Threshold of CD47 expression for NK cell and macrophage inhibition. (A) SIRPα expression on macrophages and time course of SIRPα expression on highly selected CD3⁻CD7⁺CD56⁺ primary NK cells stimulated with IL-2 (mean ± SD, four independent experiments per group, ANOVA). (B) CD47 binding to macrophages and time course of CD47 binding to highly selected CD3⁻CD7⁺CD56⁺ primary NK cells stimulated with IL-2 (mean ± SD, four independent experiments per group, ANOVA). (C and D) From the pool of B2M^−/−CIITA^−/− CD47 tg hiECs, five clones with different levels of CD47 overexpression were selected. The CD47 expression on transduced cells was always higher than the basal expression level on WT hiECs but below or above the mean of the pool. CD47 was quantified by RT-PCR (C; mean ± SD, three independent experiments per group) and fluorescence (D; mean ± SD, four independent experiments per group). (E and F) The five B2M^−/−CIITA^−/− CD47 tg hiEC clones were challenged with IL-2–activated human primary NK cells (E) or human macrophages (F). Graphs show mean ± SD and three independent replicates per group and time point; three different E:T ratios are shown. (G) A 1:1 mixture of CFSE-labeled WT and one of the B2M^−/−CIITA^−/− CD47 tg hiEC clones was injected into the peritoneum of immunodeficient NSG mice. Additionally, either IL-2–activated primary NK cells or macrophages were coinjected. After 48 h, the ratio of recovered CFSE-positive hiECs was determined (mean ± SD, triplicates in four animals per group). wt, wild-type.