Fc modification augments NK-cell activation. (A) Gating strategy for the evaluation of NK-cell activation. RhD^pos RBCs were incubated with mAbs. NK cells obtained from 3 to 4 healthy donors were added to opsonized RBCs in the presence of brefeldin A and GolgiStop, anti-CD107a (APC H7), anti-CD3 (V500), anti-CD56 (PE-Cy7), and anti- IFN-γ (AF700). Activation was measured by evaluating intracellular IFN-γ production and/or the CD107a degranulation marker expression in NK cells (CD3^– CD56^dim). (B) The bar graphs show percentage of CD107⁺ cells, IFNγ⁺, or total activated NK cells. C107⁺ cells are represented by the combined populations of Q2 and Q3, IFNγ⁺ cells by Q1 and Q2, and total activated NK cells by Q1, Q2, and Q3 combined. Data are the average of 3 replicates, and error bars denote SEM. Total NK-cell activation was calculated with subtraction of “no-Ab” control background. Data show the average of 3 replicates, and error bars denote SEM. Statistical analyses were conducted using 1-way ANOVA, followed by Dunnett multiple comparison test (GraphPad Prism v9). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Data are the average of 3 replicates, and error bars denote SEM. FSC-A: Forward scatter area; Q: Quarter; SSC-A: Side scatter area.