Figure 1.
Figure 1. Crebbp deletion leads to impairments in B-cell development. (A) An example of flow cytometry density dot plots showing B220 and IgM staining of bone marrow cells from mice older than 5 months. Plots are gated on viable single leukocytes based upon PI staining and scatter characteristics. Gating shows pre- or pro-B cells (B220low, IgM−), IgM+ B cells (B220+, IgM+), and B220high B cells that can be defined as recirculating B cells based upon expression of IgM and IgD. A notable change in the IgM+ and B220high B-cell populations can be observed across strains, with reduced numbers being associated with Crebbp deletion and being partially rescued by the EµBcl2 transgene. (B) A summary of total B-cell percentage in the bone marrow, measured as the percentage of B220+ cells among viable single cells, is shown for all strains. A 1-way ANOVA test showed a significant variance across strains in the dataset (P < .001). Post hoc testing (Tukey) revealed that this was driven by significantly higher numbers of total B cells in EµBcl2 mice compared with CbpWT/F (P = .011), CbpWT/Δ (P < .001), CbpΔ/Δ (P < .001), and CbpWT/Δ × EµBcl2 (P < .001) mice, but not between EµBcl2 and CbpΔ/Δ × EµBcl2 mice (P = .068). All other head-to-head comparisons were not significant (P > .05). (C) A summary of pre- or pro-B cells, measured as the percentage of B220low IgM− cells as a proportion of all B220+ cells, is shown for all strains from mice older than 5 months. There was no significant variance in this population across strains (1-way ANOVA, P = .652). (D) A summary of immature B-cell frequencies, measured as the percentage of B220+ IgM+ IgD− cells as a proportion of all B220+ cells, is shown for all strains. A 1-way ANOVA test showed a significant variance across the dataset (P = .034) that was driven by significantly higher frequencies in the CbpΔ/Δ × EµBcl2 strain compared with the CbpWT/Δ strain (Tukey, P = .018). No other head-to-head comparisons were statistically significant in post hoc testing (P > .05). (E) A summary of recirculating B-cell frequencies, measured as the percentage of B220+ IgM+ IgD+ cells as a proportion of all B220+ cells, is shown for all strains. One-way ANOVA showed a significant variance across the strains in the dataset (P < .001). Post hoc testing (Tukey) revealed that this was driven by a significantly higher frequency of mature B cells in EµBcl2 mice compared with CbpWT/F (P = .001), CbpWT/Δ (P < .001), CbpΔ/Δ (P < .001), CbpWT/Δ × EµBcl2 (P < .001), and CbpΔ/Δ × EµBcl2 (P = .004) mice. (F) An example of flow cytometry density dot plots showing IgD and IgM staining of splenocytes from mice older than 5 months. Plots are gated on viable single leukocytes based upon PI staining and scatter characteristics. A reduction in the frequency of immature (IgM+IgD−), transitional (IgMhiIgD+), and mature (IgM+IgD+) cells can be seen with Crebbp deletion, and this is not restored by the addition of the EµBcl2 transgene. Note that the frequencies for these populations, shown in panels I and J of and supplemental Table 2, are based upon additional gating for B220+ cells that are not shown in this figure. (G) Box plots show the B220+ cells, as a percent of all viable single cells, across the 6 strains. There was a significant variability among strains (1-way ANOVA, P = .007) that was driven by significantly lower frequencies in CbpWT/Δ compared with CbpWT/F (Tukey, P = .036) and CbpWT/Δ × EµBcl2 (Tukey, P = .048) strains. No other head-to-head comparison was significant. (H) Box plots show follicular (B220+CD21+CD23+) B cells, as a percent of B220+ cells, across the 6 strains. There was significant variance across the strains, driven by significantly lower frequencies in CbpWT/Δ, CbpΔ/Δ, CbpWT/Δ × EµBcl2, and CbpΔ/Δ × EµBcl2 mice compared with both CbpWT/F and EµBcl2 (Tukey, P < .01 for all head-to-head comparisons). (I) The frequency of immature (B220+IgM+IgD−) B cells, as a percentage of B220+ cells, are shown in box plots. There was significant variance across the strains (1-way ANOVA, P < .001) that included significantly lower frequencies in CbpWT/Δ (Tukey, P < .001) and CbpΔ/Δ (Tukey, P = .005) compared with CbpWT/F mice, and significantly lower frequencies in CbpWT/Δ × EµBcl2 (Tukey, P = .006), and CbpΔ/Δ × EµBcl2 (Tukey, P = .013) compared with EµBcl2 mice. (J) The frequencies of mature B cells (B220+IgM+IgD+), as a percentage of B220+ cells, are expressed in a box plot. There was significant variance across the strains (1-way ANOVA, P < .001) that was driven by significantly lower frequencies in CbpΔ/Δ, CbpWT/Δ × EµBcl2, and CbpΔ/Δ × EµBcl2 mice compared with both CbpWT/F and EµBcl2 mice (Tukey, P < .05 for all comparisons). There was no significant difference between CbpWT/Δ mice and either CbpWT/F (Tukey, P = .172) or EµBcl2 mice (Tukey, P = .082).

