Post-translational modification by glycosylation influences ADCC, and inhibition of N-glycan processing can enhance NK cell cytotoxicity by increasing CD16a antibody binding affinity. Here, we investigated the effects of kifunensine, a Golgi ɑ-mannosidase I inhibitor, on CD16a activation pathways and NK cell killing kinetics.

We used phospho-CD3ζ (pCD3ζ) as a proximal marker for CD16a activation. Using confocal microscopy, we found and increase in the phosphorylation and localization of Syk and CD3ζ at the ADCC immune synapse following kifunensine treatment. This increase is specific to ADCC and kifunensine does not increase natural cytotoxicity mediated by non-ADCC activating receptors. CD16a-mediated signal transduction also impacts the morphology of actin in treated cells, and sites of enhanced pCD3ζ  phosphorylation were sites of increased actin nucleation detected by confocal and super-resolution microscopy.

To determine if increased downstream signaling results in faster killing, we performed live-cell imaging and functional assays. These revealed that kifunensine-treated NK cells perform ADCC in approximately half the time and exhibit increased serial killing efficiency when compared to their untreated counterparts. Detailed analysis showed that each step involved in cytotoxicity is significantly faster in treated NK cells. Increased rates of serial killing include faster detachment from effectors following their first kill, making them more efficient killers.

In summary, our study shows that kifunensine-treated NK cells have higher levels of pCD3ζ and pSyk, reflecting increased CD16a activation and signal transduction during ADCC resulting in enhanced killing. These findings highlight the potential of glycosylation inhibition to enhance CD16a-mediated ADCC through improved signaling pathways, offering a promising approach to advance NK cell-based therapies.