BCR signaling increases CXCR4 expression, promotes B lymphocyte motility, and prevents BM egress. (A) IgM and IgD expression in unstimulated and BCR-stimulated B220⁺ cells (left and right gates, respectively). (B) CXCR4 mean fluorescence intensity (MFI) in IgM⁺ IgD^lo CD93⁺ MD4 B cells untreated (black) or stimulated with 1 mg/ml HEL (gray) for the indicated time periods. Lines indicate mean (±SEM) of three independent experiments. (C) CXCR4 surface expression in unstimulated (black) and in vivo HEL-stimulated (6 h; gray) immature (CD93⁺) MD4 B cell subsets. (D) MD4 immature IgM⁺ IgD^lo CD93⁺ B cell distribution in BM before and after HEL i.v. treatment for 6 h. (E) Quantification of immature B cell subsets in BM parenchyma (Par.), sinusoids (Sin.), and PB of MD4 mice treated with HEL for 6 h. (F) CXCR4 surface expression in WT immature IgM⁺ IgD^lo CD93⁺ B cells stimulated with PBS (black) or with HEL i.v. (gray). (right) CXCR4 MFI on unstimulated (black) and HEL-stimulated (gray) immature IgM⁺ IgD^lo CD93⁺ B cells from WT mice. (G) Quantification of immature B cell subsets in BM parenchyma, sinusoids, and PB of WT mice treated with HEL for 6 h. (H, left) Distribution of immature IgM⁺ IgD^lo CD93⁺ MD4 B cells overexpressing Bcl2 in BM parenchyma (CD19-PE⁻) and in sinusoids (CD19-PE⁺). (right) Immature CD93⁺ IgM⁺ IgD^lo B cell numbers in BM parenchyma, sinusoids, and PB of MD4.BCL2 transgenic mice treated with HEL. (E, G, and H) Bars indicate mean, and circles depict individual mice. Data are representative of two independent experiments. (I) Mean motility coefficient of MD4⁺ and MD4⁻ Cd19^Cre/+ Rosa26^ZsGreen/+ B lineage cells before and 6 h after treatment with HEL i.v. Lines depict the average mean motility coefficient calculated from datasets obtained by IVM of two to three mice. (J) Axis ratio (x/y) of MD4⁺ and MD4⁻ Cd19^Cre/+ Rosa26^ZsGreen/+ B lineage cells before and 6 h after HEL treatment i.v. Circles, unstimulated MD4⁺ B cells; squares, HEL-stimulated MD4⁺ B cells; triangles, HEL-stimulated MD4⁻ B cells; diamonds, MD4⁻ unstimulated B cells. Lines indicate mean. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.005; ****, P < 0.00005 by unpaired Student’s t test.