p53−/−, aid−/−, and wild-type littermate mice were inoculated i.p. with 2×10⁶ pfu of polyoma virus strain A2. Splenic germinal center B cells were analyzed for surface isotype expression by flow cytometry 12–20 days after inoculation. (A) FACS plots show surface Ig expression on B220+ GL7+ splenic B cells from an experiment in which cells were harvested 14 days after infection. Percentages of switched cells are depicted next to the gates. (B) Data points and bars represent individual mice and means (n=7 for wild-type, n=5 for p53−/−) of the percentages of isotype switched cells within the B220+ GL7+ splenic germinal center B-cell subset. Three independent experiments were performed using: 3, 2, and 2 wild-type mice, 1, 2, and 2 p53−/− mice, and in the second experiment (shown) 2 aid−/− mice were included. Significance was determined by the Mann-Whitney U test.

Sμ and Sγ2a LM-PCR were performed on threefold dilutions of GAPDH-normalized DNA isolated from wild-type (WT), p53−/−, and aid−/− splenic B cells that had been stimulated for 2 days with LPS+IL4 or LPS+IFNγ. Blunt DSBs in Sμ and Sγ2a were assessed by use of 5’ Sμ or 5’ Sγ2a primer, respectively, in combination with a linker specific primer. PCR products were blotted and hybridized with an internal Sμ or Sγ2a probe, respectively. (A) Sμ DSBs in cells activated for IgG1 switching. (B) Sμ DSBs in cells activated for IgG2a switching. (C) Sγ2a DSBs in cells activated for IgG2a switching. (D) Bar graph depicts means (+SEM) of densitometry measurements of autoradiographic films, normalized to WT, which was set at 1.0 (n=4 for all experiments, except for Sγ2a DSBs under IgG2a conditions, n=3.) All 3 titration lanes were scanned together. Each replicate experiment was performed on material from a separate set of mice. Two independent experiments using two sets of mice were performed. (E) Mutations in Sμ-Sγ3 junctions from WT and p53−/− splenic B cells activated to undergo IgG3 switching. Mutation frequency per 50-nucleotide segment is plotted for WT (black bars) and p53−/− (white bars). Overall mutation frequency is 3.1-fold increased in p53−/− versus WT (p = 0.00001). Total mutations/nucleotides analyzed for WT: 31/10,231; for p53: 30/3,182.

CFSE-loaded splenic B cells from p53S18A and wild-type control mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. Class switching was determined by flow cytometry for surface Ig staining. (A) Representative FACS plots are shown. Upper panel shows equal CFSE loading in wild-type mice from the same colony and p53S18A splenic B cells. Percentages of switched cells are indicated within the gates. (B) Bar graph depicts data from 4 sets of mice, which were all analyzed in one experiment. Percent CSR in p53S18A relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

CFSE-loaded splenic B cells from p53−/− and wild-type littermate mice were cultured for 2.5 days with LPS, cytokines and/or anti-δ-dextran to induce Ig class switching to the indicated isotypes. (A) Cells were analyzed for CSFE-dilution and surface Ig by flow cytometry; representative FACS plots are shown; percentages of switched cells are indicated within the gates. (B) Data from 8 experiments (8 sets of mice) were normalized to the percent of wild-type littermate (WT) switching, indicated as 100%. Mean percentages of switching in p53−/− relative to WT (+SEM) for the different isotypes are shown. Significance was determined by the one-sample t test.

CFSE-loaded wild-type splenic B cells were activated for CSR to the indicated Ig isotypes. 2.5 µM Nutlin-3 or solvent control (DMSO) was added at the start of the cultures. Class switching was assessed by flow cytometry for surface Ig after 2.5 days of culture. (A) Data from 4 experiments were normalized to the percent of switching in untreated (DMSO) splenic B cells, indicated as 100%. Mean percentages (+SEM) of switching in Nutlin-3 treated splenic B cells relative to untreated for the different isotypes are shown. Significance was determined by the one-sample t test. (B) Overlay CSFE histograms of splenic B cells activated for switching to the indicated isotypes, treated with 2.5 µM Nutlin-3 (blue profiles) or DMSO vehicle control (red profiles) are shown.

(A) Western blot analysis of Ser18 phosphorylated p53 (pSer18) in ex vivo splenic B cells, B cells stimulated for 2 days with LPS+anti-δ-dextran, or LPS+anti-δ-dextran+IFNγ. Activated splenic B cells irradiated at 5 Gy were used a positive control. Gapdh was used for protein loading control. This experiment was performed twice, using cells from one mouse each time. (B) Quantitative PCR analysis for p21 mRNA expression in WT splenic B cells stimulated for 48 h with LPS+anti-δ-dextran or LPS+anti-δ-dextran+IFNγ. Cells treated with 2.5 µM Nutlin-3 were used as a positive control, p53−/− cells as a negative control. Bar graph depicts relative p21 mRNA abundance normalized to wild-type splenic B cells stimulated with LPS+anti-δ-dextran. Significance was determined by the one-sample t test. Four independent experiments were performed, using 4 sets of mice. (C) Quantitative PCR analysis for sestrin 1 mRNA expression relative to hprt mRNA in WT splenic B cells stimulated to switch to the indicated isotypes as described in Methods. Data are from one experiment, PCR was performed in triplicate.

(A) Shown are overlay histograms of wild-type (red profile) and p53−/− (blue profile) splenic B cells loaded with the CM-H₂-DCFDA fluorescent ROS probe after activation with LPS + anti-δ-dextran + IFNγ for 48 h. Treatment with 10 mM (blue profile) and 20 mM NAC (green profile) decreases intracellular ROS in activated wild-type and p53−/− splenic B cells. (B) CFSE-loaded cells were activated for IgG2a switching and treated with 10 mM NAC. IgG2a switching was assessed by flow cytometry for surface Ig staining after 2.5 days. Representative FACS plots of 3 independent experiments are shown. Percentages of IgG2a switched cells are given within the gates. (C) CFSE-loaded wild-type splenic B cells were activated with LPS + cytokines and/or anti-δ-dextran to induce CSR to the indicated Ig isotypes. CSR was assessed by flow cytometry for surface Ig staining. Percentages of switched cells are indicated within the gates. Top row shows untreated control cultures, middle row shows cultures treated with 10 mM NAC. Bottom row shows overlay histograms of CSFE fluorescence in control cultures (red profiles) and 10 mM NAC treated cultures (blue profiles). Representative FACS plots of three experiments are shown. (D) Bar graph shows data from the 3 experiments normalized to untreated cells, shown as 100%. Means (+SEM) of percentages of switched cells in NAC-treated cutures relative to untreated cultures are shown. Significance was determined by the one-sample t test. (E) Sμ LM-PCR was performed on threefold dilutions of Gapdh-normalized DNA from wild-type and aid−/− splenic B cells activated with LPS + anti-δ-dextran with or without 10 mM NAC for 2 days. LM-PCR products were blotted and hybridized with an internal Sμ probe. Densitometry scanning was performed on all three lanes representing the threefold dilutions. Untreated sample was set at 1.0 to determine relative density. Representative result of 2 experiments is shown.