CXCR4 expression in egress-competent immature B lymphocytes. (A) CXCR4 expression in B220⁺ immature IgM⁺ IgD^lo B cells positioned in BM parenchyma (Par.) and sinusoids (Sin.). (left) IgM and IgD expression in B220⁺ BM cells. (middle) Distribution of B220⁺ CD93⁺ IgM⁺ IgD^lo B cells in BM parenchyma (CD19-PE⁻) and sinusoids (CD19-PE⁺). (right) CXCR4 expression in B220⁺ immature IgM⁺ IgD^lo B cells in parenchyma (gray) and sinusoids (black line). (B) Geometric mean fluorescence intensity (MFI) of CXCR4 surface expression in BM parenchyma and sinusoid immature CD93⁺ IgM⁺ IgD^lo B cells from C57BL/6 mice (n = 4). Bars indicate the mean; circles indicate individual mice. Data are representative of >10 independent experiments. (C) Cell migration through 5-µm transwells. Cxcr4^+/+ (gray bars) and Cxcr4^+/− (black bars) immature CD93⁺ IgM^hi IgD⁻ (left) and IgD^lo (right) cells were allowed to migrate toward 0.3 µg/ml CXCL12 for 3 h. Bars indicate mean, and circles indicate replicates. Data were pooled from two independent experiments. (D, left) CXCR4 surface expression in Mb1^Cre/+ Cxcr4^Fl/+ immature IgM⁺ IgD^lo cells overlaid with Mb1^Cre/+ Cxcr4^+/+ immature IgM⁺ IgD^lo cells in BM parenchyma. (right) CXCR4 surface expression in Ly5.1⁺ Mb1^Cre/+ Cxcr4^Fl/+ immature IgM⁺ IgD^lo cells in BM parenchyma overlaid with Ly5.2⁺ (Cxcr4^+/+) immature IgM⁺ IgD^lo cells residing in sinusoids. (E) Distribution of B220⁺ CD93⁺ IgM⁺ IgD^lo immature B cells in BM parenchyma, sinusoids, and PB of lethally irradiated mice reconstituted with a mixture of Ly5.1⁺ Mb1^Cre/+ Cxcr4^+/+ and Ly5.2⁺ WT cells (gray) or with Ly5.1⁺ Mb1^Cre/+ Cxcr4^Fl/+ and Ly5.2⁺ WT cells (black). Error bars indicate ±SEM. (F) Overexpression of truncated CXCR4 (R334X WHIM mutation) in BM cell subsets reported by GFP expression. (left) GFP expression in gated B220⁺ CD93⁺ IgM⁺ IgD^lo immature B cells. (middle) CXCR4 surface expression in gated GFP⁻ (gray) and GFP⁺ (black line) B220⁺ CD93⁺ IgM⁺ IgD^lo immature B cells. (right) Distribution of CXCR4-transduced (GFP⁺) B220⁺ CD93⁺ IgM⁺ IgD^lo immature B cells in BM parenchyma and sinusoids. (G) Ratio of the frequencies of GFP^hi immature IgD^lo and mature B cells in PB and in BM. Bars indicate mean; circles indicate individual mice. Data were pooled from three independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 by unpaired Student’s t test.