Sirpa^hu/humacrophages bind human CD47 more strongly than do Sirpa^NOD/NODmacrophages, and they also recognize mouse CD47. (A) Representative flow cytometric plots of peritoneal lavage in B6-Sirpa^W/W and B6-Sirpa^hu/hu mice. Mac-1⁺ peritoneal macrophages from B6-Sirpa^hu/hu mice express human SIRPA but not mouse SIRPA. (B) Results of dose-dependent binding assays of human CD47-Fc fusion protein to B6-Sirpa^W/W, B6-Sirpa^NOD/NOD, B6-Sirpa^hu/W, and B6-Sirpa^hu/hu mouse macrophages. Sirpa^hu/hu macrophages had the strongest binding affinity for human CD47 (Bmax, 3.02 ± 0.19), and Sirpa^hu/W or Sirpa^NOD/NOD macrophages had intermediate levels of affinity (Bmax, 1.69 ± 0.23 and 1.80 ± 0.06, respectively), whereas Sirpa^W/W macrophages did not bind to human CD47. (C) Flow cytometric analysis of macrophage binding to human CD47-Fc fusion. (D) Phagocytosis of opsonized human Lin⁻CD34⁺CD38⁻ HSCs by mouse macrophages. The phagocytic index was calculated as the number of engulfed human cells per 100 macrophages. Data are mean ± standard deviation. (E) Dose-dependent binding of mouse CD47-Fc to macrophages. Sirpa^hu/hu macrophages had moderate binding affinity for mouse CD47 (Bmax, 0.29 ± 0.02). Chinese hamster ovary (CHO) cells that did not express mouse or human SIRPA were used as a negative control. *P < .05, **P < .01. SSC, side scatter.