We are building techniques and protocols to localize Fc gamma RIIIa (CD16) using MINFLUX. CD16 is the primary activating antibody receptor on the surface of NK cells and understanding how antibody phenotype alters antigen spacing and interaction with CD16 will shed light on the mechanisms guiding cellular cytotoxicity. The precision of MINFLUX promises the most detailed images of the the NK cell ADCC synapse yet seen.

After antibody receptors bind to immune complexes on the cell surface, intracellular kinases, such as Syk and Zap70, are then primed to phosphorylate activating domains associated with CD16, which initiates signaling cascades necessary for cytotoxicity to occur. Using 2-color labeling with MINFLUX and imaging in 3D, we will determine if immune complex geometry influences interaction with early signaling kinases and correlates with ADCC.

Upon recognition of a virally infected cell, the antibody receptor CD16 forms clusters and moves dynamically within the developing immune synapse of NK cells, eventually leading to toxin deposition to the target. We are exploring how antibody phenotypes, such as immune complex geometry, alter the movement of single CD16 molecules within active synapses, and finding ways to quantify this data to extract correlates with ADCC activity.

How does a cell distinguish between an unbound IgG and an opsonized target? Generally, antibody clustering due to cross linking within immune complexes is thought to increase FcGR binding through avidity effects. However, there are several lines of evidence that suggest the process of FcGR-based activation is more organized. We are exploring how antigen binding alters FcGR affinity through allosteric changes in the Fc and exploring how FcGR receptors are organized within the NK cell immune synapse.

Human IgG1 is naturally bivalent, in that it has two equivalent antigen binding arms. The bivalent nature of IgG can facilitate the clustering of antigen to promote clearance by effector cells. In some cases, especially with multivalent viral spikes, an IgG can binding bivalently to a single antigen. We are exploring how this contributes to FcGR binding and cellular cytotoxicity and if bivalency contributes to FcGR organization within the immune synapse.