M40 deficiency leads to an expansion of phenotypic and functional HSCs. (A) Frequency of LT-HSCs in WT and M40^−/− mice as determined by flow cytometry. LT-HSC is defined as CD34⁻Flk2⁻CD150⁺LSK, and representative plots and gating strategy are shown. Each symbol represents an individual mouse. P value was determined by the 2-tailed Student t test. (B) Functional HSC frequency in WT and M40^−/− mice as determined by limiting dilution BMT. Percentage of donor-derived cells in peripheral blood 16 weeks after BMT were analyzed by flow cytometry. Data from 3 to 5 independent experiments were combined, and reconstitution frequency and statistical analysis were calculated using L-Calc software. The results are presented as number of positively engrafted mice vs total number of mice analyzed for the indicated doses. Positive engraftment was defined as >1% donor-derived cells in the peripheral blood. (C-D) Donor chimerism in total leukocytes (C) and different lineages (D) in peripheral blood of the recipient mice transplanted with 3 × 10⁴ and 1 × 10⁵ donor cells along with competitors are shown. Each symbol represents an individual mouse; horizontal lines indicate the mean value in each group. P values were calculated using the 2-tailed Student t test. *P < .05; **P < .01; ***P < .005. 1SE, 1 standard deviation; CRU, competitive repopulating unit.