Soluble FLT3 ligand is increased, but the myeloproliferative phenotype of Flt3^ITD/ITD mice is FL-independent. (A) FL serum levels in Flt3^+/+, Flt3^+/ITD, Flt3^ITD/ITD, and Flt3^−/− mice, on an Fl^+/+ or Fl^−/− background, as determined by ELISA. Mean (SD) values of 2-7 mice (8-10 weeks old) investigated per genotype. (B) Representative FACS analysis of FLT3 expression in LSK CD34⁺ lymphoid-primed multipotent progenitors (LMPPs) and LKS⁻ CD41⁻CD16/32^+/− myeloid progenitors in BM of Flt3^+/+ and Flt3^ITD/ITD mice. Numbers in the graph represent mean frequencies (of total BM cells) of cell populations within indicated quadrants, with 2-5 mice analyzed of each genotype. (C-E) Analysis of BM GMPs (C), BM LSKs (D), and Mac1^low/+ c-Kit^low/+ myelomonocytic immature cells in spleen and BM (E) of Flt3^+/+, Flt3^+/ITD, and Flt3^ITD/ITD mice on a WT Fl^+/+ or Fl^−/− background. Mean (SD) results from 5-15 mice (8-9 weeks old) of each genotype. *P < .05. ***P < .001. (F) In vitro cytokine (FL and IL-3) responsiveness of LSK cells purified from BM of WT and FLT3-ITD mice. Clonal growth was scored after 8 days of culture. Mean (SD) values from 2 experiments, with 120 cells plated per group in each experiment. Open bars represent cloning frequencies; and black bars, frequency of high proliferative clones (covering > 50% of the well). (G) A total of 0.3 × 10⁶ unfractionated BM cells from Flt3^ITD/ITD and Flt3^ITD/ITDxFl^−/− mice were cultured in methylcellulose with 50 ng/mL G-CSF, in the absence of any further additions (control), together with the vehicle (dimethyl sulfoxide [DMSO]) or 50nM CEP701, and scored for colony formation 7 days later. Mean (SD) results from 2 independent experiments.