Fig. 1

The defect of B cell development in Lf^−/− mice is both cell autonomous and is associated with the bone marrow microenvironment. a Lactoferrin deficiency leads to imbalance of hematopoiesis. Cells were isolated from the bone marrow (BM), peripheral blood (PB), and spleens (SP) of Lf^−/− mice and WT littermates. Frequencies of indicated cells were identified by flow cytometry. All immune cells were firstly gated on CD45⁺. Each group has 11 mice. b Splenic B cells were sorted from WT and Lf^−/− mice. The amount of apoptosis cells was detected by flow cytometer. Representative data from three independent experiments are shown. c, e Representative strategy of flow analysis of c HSC, CLP, CMP and e pre-pro-B, pro-B, pre-B, immature B cells. Cells were isolated from the mice bone marrow. d, f Frequencies of d HSC (Lin⁻ IL7R⁻ C-kit⁺ Sca-1⁺), CLP (Lin⁻ IL7R⁺ C-kit^lo Sca-1^lo), CMP (Lin⁻ IL7R⁻ C-kit⁺ Sca-1⁻) and f pre-pro-B (AA4.1⁺B220⁺CD19⁻CD24⁻), pro-B (B220⁺CD43⁺IgM⁻), pre-B (B220⁺CD43⁻IgM⁻), immature B (B220⁺IgM⁺) cells were identified by flow cytometry. Each group has 11 mice. (note, CD117 is C-kit, CD93 is AA4.1.) g mRNA expression of lactoferrin in distinct stages of developing B cells from WT mouse bone marrow was evaluated by RT-qPCR. h, i In vitro B cell differentiation experiment: purified pre-pro-B cells from Lf^−/− or WT mice were cocultured with OP9 stromal cells in the presence of IL-7 (10 ng/ml), SCF (5 ng/ml), and Flt3L (5 ng/ml) for 9 days. h Representative data from eight specimens each group are shown. i The proportions of pro-B cells generated were then determined by flow cytometric analysis. j Bone marrow transplantation experiment: bone marrow cells from either WT or Lf^−/− (CD45.2⁺) mice with bone marrow from syngenic mice (CD45.1⁺) were mixed at a 1:1 ratio. The recipient WT mice (CD45.1⁺) were irradiated in fractionated doses (5 Gy × 2), and 16 h later, the recipient mice were injected with mixed cells (2 × 10⁶ cells). After 6 weeks, the recipient mice were killed to prepare the bone marrow single-cell suspension, and the B cell proportion of each stage of B cell differentiation was analyzed by flow cytometry. Representative data from six mice each group are shown. k Bone marrow transplantation experiment: bone marrow cells from CD45.2⁺ WT mice or from CD45.1⁺ WT mice were mixed at a 1:1 ratio, while WT or Lf^−/− mice with CD45.2⁺ genetic background were used as recipient mice. The rest of the operation was the same as j. Representative data from six mice each group are shown