Figure 6 Increased spontaneous... | American Society of Hematology

Figure 6

$Figure 6. Increased spontaneous production of anti-TNP and anti-phosphocholine IgM Abs in B/WcKO mice. (A-B) Serum from naive WT, B/WcKO, and WKO mice was tested for IgM binding to TNP (A) and phosphocholine (B). Mean ± SEM values of 5 mice per group are shown. Significance was assessed by 2-way ANOVA and Bonferroni posttest analysis. ***P < .001 and ****P < .0001. (C) The proportion of NP-specific CD19+ splenic B cells in WT, WKO, and B/WcKO mice was analyzed by FACS by staining for cells reactive to PE-labeled NP hapten. Differences were not significant (NS) with 1-way ANOVA and Bonferroni posttest analysis. (D) The relative proportion of peritoneal CD19+ CD5+ B1 cells among peritoneal CD19+ B cells was assessed by flow cytometry in WT, WKO, and B/WcKO mice. The mean ± SEM for 4 mice per group is shown. Differences were not significant (NS) with 1-way ANOVA and Bonferroni posttest analysis. (E) Het-B/WcKO female mice were tested for the proportion of WASp+ versus WASp− cells within follicular B cell, MZ, GC, and PC B-cell compartments. The mean ± SEM values of 4-8 mice per group are shown. Statistical significance was assessed by 2-way ANOVA and Bonferroni posttest analysis. ****P < .0001. (F) Splenocytes of WT, B/WcKO, and WKO mice were stimulated in vitro with CpG (ODN 1826, 1.25μM final concentration). Five days later, generation of intracytoplasmic IgG+ (icIgGhi) plasmablasts was measured by flow cytometry. Bars indicate the mean ± SEM of icIgGhi plasmablasts generated in vitro from individual mice. Statistical significance was assessed by 1-way ANOVA and Bonferroni posttest analysis. *P < .05 and **P < .01. (G) HetB/WcKO mice were injected intraperitoneally with 2 mg of BrdU. Twelve hours later, BrdU incorporation in WASp+ versus WASp− fractions of follicular B cells (left) and GC B cells (right) was measured by BrdU-specific Ab. Data of individual mice are shown. Statistical significance was assessed by Student t test. *P < .05 and **P < .01. (H) Apoptosis of GC B cells was assessed by annexin V labeling gating on CD19+ FAS+ GL7+ GC B cells from WT, B/WcKO, and WKO mice.$

Increased spontaneous production of anti-TNP and anti-phosphocholine IgM Abs in B/WcKO mice. (A-B) Serum from naive WT, B/WcKO, and WKO mice was tested for IgM binding to TNP (A) and phosphocholine (B). Mean ± SEM values of 5 mice per group are shown. Significance was assessed by 2-way ANOVA and Bonferroni posttest analysis. ***P < .001 and ****P < .0001. (C) The proportion of NP-specific CD19⁺ splenic B cells in WT, WKO, and B/WcKO mice was analyzed by FACS by staining for cells reactive to PE-labeled NP hapten. Differences were not significant (NS) with 1-way ANOVA and Bonferroni posttest analysis. (D) The relative proportion of peritoneal CD19⁺ CD5⁺ B1 cells among peritoneal CD19⁺ B cells was assessed by flow cytometry in WT, WKO, and B/WcKO mice. The mean ± SEM for 4 mice per group is shown. Differences were not significant (NS) with 1-way ANOVA and Bonferroni posttest analysis. (E) Het-B/WcKO female mice were tested for the proportion of WASp⁺ versus WASp⁻ cells within follicular B cell, MZ, GC, and PC B-cell compartments. The mean ± SEM values of 4-8 mice per group are shown. Statistical significance was assessed by 2-way ANOVA and Bonferroni posttest analysis. ****P < .0001. (F) Splenocytes of WT, B/WcKO, and WKO mice were stimulated in vitro with CpG (ODN 1826, 1.25μM final concentration). Five days later, generation of intracytoplasmic IgG⁺ (icIgG^hi) plasmablasts was measured by flow cytometry. Bars indicate the mean ± SEM of icIgG^hi plasmablasts generated in vitro from individual mice. Statistical significance was assessed by 1-way ANOVA and Bonferroni posttest analysis. *P < .05 and **P < .01. (G) HetB/WcKO mice were injected intraperitoneally with 2 mg of BrdU. Twelve hours later, BrdU incorporation in WASp⁺ versus WASp⁻ fractions of follicular B cells (left) and GC B cells (right) was measured by BrdU-specific Ab. Data of individual mice are shown. Statistical significance was assessed by Student t test. *P < .05 and **P < .01. (H) Apoptosis of GC B cells was assessed by annexin V labeling gating on CD19⁺ FAS⁺ GL7⁺ GC B cells from WT, B/WcKO, and WKO mice.

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