Clone rtk2758

The following antibodies were used in this study: BioLegend—anti-NKp46 mAb (clone 29A1.4), anti-CD3ε (clone145–2C11), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-CD27 (clone LG.3A10), anti-NKG2D (clone C7), anti-CD107a/LAMP-1 (clone 1D4B); anti-CD11b (eBioscience, clone M1/70); anti-human NKp46 (461-G1,[26 (link)]); mouse IgG1, κ control for injections, rat IgG2a, κ isotype control for FACS (BioLegend, clone RTK2758). The Production of mNKp46-Ig, LIR1-Ig and mNKG2D-Ig as previously described [25 (link),27 (link),28 (link)].

CD14^brightCD16- monocytes (2.5 × 10⁵/500 μL) from the peripheral blood of four healthy volunteers were incubated with 25 ng/mL IL-10. In some experiments, anti-IL-10 receptor antibody (5 μg/mL) (clone 3F9, Biolegend) or rat IgG2aκ (5 μg/mL) to an irrelevant antigen (clone RTK2758, Biolegend) was added to the cultures. CD16 expression on monocytes was then measured.

Employing flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA), neutrophils isolated from bone marrow were gated as Ly6G⁺ cells (PE anti-mouse Ly6G, 1A8, BioLegend, San Diego, CA, USA) and then LFA-1⁺ (PE anti-mouse CD11a/CD18, BioLegend, San Diego, CA, USA) neutrophils were quantified within this population using CellQuest Pro Pro v5.2.1 software (Becton Dickinson, San Jose, CA, USA). κ isotype control (PE Rat IgG1, RTK2071, BioLegend, San Diego, CA, USA) was run in parallel. In experiments verifying purity of isolated platelets, the following antibodies were used—anti-mouse PE CD41 antibody (clone MWReg30; BioLegend, San Diego, CA, USA), PE Ly-6G antibody (clone 1A8-Ly6g; eBioscience, San Diego, CA, USA), PE F4/80 antibody (clone BM8; eBioscience, San Diego, CA, USA), Alexa Fluor 647 Ly-6G antibody (clone 1A8; BioLegend, San Diego, CA, USA), PE IgG2a, κ isotype control antibody (clone MOPC-173; BioLegend, San Diego, CA, USA), PE IgG2a, κ isotype control antibody (clone RTK2758; BioLegend, San Diego, CA, USA).

To assess the importance of LFA-1, CTLs were pre-incubated with LFA-1 blocking antibody (20 µg/mL, Clone M17/4, BioXCell) or an IgG2aκ isotype control antibody (20 µg/mL, Clone RTK2758, BioLegend) at 37 °C for 5 min before the addition of target cells/stimulatory beads. The final concentration of antibodies during the assay was 10 µg/mL. Human LFA-1 was blocked using the anti-CD18 clone TS1/18 (20 µg/mL, Invitrogen), with a mouse IgG2a antibody (Sigma) serving as an isotype control. To induce T-cell activation independently of the TCR, CTLs were pre-incubated with phorbol myristate acetate (PMA, 20 ng/mL, Sigma Aldrich) and the Ca²⁺ ionophore A23187 (2 µM, Tocris Bioscience) at 37 °C for 5 min before the addition of target cells/stimulatory beads, yielding final concentrations of 10 ng/mL PMA and 1 µM A23187. Additional information about antibodies may be found in Supplementary Table 1.