CD16a pairs form the basal molecular subunit for the NK-cell ADCC lytic synapse

NK cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. While antibody-induced clustering of FcγRIIIa (CD16a) is thought to drive ADCC, the molecular basis for this activity has not been fully described. We used MINFLUX nanoscopy to map the spatial distribution of CD16a within the NK-cell ADCC immune synapse. In both resting and NK cells activated on supported lipid bilayers by Trastuzumab, we detected pairs of CD16a molecules approximately 18 nm apart that could be homodimers. NK-cell activation results in a modest increase of clusters of 4 or more CD16a localizations without a change in cluster characteristics, while CD16a pair distances do not significantly change, suggesting that subtle structural changes underpin ADCC-based activation. Our results provide the highest spatial resolution yet described for CD16a imaging, offering insight into how CD16a organization within the immune synapse could influence ADCC activity.