Fig 2: Reduced in vitro class switch in B cells from P2KO vs. WT mice. B cells were isolated from the spleens of young and old WT and P2KO mice (6 mice/group) by magnetic sorting. B cells (106/ml) were stimulated for 1–7 days with 1 μg/ml of LPS, then mRNA was extracted and qPCR performed to evaluate mRNA expression of E47 (A) and Pax5 (B) (at day 1), AID (C) (at day 5), and Prdm1 (Blimp-1) (D) (at day 4). Results show qPCR values (2−ΔΔCt). At day 5, cells are harvested to evaluate frequencies of CD138+ cells by flow cytometry (F). At day 7, supernatants were collected to measure IgG3 secretion by ELISA (E). Mean comparisons between groups were performed by two-way ANOVA followed by Bonferroni's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: IgG1 CSR primarily relies on c-NHEJ, whereas IgA CSR is more dependent on A-EJ. a Spleen B cells isolated from 53BP1 KO or WT littermates were induced for ex vivo IgG1, IgG2b, IgG3, or IgA CSR for 4 days. 53BP1-/- B cells (deficient in c-NHEJ) exhibited a less severe defect in IgA CSR (~ 60% reduction) compared with IgG CSR (~ 80–90% reduction). Data represent two independent experiments each with three to four mice per group. b A-196 (10 µM) was used to inhibit c-NHEJ during the ex vivo CSR of WT spleen B cells and A-197 is the control compound. Inhibition of c-NHEJ markedly reduced IgG1 CSR, but not IgA CSR (n = 4 samples per group). Data represent three independent experiments. c, d The switch region sequence of Sµ on the IgH locus of C57BL/6 background mice were compared with that of c Sa or d S?1. Dot Matrix analysis of the mouse switch regions. The dots represent homologies between two switch regions with a search length of 30 bp and defined percentage of identity. Data in a and b were presented as mean ± SEM and were analyzed using two-tailed unpaired Student’s t-test. ***p < 0.001; NS, not significant

Fig 4: Usp22 KO mice exhibited defective IgG/IgE CSR but not IgA CSR. a NP25-CGG plus alum was intraperitoneally injected into CD19-cre Usp22 KO or WT littermate mice. At d22 post immunization, mice were subjected to NP-specific IgG+ ELISPOT assay to measure the amount of NP-specific IgG+ antibody secreting cells (ASC) in the spleen (n = 7 mice per group). Data were combined from two independent experiments. b Rotavirus (RV) was orally administered to CD19-cre-Usp22 KO or WT littermates. Fecal samples were collected at the indicated time-points post-RV infection, followed by fecal supernatant preparation. The 1:2 dilution of fecal supernatant was used for fecal anti-RV IgA ELISA assay. Data was analyzed by two-way ANOVA. c ELISPOT assays were performed at d23 post-RV infection, to measure the numbers of IgA+ RV-specific antibody secreting cells in lamina propria (LP) and bone marrow (BM), respectively. Data in b and c represent two independent experiments each with three mice per group. d Splenic B cells from CD19-cre-Usp22 KO or WT littermates were ex vivo induced to switch to IgG1, IgG2b, IgG3, IgE, and IgA (n = 3 mice per group). e Same as d, except that Mb1-cre Usp22 KO mice were used (n = 3–4 mice per group). Data in d and e represent three independent experiments; data in a and c–e were analyzed using two-tailed unpaired Student’s t-test. Data were presented as mean ± SEM. *p < 0.05 and ***p < 0.001; NS, not significant