ELISA-Based NK Cell Activation Assay

ELISA-based antibody-dependent NK cell activation assays were performed^{18 (link),62 (link)}. ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with PPD (300 ng/well) or BSA as a negative control at 4°C for 16 hrs. Plasma (at 1:100, 1:1000, 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 (5x10⁴ per well), anti-CD107a-phycoerythrin (PE)-Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added and incubated for 5 hrs at 37°C. Cells were stained for surface markers using anti-CD16–allophycocyanin (APC)-Cy7 (BD), anti-CD56-PE-Cy7 (BD), and anti-CD3-AlexaFluor 700 (BD), and intracellularly with anti-IFNγ-APC (BD) and anti-MIP1β-PE (BD) using Fix and Perm A and B solutions (ThermoFisher). NK cells were defined as CD3- and CD16/56+ (Extended data Figure 8C). NK cell activation assays were performed in across dilutions stated above using cells from four healthy HIV negative donors.