Figure 1

B7-H4 promotes the differentiation of LICs to inhibit leukemogenesis by downregulating the RCOR2 level. (a) Survival was compared between the recipients transplanted with MLL-AF9-infected WT and B7-H4-null (KO) hematopoietic stem/progenitors upon primary transplantation (first, n=7–8, log-rank test, first panel), and mice receiving 10 000 WT or B7-H4-null YFP⁺ door leukemia cells from the primary (second, second panel) and secondary (third, third panel) recipients (n=5–6, log-rank test). Limiting dilution assays comparing the frequencies of LICs in WT and B7-H4-null BM cells. The indicated numbers of YFP⁺ leukemia cells isolated from secondary recipients were co-injected with 2 × 10⁵ competitor cells into lethally irradiated recipients (fourth panel). The competitive repopulating units (CRUs) were evaluated using L-Calc software and indicated. (b) Representative flow cytometric analysis of the YFP⁺Mac-1⁺Gr-1⁺ and YFP⁺Mac-1⁺Gr-1^- leukemia cells in the peripheral blood of primary recipients receiving transplants of MLL-AF9-transduced WT or B7-H4-null hematopoietic stem/progenitors (first panel), and qualification of frequencies of YFP⁺Mac-1⁺Gr-1⁺ and YFP⁺Mac-1⁺Gr-1⁻ leukemia cells in the peripheral blood (PB) of primary, secondary and tertiary recipients are shown (n=5, second panel). (c) Potential candidate genes related to transcription factors (important for stemness) and myeloid differentiation were validated in fluorescence activated cell sorting-purified WT or B7-H4-null YFP⁺Mac-1⁺c-Kit⁺ LICs using quantitative Reverse transcription-polymerase chain reaction analysis (n=3, first panel), and protein levels of RCOR2 in WT and B7-H4-null BM AML cells were measured using immunoblotting (n=3, second panel). Rcor2 was silenced in B7-H4-null AML cells using shRcor2#2, which were transplanted into the recipient mice. The survival was analyzed among the mice receiving WT cells, B7-H4-null cells and Rcor2-knockdown B7-H4-null cells (n=12–18, log-rank test, third panel). mRNA, messenger RNA.