Figure 4.
Absence of B cell immune responses in Pax5R31Q/E242* and Pax5R31Q/−mice. (A) PTEN expression in lymph node FO B cells was determined by intracellular staining with an anti-PTEN antibody (left). The median fluorescence intensity (MFI) of PTEN expression in Pax5R31Q/− B cells is shown as mean value with SEM relative to the that of Pax5+/+ B cells (set to 1; left; n ≥ 6 per genotype). (B) Impaired PI3K-AKT signaling in Pax5R31Q/− B cells. FO B cells from Pax5R31Q/− and Pax5+/+ lymph nodes were either left untreated (−) or stimulated (+) for 30 min with anti-IgM before intracellular staining with an antibody detecting AKT phosphorylation at Ser473 (left). The MFI of phosphorylated AKT is shown as mean value with SEM relative to that of the Pax5+/+ B cells (set to 1; left; n ≥ 5 per genotype and condition). (C) NP+ plasma cells (NP29-PE+NP14-CGG-Alexa-Fluor-488+CD138hiTACIhi) in the spleen of the Pax5+/+, and Pax5R31Q/− mice on day 7 after immunization with NP-Ficoll. Absolute cell numbers are shown as mean values with SEM (n = 5 per genotype). (D) Serum titers of NP-specific IgM and IgG antibodies 14 d after NP-Ficoll immunization. The antibody concentrations were measured by ELISA by using NP24-BSA–coated plates for capturing NP-specific IgM and IgG antibodies and are shown as mean values with SEM (n ≥ 7 per genotype). (E) GC B cell differentiation in the spleen of Pax5+/+, Pax5+/−, and Pax5R31Q/– mice on day 14 after immunization with NP-KLH in alum. GC B cells (CD19+B220+Fas+GL7+) were identified by flow cytometry (left). Numbers refer to the percentage of cells in the indicated gate. Absolute cell numbers are shown as mean values with SEM (right; n ≥ 12 per genotype). (F) NP+ plasma cells in the spleen of the indicated genotypes on day 14 after NP-KLH immunization. Absolute cell numbers are shown as mean values with SEM (n ≥ 5 per genotype). (G) Serum titers of NP-specific IgG and IgG1 antibodies 14 d after NP-KLH immunization. The antibody concentrations were measured by ELISA by using NP24-BSA– or NP7-BSA–coated plates for capturing NP-specific IgG and IgG1 antibodies, respectively. NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 monoclonal antibody. NP-specific IgG and IgG1 antibodies are shown as mean values with SEM (n ≥ 6 per genotype). Unpaired t test (A, C, and D), two-way ANOVA with Tukey’s multiple comparisons test (B), ANOVA with Dunnett’s or Dunnett’s T3 multiple comparisons test (E–G); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For detailed statistical information (A–G), see Table S6. Each dot (A–G) corresponds to one mouse.

Absence of B cell immune responses in Pax5 R31Q/E242 * and Pax5 R31Q/− mice. (A) PTEN expression in lymph node FO B cells was determined by intracellular staining with an anti-PTEN antibody (left). The median fluorescence intensity (MFI) of PTEN expression in Pax5R31Q/− B cells is shown as mean value with SEM relative to the that of Pax5+/+ B cells (set to 1; left; n ≥ 6 per genotype). (B) Impaired PI3K-AKT signaling in Pax5R31Q/− B cells. FO B cells from Pax5R31Q/− and Pax5+/+ lymph nodes were either left untreated (−) or stimulated (+) for 30 min with anti-IgM before intracellular staining with an antibody detecting AKT phosphorylation at Ser473 (left). The MFI of phosphorylated AKT is shown as mean value with SEM relative to that of the Pax5+/+ B cells (set to 1; left; n ≥ 5 per genotype and condition). (C) NP+ plasma cells (NP29-PE+NP14-CGG-Alexa-Fluor-488+CD138hiTACIhi) in the spleen of the Pax5+/+, and Pax5R31Q/− mice on day 7 after immunization with NP-Ficoll. Absolute cell numbers are shown as mean values with SEM (n = 5 per genotype). (D) Serum titers of NP-specific IgM and IgG antibodies 14 d after NP-Ficoll immunization. The antibody concentrations were measured by ELISA by using NP24-BSA–coated plates for capturing NP-specific IgM and IgG antibodies and are shown as mean values with SEM (n ≥ 7 per genotype). (E) GC B cell differentiation in the spleen of Pax5+/+, Pax5+/−, and Pax5R31Q/– mice on day 14 after immunization with NP-KLH in alum. GC B cells (CD19+B220+Fas+GL7+) were identified by flow cytometry (left). Numbers refer to the percentage of cells in the indicated gate. Absolute cell numbers are shown as mean values with SEM (right; n ≥ 12 per genotype). (F) NP+ plasma cells in the spleen of the indicated genotypes on day 14 after NP-KLH immunization. Absolute cell numbers are shown as mean values with SEM (n ≥ 5 per genotype). (G) Serum titers of NP-specific IgG and IgG1 antibodies 14 d after NP-KLH immunization. The antibody concentrations were measured by ELISA by using NP24-BSA– or NP7-BSA–coated plates for capturing NP-specific IgG and IgG1 antibodies, respectively. NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 monoclonal antibody. NP-specific IgG and IgG1 antibodies are shown as mean values with SEM (n ≥ 6 per genotype). Unpaired t test (A, C, and D), two-way ANOVA with Tukey’s multiple comparisons test (B), ANOVA with Dunnett’s or Dunnett’s T3 multiple comparisons test (E–G); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For detailed statistical information (A–G), see Table S6. Each dot (A–G) corresponds to one mouse.

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