FIGURE 4.

NK cell activation, IFN-γ, and TNF-α release of the four leading ΔB7-H6 SEED-PGLALA immunoligands. Human purified NK cells were cocultured for 24 h with A431 cells at an E:T ratio of 5:1 prior to analysis of NK cell activation (A), IFN-γ (B), and TNF-α (C) release. Affinity-matured ΔB7-H6 NK cell engagers and oa_hu225-SEED-PGLALA were compared with wild-type ΔB7-H6_wt-SEED-PGLALA (all at 85 nM). Percentage of CD69⁺ NK cells was determined by double-staining of viable NK cells with CD56-PE and CD69-allophycocyanin using flow cytometric analyses. Human cytokine HTRF kits were used for the quantification of IFN-γ and TNF-α release of NK cells. Graphs show box and whisker plots as superimposition with dot plots of six (A) and eight (B and C) individual experiments, respectively. ***p < 0.001, **p < 0.01, *p < 0.05. n.s., not significant compared with ΔB7-H6_wt-SEED-PGLALA.