American Society of Hematology

Figure 2.

$Figure 2. Chemokine receptor expression and chemokine responsiveness of wt and CD19-/- B-cell precursors. Wild-type and CD19-/- B-cell precursors were grown in BM cultures. (A) B-cell precursors were fractionated into pro-/pre- (IgM-), immature (IgM+IgD-), and transitional (IgM+IgD+) populations. Mature B cells were isolated from spleen. Cells were lysed rapidly, and the expression of CXCR4 and CXCR5 was determined for each sorted population by RT-PCR. Results shown are representative of 5 different experiments. Actin blot is shown as an internal control gene expression. (B) Quantification of CXCR4 and CXCR5 expression. Results are presented as a semiquantitative estimate, where signal intensity of CXCR4 or CXCR5 products was normalized to that of β-actin. □ indicates wt; ▦, CD19-/-. Results of 5 experiments are presented as mean ± SE. Asterisk indicates values that are statistically different (P < .05). (C) Total cell migration of wt and CD19-/- B-cell precursors. Cells grown in BM cultures were tested for migration capabilities to BLC (2 mg/mL, left, ▦) or to SDF1-α (250 ng/mL, right, ▦) using transwell inserts. □ indicates medium. Migrating cells were counted by FACS, and the percentage of migration was calculated as: no. migrating cells/no. loaded cells × 100. Results are expressed as mean ± SE of 3 different experiments. (D) Relative proportion of pro-/pre-, immature, and transitional populations in migrating cells. Cells migrating to medium (spontaneous), BLC, or SDF1-α were stained for B220 IgM and IgD to determine the relative proportions of pro-/pre- (□), immature (▦), and transitional cells (▪) by FACS analysis. Results are shown in bars as percentage of cells of each population. Results shown are representative of 3 different experiments. (E) Migration of IgM+ precursors to the spleen. CD19-deficient and wt B-cell precursors grown in BM cultures were labeled with CFSE and injected intravenously into recipient B10D2 mice (total injected cells were adjusted to include 5 × 106 IgM+ cells/mouse). Six hours after transfer, spleens of reconstituted mice were removed, and spleen cells were stained for B220 and IgM. Cells were analyzed by FACS to quantify the number of CFSE-labeled IgM+ cells. Results are presented as the number of CFSE-labeled IgM+ cells/106 spleen cells and are the mean ± SE of 3 different mice. Asterisk indicates values that are statistically different (P < .05). (F) Chemokine-induced migration of splenic B cells. Spleen cells from wt and CD19-/- mice were tested for migration response to SDF1-α (right, ▦) and to BLC (left, ▦) using a transwell system. □ indicates medium. Migrating cells were stained for B220 and IgM to determine the relative proportion of B cells. Results are presented in a migration index and are calculated as: no. migrating B cells/no. loaded B cells × 100. Results are expressed as mean ± SE of 3 different experiments.$

Chemokine receptor expression and chemokine responsiveness of wt and CD19^-/- B-cell precursors. Wild-type and CD19^-/- B-cell precursors were grown in BM cultures. (A) B-cell precursors were fractionated into pro-/pre- (IgM^-), immature (IgM⁺IgD^-), and transitional (IgM⁺IgD⁺) populations. Mature B cells were isolated from spleen. Cells were lysed rapidly, and the expression of CXCR4 and CXCR5 was determined for each sorted population by RT-PCR. Results shown are representative of 5 different experiments. Actin blot is shown as an internal control gene expression. (B) Quantification of CXCR4 and CXCR5 expression. Results are presented as a semiquantitative estimate, where signal intensity of CXCR4 or CXCR5 products was normalized to that of β-actin. □ indicates wt; ▦, CD19^-/-. Results of 5 experiments are presented as mean ± SE. Asterisk indicates values that are statistically different (P < .05). (C) Total cell migration of wt and CD19^-/- B-cell precursors. Cells grown in BM cultures were tested for migration capabilities to BLC (2 mg/mL, left, ▦) or to SDF1-α (250 ng/mL, right, ▦) using transwell inserts. □ indicates medium. Migrating cells were counted by FACS, and the percentage of migration was calculated as: no. migrating cells/no. loaded cells × 100. Results are expressed as mean ± SE of 3 different experiments. (D) Relative proportion of pro-/pre-, immature, and transitional populations in migrating cells. Cells migrating to medium (spontaneous), BLC, or SDF1-α were stained for B220 IgM and IgD to determine the relative proportions of pro-/pre- (□), immature (▦), and transitional cells (▪) by FACS analysis. Results are shown in bars as percentage of cells of each population. Results shown are representative of 3 different experiments. (E) Migration of IgM⁺ precursors to the spleen. CD19-deficient and wt B-cell precursors grown in BM cultures were labeled with CFSE and injected intravenously into recipient B10D2 mice (total injected cells were adjusted to include 5 × 10⁶ IgM⁺ cells/mouse). Six hours after transfer, spleens of reconstituted mice were removed, and spleen cells were stained for B220 and IgM. Cells were analyzed by FACS to quantify the number of CFSE-labeled IgM⁺ cells. Results are presented as the number of CFSE-labeled IgM⁺ cells/10⁶ spleen cells and are the mean ± SE of 3 different mice. Asterisk indicates values that are statistically different (P < .05). (F) Chemokine-induced migration of splenic B cells. Spleen cells from wt and CD19^-/- mice were tested for migration response to SDF1-α (right, ▦) and to BLC (left, ▦) using a transwell system. □ indicates medium. Migrating cells were stained for B220 and IgM to determine the relative proportion of B cells. Results are presented in a migration index and are calculated as: no. migrating B cells/no. loaded B cells × 100. Results are expressed as mean ± SE of 3 different experiments.

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