Crebbp deletion leads to impairments in B-cell development. (A) An example of flow cytometry density dot plots showing B220 and IgM staining of bone marrow cells from mice older than 5 months. Plots are gated on viable single leukocytes based upon PI staining and scatter characteristics. Gating shows pre- or pro-B cells (B220low, IgM−), IgM+ B cells (B220+, IgM+), and B220high B cells that can be defined as recirculating B cells based upon expression of IgM and IgD. A notable change in the IgM+ and B220high B-cell populations can be observed across strains, with reduced numbers being associated with Crebbp deletion and being partially rescued by the EµBcl2 transgene. (B) A summary of total B-cell percentage in the bone marrow, measured as the percentage of B220+ cells among viable single cells, is shown for all strains. A 1-way ANOVA test showed a significant variance across strains in the dataset (P < .001). Post hoc testing (Tukey) revealed that this was driven by significantly higher numbers of total B cells in EµBcl2 mice compared with CbpWT/F (P = .011), CbpWT/Δ (P < .001), CbpΔ/Δ (P < .001), and CbpWT/Δ × EµBcl2 (P < .001) mice, but not between EµBcl2 and CbpΔ/Δ × EµBcl2 mice (P = .068). All other head-to-head comparisons were not significant (P > .05). (C) A summary of pre- or pro-B cells, measured as the percentage of B220low IgM− cells as a proportion of all B220+ cells, is shown for all strains from mice older than 5 months. There was no significant variance in this population across strains (1-way ANOVA, P = .652). (D) A summary of immature B-cell frequencies, measured as the percentage of B220+ IgM+ IgD− cells as a proportion of all B220+ cells, is shown for all strains. A 1-way ANOVA test showed a significant variance across the dataset (P = .034) that was driven by significantly higher frequencies in the CbpΔ/Δ × EµBcl2 strain compared with the CbpWT/Δ strain (Tukey, P = .018). No other head-to-head comparisons were statistically significant in post hoc testing (P > .05). (E) A summary of recirculating B-cell frequencies, measured as the percentage of B220+ IgM+ IgD+ cells as a proportion of all B220+ cells, is shown for all strains. One-way ANOVA showed a significant variance across the strains in the dataset (P < .001). Post hoc testing (Tukey) revealed that this was driven by a significantly higher frequency of mature B cells in EµBcl2 mice compared with CbpWT/F (P = .001), CbpWT/Δ (P < .001), CbpΔ/Δ (P < .001), CbpWT/Δ × EµBcl2 (P < .001), and CbpΔ/Δ × EµBcl2 (P = .004) mice. (F) An example of flow cytometry density dot plots showing IgD and IgM staining of splenocytes from mice older than 5 months. Plots are gated on viable single leukocytes based upon PI staining and scatter characteristics. A reduction in the frequency of immature (IgM+IgD−), transitional (IgMhiIgD+), and mature (IgM+IgD+) cells can be seen with Crebbp deletion, and this is not restored by the addition of the EµBcl2 transgene. Note that the frequencies for these populations, shown in panels I and J of and supplemental Table 2, are based upon additional gating for B220+ cells that are not shown in this figure. (G) Box plots show the B220+ cells, as a percent of all viable single cells, across the 6 strains. There was a significant variability among strains (1-way ANOVA, P = .007) that was driven by significantly lower frequencies in CbpWT/Δ compared with CbpWT/F (Tukey, P = .036) and CbpWT/Δ × EµBcl2 (Tukey, P = .048) strains. No other head-to-head comparison was significant. (H) Box plots show follicular (B220+CD21+CD23+) B cells, as a percent of B220+ cells, across the 6 strains. There was significant variance across the strains, driven by significantly lower frequencies in CbpWT/Δ, CbpΔ/Δ, CbpWT/Δ × EµBcl2, and CbpΔ/Δ × EµBcl2 mice compared with both CbpWT/F and EµBcl2 (Tukey, P < .01 for all head-to-head comparisons). (I) The frequency of immature (B220+IgM+IgD−) B cells, as a percentage of B220+ cells, are shown in box plots. There was significant variance across the strains (1-way ANOVA, P < .001) that included significantly lower frequencies in CbpWT/Δ (Tukey, P < .001) and CbpΔ/Δ (Tukey, P = .005) compared with CbpWT/F mice, and significantly lower frequencies in CbpWT/Δ × EµBcl2 (Tukey, P = .006), and CbpΔ/Δ × EµBcl2 (Tukey, P = .013) compared with EµBcl2 mice. (J) The frequencies of mature B cells (B220+IgM+IgD+), as a percentage of B220+ cells, are expressed in a box plot. There was significant variance across the strains (1-way ANOVA, P < .001) that was driven by significantly lower frequencies in CbpΔ/Δ, CbpWT/Δ × EµBcl2, and CbpΔ/Δ × EµBcl2 mice compared with both CbpWT/F and EµBcl2 mice (Tukey, P < .05 for all comparisons). There was no significant difference between CbpWT/Δ mice and either CbpWT/F (Tukey, P = .172) or EµBcl2 mice (Tukey, P = .082).

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