Perm b

We used a modified ELISA-based protocol for detection of CD107a as a surrogate marker of NK cell–mediated cytolysis^{[40 (link)]}. This assay has previously been shown to be associated with FcγRIIIa binding capacity of antigen-specific antibodies^{[41 , 42 (link)]}. A 96-well plate was coated overnight at 4°C with 150ng of recombinant protein per well,. 2% BSA blocked plates were used as antigen controls. The next day the plates were washed 6 times with PBS, 50μl of plasma (diluted 1:100) was added to each well, and incubated at 37°C for 2 hours. HIV negative plasma samples or media alone were used as negative controls, while HIVIG (pooled HIV Immunoglobulin G, NIH AIDS Reagents Program) was used as a positive control. The plates were washed and 5 × 10⁴ NK cells enriched via negative selection from healthy blood donors (RosetteSep, Stemcell Technologies,) were added to each well in the presence of Brefeldin A (Biolegend), Golgi stop, and anti-CD107a-PE-Cy5 (BD Biosciences). The plate was incubated for 5 hours at 37°C and 5% CO₂. Following incubation, cells were stained with anti-CD3-AF700, anti-CD56-PE-Cy7, anti-CD16-APC (BD), fixed with Perm A, permeabilized using Perm B (Invitrogen), and stained with anti-IFNγ-APC and anti-MIP1β-PE (BD). The cells were then fixed with 2% paraformaldehyde and analyzed by flow cytometry (gating strategy shown in Supp Figure 2).