The protective effect of Cd47 overexpression against NK cell and macrophage killing is mediated through Sirpα. (A) Sirpα expression on C57BL/6 macrophages and time course of Sirpα expression on C57BL/6 NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (B) Cd47 binding to C57BL/6 macrophages and time course of Cd47 binding to C57BL/6 NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (C) Sirpα expression on naive C57BL/6 NK cells and 48 h after in vivo stimulation with i.p. mouse IL-2 (mean ± SD, six independent experiments per group, Student’s t test). (D–I) A 1:1 mixture of CFSE-labeled WT and B2m^−/−Ciita^−/− Cd47 tg miPSCs was injected into the peritoneum of syngeneic C57BL/6 mice and after 48 h, and the ratio of recovered CFSE-positive miPSCs was determined (mean ± SD, triplicates in four animals per group). Animals after NK cell depletion received an anti-Cd47 blocking antibody (clone BE0270) with the miPSC injection (D). Animals after macrophage depletion received an anti-Cd47 blocking antibody with the miPSC injection (E) and additionally mouse IL-2 to activate NK cells in vivo (F). Animals after NK cell depletion received an anti-Sirpα blocking antibody (clone P84; G). Animals after macrophage depletion received an anti-Sirpα blocking antibody (H) and additionally mouse IL-2 to activate NK cells in vivo (I). (J) Sirpα expression on Sirpa^−/− macrophages and time course of Sirpα expression on Sirpa^−/− NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (K) Cd47 binding to Sirpa^−/− macrophages and time course of Cd47 binding to Sirpa^−/− NK cells stimulated with mouse IL-2 (mean ± SD, four independent experiments per group, ANOVA). (L–O) In some Sirpa^−/− mice, NK cells were depleted (L). In other Sirpa^−/− mice, macrophages were depleted, and NK cells were stimulated with mouse IL-2 (M); some animals in addition received a blocking antibody for Sirpα (N) or Cd47 (O). Graphs show mean ± SD and triplicates in four animals per group. (P) WT, B2m^−/−Ciita^−/−, and B2m^−/−Ciita^−/− Cd47 tg miECs were challenged with Sirpa^−/− macrophages (mean ± SD, three independent replicates per group and time point, and three different E:T ratios). (Q)B2m^−/−Ciita^−/− Cd47 tg miECs were challenged with mouse IL-2–stimulated C57BL/6 NK cells or Sirpa^−/− NK cells. In some groups, an anti-Cd47 or anti-Sirpα blocking antibody was used (mean ± SD, three independent replicates per group and time point, and three different E:T ratios). (R) IFNγ ELISpot assays with B2m^−/−Ciita^−/− Cd47 tg miEC target cells and either mouse IL-2–stimulated C57BL/6 or Sirpa^−/− NK cells. Yac-1 served as controls (boxes show 25th to 75th percentile with median, and whiskers show minimum to maximum; six independent samples per group, Student’s t test). wt, wild-type.