Importance of both hematopoietic and nonhematopoietic CD47 for homeostasis of splenic CD4⁺ cDCs and DN cDCs. (A) Splenocytes from WT or CD47 KO mice were stained as in Figure 1A, and the expression of CD4 and CD8 on cDCs was analyzed by 5-color flow cytometry (left panel). The relative numbers of CD8⁺, CD4⁺, and DN cDCs are expressed as a percentage of PI-negative cDCs in each plot. The absolute numbers of CD8⁺, CD4⁺, and DN cDCs in the spleen were also determined (right panel); data are means ± SE from 3 mice per group and are representative of 3 independent experiments. *P < .05, **P < .01 (Student t test). (B) B6-Ly5.1 mice were subjected to sublethal irradiation and then reconstituted with 5 × 10⁶ BM cells from either WT or CD47 KO mice on day 0 (BMT). MDPCl₂ entrapped in liposomes (Lipo-MDP) was administrated intravenously on days −2, 3, 6, and 11, as indicated in the top panel, to prevent initial rejection of CD47 KO donor cells by recipient macrophages. Splenocytes were prepared from recipient mice on day 28 and stained as in Figure 3A. The expression of CD4 and CD8 on cDCs was analyzed by 6-color flow cytometry (bottom left panel). The relative numbers of CD8⁺, CD4⁺, and DN cDCs are expressed as a percentage of Ly5.1⁻ cDCs on each plot. The percentages of CD8⁺, CD4⁺, and DN cDCs among total PI-negative Ly5.1⁻ cells were also determined (right panel); data are means ± SE from 5 or 6 mice per group in 2 independent experiments. **P < .01 (Student t test). (C) WT or CD47 KO mice were subjected to lethal irradiation and reconstituted with 5 × 10⁶ BM cells from B6-Ly5.1 mice. Eight weeks after transplantation, splenocytes were prepared from recipient mice and stained as in panel B. The expression of CD4 and CD8 on cDCs was analyzed by 6-color flow cytometry (left panel). The relative numbers of CD8⁺, CD4⁺, and DN cDCs are expressed as a percentage of total cDCs in each plot. The percentages of CD8⁺, CD4⁺, and DN cDCs among total splenocytes were also determined (right panel); data are means ± SE from 3 mice per group and are representative of 2 independent experiments. *P < .05, **P < .01 (Student t test).