Figure 3.

PLZF negatively regulates myeloid development in vivo as assessed in the NOD/SCID xenotransplant system. (A) Effect of PLZF on CD45⁺ human cell engraftment. Proportion of GFP⁺ BMCs in individual mice each denoted with a symbol at 8 wk post-transplant (mean marked as solid lines) compared with mean GFP positivity of input HSCs prior to transplantation (broken lines). Lin⁻ CB cells were transduced with control (clear triangles), PLZF knockdown (PLZF^KD), or CEP-PLZF (PLZF^OX) vectors (black triangles) and transplanted into NOD/SCID mice. The level of transduction in three independent experiments ranged from 5% to 12%; as a result, mice were transplanted with a mixture of transduced and nontransduced cells. Thus, equivalent means in the values of the pretransplant and post-transplant percentage of GFP in each experiment indicates a lack of competitive advantage of GFP⁺ cells relative to nontransduced cells, while increased or decreased percentage of GFP⁺ indicates competitive advantage or disadvantage, respectively, in vivo. (B) Frequency of human myeloid BMCs in mice transplanted with PLZF^KD cells (right) and a representative flow analysis (left). The frequency of granulocytes was calculated as the proportion of SSC^hi CD33^lo cells within the GFP⁺ CD45⁺ graft and monocytes as SSC^lo CD33^hi cells within the GFP⁺ CD45⁺ graft; the remaining cells were SSC^lo CD33⁻ lymphocytes. To obtain enough cells for accurate flow cytometric analysis, bone marrow was pooled from four to eight mice. Data are expressed as mean ± SEM of five independent experiments. (**) P < 0.01. (C) Expansion of the absolute number of human granulocytes, monocytes, and lymphoid cells in mice transplanted with PLZF^KD cells normalized to controls. The absolute number was calculated by multiplying the frequency of each cell type by the number of GFP⁺ CD45⁺ cells in both femurs, tibiae, and pelvis. Data are expressed as mean ± SEM of five independent experiments. (*) P < 0.02. (D) Representative analysis of the proportion of CD11b⁺ CD16⁺ human neutrophils in the GFP⁺ fraction of mice engrafted with Lin⁻ CB cells transduced with control, PLZF^KD, or PLZF^OX viruses. To obtain enough cells for accurate flow cytometric analysis, bone marrow was pooled from four to eight mice.