CD47 overexpression in K562 cells . (A) CD47 overexpression was achieved with lentiviral particles and the relative surface expression on K562 CD47 tg was assessed in flow cytometry (mean ± SD, three independent samples per group, Student’s t test). (B–D) CD3⁻CD7⁺CD56⁺ primary NK (B), NKL (C), or NK92 (D) cells were stimulated with IL-2 for 72 h before they were added to Fluc⁺ K562 or K562 CD47 tg, and target cell killing was assessed in BLI assays (mean ± SD, three independent samples per group, Student’s t test). (E) Fluc⁺ K562 cells were transplanted into NSG mice, which received adoptive transfer of IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells. Cell survival was longitudinally monitored using BLI (five mice; each line represents one mouse). (F–G) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice, and cell survival was longitudinally monitored using BLI (five mice per graph; each line represents one mouse). IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells were used as effector cells without blocking CD47 (F) or with CD47 antibody block and FcR block (G; clone B6.H12). (H and I) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice with (H) or without (I) CD47 antibody block but without transfer of human NK cells (three mice per graph; each line represents one mouse). (J) Fluc⁺ K562 CD47 tg cells were transplanted into NSG mice that received IL-2–treated CD3⁻CD7⁺CD56⁺ primary NK cells (five mice; each line represents one mouse). Free Fab fragments of the CD47 antibody (clone B6.H12; H) were used to block CD47 interactions. (K) IL-2–treated NK92 effector cells were transferred in conjunction with the CD47 blocking antibody in mice transplanted with Fluc⁺ K562 CD47 tg cells (five mice; each line represents one mouse).