Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC

Abstract Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N -glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N -glycan processing increased ADCC. We reduced N -glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N -glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N -glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this “affinity profiling” approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.