Ebf2⁺cell depletion accelerates AML development. (A) Experimental design. Eight- to 10-week-old Ebf2-Cre^ER x Rosa26-^loxpStop^loxp-Dta and control single-transgenic mice were injected with TAM to induce specific deletion of Ebf2⁺ cells either 1 month prior to or 1 week after the transplantation of CD45.1⁺MLL-AF9 AML cells without irradiation. Survival of the mice was monitored after transplantation. BM stromal cells from the control and AML mice were collected and analyzed by FACS at the onset of AML. (B) Predepletion of Ebf2⁺ cells led to reduced survival of depleted mice compared with that of nondepleted recipient mice after transplantation of AML cells. Because the TAM injection was given just 1 week after AML cell transplantation, all recipients were male mice to avoid TAM-induced effects. Survival differences were tested using a log-rank (Mantel-Cox) test in Prism 6.0. (C) Deletion of Ebf2⁺ cells after AML cell transplantation caused shorter latency and reduced survival of Ebf2⁺ cell–depleted recipients compared with nondepleted recipient mice. The difference in survival rate was tested using a log-rank (Mantel-Cox) test in Prism 6.0. (D) Representative FACS profiles showing AML engraftment and host-derived total cells and blood lineages, including T (CD4⁺/CD8⁺), B (CD19⁺), natural killer (NK1.1⁺), and myeloid (CD11B⁺GR1⁺) cells 22 days after transplantation. (E) Increased total AML cell engraftment in the PB of the Ebf2⁺ cell–depleted recipient. (F-G) Homing of AML cells into Ebf2⁺ cell–depleted recipient mice after transplantation. CD45.1⁺ AML cells (10 million per mouse) were transplanted into Ebf2⁺ cell–nondepleted (n = 5) and Ebf2⁺ cell–depleted (n = 4) mice via tail vein injection 1 month after TAM treatment. Blood, femur, and spleen of recipients were harvested 3 hours after transplantation, and homing of donor CD45.1⁺ AML cells were examined by FACS based on CD45.1 expression. (F) Representative FACS profiles showing AML cells in PB, BM, and spleen after transplantation. The numbers in the panels are the frequencies of donor AML cells among the total number of nucleated cells in each organ. (G) Frequencies of donor cells among the total number of nucleated cells in recipient PB, BM, and spleen. Differences were determined using an unpaired parametric Student t test or Mann-Whitney U test. Horizontal bars represent mean values in panels E,G. Data are from 2 independent experiments.