Figure 1
TLR2 agonist treatment leads to increased bone marrow phenotypic HSC frequency but loss of bone marrow repopulating activity. WT mice (6–8 weeks old) were treated with PAM3CSK4 (100 μg intraperitoneally (i.p.) q48 h × 3 doses, analyzed 24 h after final dose). Shown are the frequency (a) and absolute numbers (b) of bone marrow HSPCs as determined by flow cytometry using the gating strategy outlined in Supplementary Figure 1 (n= 12–13 mice per treatment group). (c) Whole bone marrow from PAM3CSK4-treated mice or water-treated controls (CD45.1) was transplanted in a 1:1 ratio with untreated competitor bone marrow (CD45.2) into lethally irradiated recipients (CD45.1/CD45.2). Shown are the frequency of donor cells (CD45.1+ single positive), including overall leukocytes, B cells (B220+), T cells (CD3e+) and neutrophils (Gr-1 hi), in the peripheral blood of primary recipients over time. (d) At 24 weeks post transplant, bone marrow from primary recipients was pooled and transplanted into secondary recipients. Shown is the frequency of donor cells (CD45.1+ single positive) in the peripheral blood of these secondary recipients over time. Data represent 8–12 recipient mice per treatment group from 2 independent experiments. *P<0.05; **P<0.01. (e) To assess homing, 2 × 106 whole bone marrow cells from PAM3CSK4-treated mice or water controls were transplanted into lethally irradiated recipients. Shown are the total number of KSL cells detected from the long bones (femurs, tibias, humerii) and pelvis 16 h later (n= 5 mice per group). For all panels, error bars represent mean±s.e.m., and P-values were determined by the two-tailed Student’s t-test.
