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. 2014 Aug 4;111(33):12151–12156. doi: 10.1073/pnas.1407370111

Fig. 4.

Fig. 4.

Overexpression of Cbfβ in Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells restored B-1 and MZ cell engraftment. (A) Cbfβ expression in WT fetal-liver HSCs and B-1 progenitors by quantitative PCR is shown. Internal control: β-actin = 1.0. (B) Tek-GFP/Cbfβ expression within E15.5 fetal-liver AA4.1+CD19+B220dim B-1 progenitor cells in Cbfβ−/−:Tek-GFP/Cbfβ embryos. Red line, WT; blue line, Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver B-1 progenitor cells. (C) Relative Cbfβ expression in E9.5 WT CD31+Tie2+ HECs, E9.5 Cbfβ−/−:Tek-GFP/Cbfβ HECs, E14.5 WT FL B-1progenitors, and E14.5 Cbfβ−/−:Tek-GFP/Cbfβ FL B-1 progenitors, compared with E9.5 WT HECs (=1). Open bar, WT; filled bar, Cbfβ−/−:Tek-GFP/Cbfβ. (D) Experimental design for rescuing Cbfβ expression in Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells. (E) Peritoneal cells of recipient mice at 12 wk after transplantation with fetal-liver cells from WT (Top) or Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells expressing empty vector (Middle) or Cbfβ (Bottom), respectively. (F) Donor-derived B-cell subsets in the recipient peritoneal cavity (Left) and spleen (Right) are depicted. Donor cells were WT fetal-liver MNC cells (left bars), Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells (center bars), and Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells infected with Cbfβ expressing retrovirus (rescue, right bars), in each panel. (G) IgM secretions from B-1 or B-2 cells stimulated with phosphorylcholine were detected by Elispot assay: (a) 1,000 B-1 cells from the recipient mice transplanted with WT fetal-liver cells with empty vector, (b) 650 B-1 cells derived from Cbfβ−/−:Tek-GFP/Cbfβ fetal-liver cells with Cbfβ expression vector, and (c) 1,000 B-2 cells derived from WT fetal-liver cells with empty vector were initially plated. (H) The numbers of spots per 1,000 B-1 or B-2 cells derived from recipient mice in Elispot assay (G, a, b, and c) are depicted. There was no significant difference between B-1 cells from WT fetal liver with empty vector and from Cbfβ−/−:Tek-GFP/Cbfβ fetal liver with Cbfβ expression vector. The number of IgM-secreting B-2 cells was significantly less than that of B-1 cells from WT or Cbfβ−/−:Tek-GFP/Cbfβ with Cbfβ expression vector (<0.05).