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4 protocols using tnfα mab11

1

Polychromatic Cytometry and Cell Sorting

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Polychromatic flow cytometry and cell sorting were performed on stained mononuclear cells as previously described15 (link). Antibodies against the following antigens were used for staining at predetermined concentrations: CD4 (clone OKT4), CD8 (SK1), IFNγ (4S.B3), IL-17 (eBio64DEC17), IL-21 (3A3-N2), IL-22 (IL22JOP), TNFα (MAb11), and PD-1 (J105) from eBioscience; CCR5 (3A9), CD3 (SP34-2), CD8 (SK1), CD16 (HI149), CD20 (2H7), CD23 (M-L233), CD45 (D058-1283), CD123 (7G3), and HLA-DR (L243) from BD; c-Kit (104D2), CD1a (HI149), CD11c (3.9), CD14 (M5E2), CD34 (561), CD95 (DX2), and IL-2 (MQ1-17H12) from Biolegend; CD127 (eBioRDR5) and CD218a (H44) from Thermo; NKp44 (2.9) from Miltenyi; and CD28 (CD28.2) from Beckman Coulter. CD4+ and CD8+ TM were defined as CD95+ singlet, clean, live, CD3+ lymphocytes. NKp44+ ILC3s were defined as CD45+ singlet, clean, live, lineage-negative (CD1a, CD3, CD8, CD11c, CD14, CD16, CD20, CD23, CD34, CD123), NKp44+CD127+CD218+ lymphocytes. Positive/negative gating based on clearly grouped populations, historical-determined expression, and the use of internal controls (Supplementary Fig. 8). A threshold of 100 collected events in the parent population was utilized for all subset expression analysis.
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2

Phenotyping of Expanded Natural Killer Cells

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The phenotype of eNK cells was analyzed weekly using flow cytometry. The following mouse anti-human antibodies were used for staining: CD16 (3G8), CD56 (NCAM 16.2), NKG2D (1D11), DNAM-1 (DX11), CD3 (HIT3a) from BD PharMingen; CXCR1 (8F1), CXCR3 (CEW33D), CXCR4 (L276F12), CXCR6 (K041E5), CCR4 (L291H4), CCR5 (J418F1), CCR6 (G034E3), CCR9 (L053E8), CX3CR1 (2A9-1), Fas-L (NOK-1), IFNγ (B27) from BioLegend; CD62L (DREG-56), intracellular Perforin (dG9), Granzyme B (GB11), and TNF-α (MAb11) from eBioscience. For surface staining, NK cells were resuspended in FACS buffer (0.5% FBS and 2 mM EDTA in PBS), blocked with 10% mouse serum for 30 min, then incubated with the indicated antibodies for 30 min at 4°C using concentrations recommended by the manufacturers. For subsequent intracellular staining, NK cells were stained using the BD Cytofix/Cytoperm Kit following manufacturer protocol. Data was acquired using a BD FACS Accuri C6 and analyzed using FlowJo v10.2 software. Isotype IgG antibodies were used as negative controls. Human NK cells were defined as CD16+, CD56+ and CD3. Mouse anti-human HLA-ABC (W6/32), MICA/B (6D4), CD155 (2H7) and PD-L1 (MIH1) from eBioscience, ULBP-1 (170818) from R&D, and Fas (DX2) from BioLegend were used to determine MHC-I, NKG2D ligand and DNAM-1 ligand expression on TC106 with surface staining performed as above for NK cells.
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3

Polychromatic Cytometry and Cell Sorting

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Polychromatic flow cytometry and cell sorting were performed on stained mononuclear cells as previously described15 (link). Antibodies against the following antigens were used for staining at predetermined concentrations: CD4 (clone OKT4), CD8 (SK1), IFNγ (4S.B3), IL-17 (eBio64DEC17), IL-21 (3A3-N2), IL-22 (IL22JOP), TNFα (MAb11), and PD-1 (J105) from eBioscience; CCR5 (3A9), CD3 (SP34-2), CD8 (SK1), CD16 (HI149), CD20 (2H7), CD23 (M-L233), CD45 (D058-1283), CD123 (7G3), and HLA-DR (L243) from BD; c-Kit (104D2), CD1a (HI149), CD11c (3.9), CD14 (M5E2), CD34 (561), CD95 (DX2), and IL-2 (MQ1-17H12) from Biolegend; CD127 (eBioRDR5) and CD218a (H44) from Thermo; NKp44 (2.9) from Miltenyi; and CD28 (CD28.2) from Beckman Coulter. CD4+ and CD8+ TM were defined as CD95+ singlet, clean, live, CD3+ lymphocytes. NKp44+ ILC3s were defined as CD45+ singlet, clean, live, lineage-negative (CD1a, CD3, CD8, CD11c, CD14, CD16, CD20, CD23, CD34, CD123), NKp44+CD127+CD218+ lymphocytes. Positive/negative gating based on clearly grouped populations, historical-determined expression, and the use of internal controls (Supplementary Fig. 8). A threshold of 100 collected events in the parent population was utilized for all subset expression analysis.
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4

Activation of CD134 in PBMCs

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Stimulated PBMCs were further co-cultured with an isotype control (cetuximab, Southampton General Hospital pharmacy), anti-CD134 (SAP25–29 h1, in-house), deglycosylated anti-CD134 (in-house), anti-CD134 F(ab′)2 (in-house, produced as previously described37 (link)), multimeric CD134L (Caltag) or control ligand (Caltag) for 6 hours. For IFNγ (B27, BD Bioscience) and TNFα (Mab11, eBioscience) intracellular staining, human cells were fixed and permeabilised as per manufacturer’s protocol (BD Bioscience) after 6 hours co-culture with brefeldin A (BD Bioscience). For assessment of NK degranulation, PBMCs were incubated with CD107a-PE (H4A3, BD Bioscience) and monensin (BD Bioscience) for 6 hours.
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