Reconstitution of B-lineage development in MBS-treated BtkTec^–/– mice. BM, splenocytes, peripheral blood, and peritoneal cells were harvested from BtkTec^–/– recipient mice 3 to 5 months after transplantation, evaluated by flow cytometry, and characterized based on “Hardy Fraction” stages of B-cell development. (A) BM (harvested 21 weeks after transplantation) was stained for B220, CD43, and IgM. Levels of Fractions A to C (pro-B) and Fraction D (pre-B), presented as percentages of the B220⁺ B-cell compartment, are shown in the upper panel (CD43/B220, gated on IgM^– cells). Fractions E to F (immature B, mature B) are shown in the lower panel (IgM+/B220, gated on live cells as determined by forward and side scatter profiles), and also as percentages of B220⁺ cells. (B) Splenocytes (harvested 15 weeks after transplantation) were stained for B220, IgM, and IgD. B220⁺ cells, shown as percentages of total splenocytes, are shown in the upper panel (IgM/B220, gated on live cells). Late-stage splenic B-cell profiles are shown in the lower panel (IgM/IgD, gated on B220⁺ cells); the sequence of maturation is from Fraction FIII (IgM^hi/IgD^lo) to FII (IgM^hi/IgD^hi) to FI (IgM^lo/IgD^hi) as percentages of B220⁺ B cells. (C) Peripheral blood (collected 21 weeks after transplantation) was analyzed by the same parameters as for splenocytes. (D) Representative plots of peritoneal cells collected from BtkTec^–/– transplant recipients 21 weeks after transplantation and stained for IgM, Mac1, and CD5 are shown. Total B1 cells are shown in the upper panel (IgM/Mac1, gated on live cells); percentages are relative to total peritoneal cells. Further resolution into CD5⁺ B1a and CD5^– B1b, also presented as a percentage of the total peritoneal population, is shown in the lower panel (CD5/Mac1, gated on IgM^– cells). Representative data from 2 of 3 independent experiments are shown.