barnes S., de Jong E., Nguyen M., Cheung L.C., Buzzai A., Wagner T., Wylie B., Fernandez S., Cruickshank M., Endersby R., Kees U, & Waithman J. (2020). Identifying the optimal donor for natural killer cell adoptive therapy to treat paediatric B‐ and T‐cell acute lymphoblastic leukaemia. Clinical & Translational Immunology, 9(7), e1151.

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Characterization of Resting and Activated NK Cells

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The fine characterization of surface markers of resting and IL-2 activated NK cells was performed using the following monoclonal antibodies (mAbs) generated in our laboratory (Department of Molecular and Translational Medicine, University of Brescia), or in the laboratory directed by A. Moretta (Laboratory of Molecular Immunology, DIMES, University of Genoa): BAB281 (IgG1, anti-NKp46); AZ20 (IgG1, anti-NKp30); ON72 (IgG1, anti-NKG2D); C127 and SUS142 (IgG1 and IgG2b respectively, anti-CD16); C227 (IgG1, anti-CD69); 11PB6 (IgG1, anti-KIR2DL1/S1); GL183 (IgG1, anti-KIR2DL2/L3/S2); AZ158 (IgG2a, anti-KIR3DL1/S1/L2); Z199 (IgG2b, anti-CD94/NKG2A); anti-XA147 (IgM, anti-CD57); A6/136 (IgM anti-HLA-I).
The commercially available antibodies used in this study are: anti-CXCR1 (IgG1, Santa Cruz biotechnologies, Santa Cruz, CA; USA); anti-NKG2C (IgG2b R&D Systems, MN, USA); anti-hCCR7 (IgG2a R&D Systems, MN, USA); anti-CD62L (IgG1 R&D Systems, MN, USA); anti-CD107a PE-labeled; anti-IFN-γ PE-(IgG1, BD-Biosciences, Pharmingen CA, USA); mixture of FITC-labeled CD3 plus PC5-labeled CD56, FITC-labeled CD14 and FITC-labeled CD20 (Beckman Coulter, Immunotech, Marseille, France); anti-human Ki-67 antigen (IgG1, Dako, Denmark A/S); Annexin V PE (BD-Biosciences, Pharmingen CA, USA); anti-human Perforin/R-PE (Ancell) and anti-human Granzyme/ R-PE (Enzo-Life Sciences).

Lougaris V., Patrizi O., Baronio M., Tabellini G., Tampella G., Damiati E., Frede N., van der Meer J.W.M., Fliegauf M., Grimbacher B., Parolini S, & Plebani A. (2017). NFKB1 regulates human NK cell maturation and effector functions. Clinical immunology (Orlando, Fla.), 175.

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