Antibody-Dependent NK-Cell Activation Assay

ELISA-based, antibody-dependent, NK-cell activation assays were performed^{17 (link),61 (link)}. ELISA plates (ThermoFisher NUNC MaxiSorp flat bottom) were coated with PPD (300 ng per well) or BSA as a negative control at 4 °C for 16 h. Plasma (at 1:100, 1:1,000, 1:10,000 dilutions in PBS) was added to each well. NK cells were isolated from whole blood from healthy HIV-negative donors with RosetteSep (Stem Cell Technologies). NK cells (5 × 10⁴ per well), anti-CD107a-phycoerythrin-Cy5 (BD Biosciences), Brefeldin A (10 mg ml^–1) (Sigma) and GolgiStop (BD Biosciences) were added and incubated for 5 h at 37 °C. Cells were stained for surface markers using anti-CD16–allophycocyanin-Cy7 (BD), anti-CD56-phycoerythrin-Cy7 (BD) and anti-CD3-AlexaFluor 700 (BD Biosciences), and intracellularly with anti-IFN-γ-APC (BD Biosciences) and anti-macrophage inflammatory protein-1β-phycoerythrin (BD Biosciences) using Fix and Perm A and B solutions (ThermoFisher). NK cells were defined as CD3^– and CD16/56⁺ (Extended data Fig. 8c). NK-cell activation assays were performed across the dilutions stated above using cells from four healthy HIV-negative donors.