American Society of Hematology

Figure 4

$Figure 4. A1 knockdown impairs B-cell responsiveness. Flow cytometric analysis of splenocytes was performed by staining single-cell suspensions derived from mice of the indicated genotypes with cell surface-marker–specific antibodies in the different subfractions, positive or negative for Venus or eGFP. (A) Early B-cell development was analyzed in the bone marrow of VVA1 mice by flow cytometric analysis, using cell surface-marker–specific antibodies to identify CD19+c-Kit+ pro-B cells, CD19+CD25+ pre-B cells, IgM+IgD− immature B cells, and IgMlowIgD+ recirculating mature B cells. (B) Analysis of early B-cell development in DT and relevant control mice performed as in panel A. (C) B-cell distribution in spleens of VVA1 and relevant control mice. Transitional type 1 (T1) B cells (IgM+IgD−), transitional type 2 (T2) B cells (IgM+IgD+), and follicular (FO) B cells (IgMlowIgD+). (D) B-cell distribution in spleens of DT and relevant control mice performed as in panel C. Data are mean ± SEM of n = 4 to 8 animals per genotype. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with WT or single-transgenic controls; **P ≤ .01, compared with WT or single-transgenic controls. (E) T2 or (F) FO B cells from DT mice were sorted based on eGFP as well as levels of surface IgM and IgD expression (T2, IgM+IgD+; FO, IgMlowIgD+) and cultured in the presence of graded concentrations of plate-bound IgM F(ab′)2 fragments. Cell viability was assessed over time by 7-amino-actinomycin D exclusion and flow cytometric analysis. The extent of apoptosis induced specifically by BCR ligation was calculated by the following equation: (induced apoptosis/spontaneous cell death) × 100. Data are mean ± SEM of 5 independent experiments performed in duplicates. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with DT− or WT controls; **P ≤ .01, compared with DT− or WT controls. Total spleen cells from WT, TREA1, DT (G) or VVA1.2 mice (H), labeled with CDP proliferation dye were incubated with the indicated mitogens (anti-CD40 mAb, 1 μg/mL; CpG oligonucleotide, 100nM; anti-IgM F(ab′)2 fragments, 2 μg/mL). The percentages of CD19+ cells that underwent cell division were assessed in cells expressing different eGFP levels after 72 hours by flow cytometric analysis. Data are mean ± SEM of 3 independent experiments performed in duplicates. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with WT or single-transgenic mice; **P ≤ .01 compared with WT or single-transgenic mice.$

A1 knockdown impairs B-cell responsiveness. Flow cytometric analysis of splenocytes was performed by staining single-cell suspensions derived from mice of the indicated genotypes with cell surface-marker–specific antibodies in the different subfractions, positive or negative for Venus or eGFP. (A) Early B-cell development was analyzed in the bone marrow of VVA1 mice by flow cytometric analysis, using cell surface-marker–specific antibodies to identify CD19⁺c-Kit⁺ pro-B cells, CD19⁺CD25⁺ pre-B cells, IgM⁺IgD⁻ immature B cells, and IgM^lowIgD⁺ recirculating mature B cells. (B) Analysis of early B-cell development in DT and relevant control mice performed as in panel A. (C) B-cell distribution in spleens of VVA1 and relevant control mice. Transitional type 1 (T1) B cells (IgM⁺IgD⁻), transitional type 2 (T2) B cells (IgM⁺IgD⁺), and follicular (FO) B cells (IgM^lowIgD⁺). (D) B-cell distribution in spleens of DT and relevant control mice performed as in panel C. Data are mean ± SEM of n = 4 to 8 animals per genotype. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with WT or single-transgenic controls; **P ≤ .01, compared with WT or single-transgenic controls. (E) T2 or (F) FO B cells from DT mice were sorted based on eGFP as well as levels of surface IgM and IgD expression (T2, IgM⁺IgD⁺; FO, IgM^lowIgD⁺) and cultured in the presence of graded concentrations of plate-bound IgM F(ab′)2 fragments. Cell viability was assessed over time by 7-amino-actinomycin D exclusion and flow cytometric analysis. The extent of apoptosis induced specifically by BCR ligation was calculated by the following equation: (induced apoptosis/spontaneous cell death) × 100. Data are mean ± SEM of 5 independent experiments performed in duplicates. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with DT⁻ or WT controls; **P ≤ .01, compared with DT⁻ or WT controls. Total spleen cells from WT, TREA1, DT (G) or VVA1.2 mice (H), labeled with CDP proliferation dye were incubated with the indicated mitogens (anti-CD40 mAb, 1 μg/mL; CpG oligonucleotide, 100nM; anti-IgM F(ab′)2 fragments, 2 μg/mL). The percentages of CD19⁺ cells that underwent cell division were assessed in cells expressing different eGFP levels after 72 hours by flow cytometric analysis. Data are mean ± SEM of 3 independent experiments performed in duplicates. ANOVA followed by Fisher PLSD posthoc test: *P ≤ .05, compared with WT or single-transgenic mice; **P ≤ .01 compared with WT or single-transgenic mice.

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