The membrane distal domain of CD16a shapes the NK cell ADCC immune synapse 2309381

Introduction Natural Killer (NK) cells are a powerful and versatile part of the human immune system. The molecular mechanisms of their antibody reliant response, antibody dependent cellular cytotoxicity (ADCC), are not fully understood. The cell surface receptor CD16a is known to bind to IgG at the membrane proximal domain (D2), but the role of the membrane distal domain (D1) is unknown. Methods Using two nanobodies (C21 and C28) that bind to CD16a D1 at distinct epitopes without blocking IgG binding, we were able to probe the role of the CD16a D1 in shaping the NK cell immune synapse during ADCC. Using real-time ADCC analysis, we examined how binding of these nanobodies altered NK cell ADCC activity toward Her2+ SKOV3 cells treated with Trastuzumab. We subsequently used confocal and stimulated emission-depletion (STED) imaging of NK cell immune synapses on supported lipid bilayers to determine the role of CD16a D1 in synapse structure and CD3-zeta (CD3z) phosphorylation. Results We found that ADCC activity is disrupted in a dose-dependent manner in the presence of C28 but not C21. Our STED imaging and analysis revealed that ADCC ablation was due to disruption of CD3z phosphorylation, disruption of normal NK cell attachment, and alteration of actin synaptic structure. Conclusion Our data support the hypothesis that CD16a D1 may be involved in signaling with an unknown co-activating receptor/protein, explaining why blocking it has negative effects on ADCC and synapse formation. Our data could point to a universal function of membrane distal domains, which are structurally conserved across all Ig receptors. Further, our findings have implications for antibody and cellular therapeutic engineering efforts aimed at enhancing in vivo NK cell ADCC activity during treatment for broad types of cancer and viral infections.