Fig 2: IgG subclass distribution in the immune seraIgG subclass (IgG1, IgG2a, IgG2b, and IgG3) distribution of sera from mice vaccinated with RBD and its conjugates, RBD-EcoCRM-CC and RBD-EcoCRM-RC, formulated with Alhydrogel or AS01. Immune sera collected on day 35 after two vaccinations (days 0 and 21) were analyzed by ELISA using pooled samples for each group using isotype-specific goat-anti-mouse secondary antibodies from Southern Biotech, Birmingham AL.

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 Sα or d Sγ1. 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: Impaired γH2AX levels in spleen B cells from CD19-cre-Usp22 KO mice. a AID mRNA measured by qPCR of d3-LPS stimulated spleen B cells from CD19-cre-Usp22 KO or WT littermates (left panel; n = 5 mice per group). AID blot analysis of d3-LPS stimulated spleen B cells from Mb1-cre or CD19-cre Usp22 KO or their WT littermates (right panel; n = 2–3 mice per group); AID-KO cells were used as control and data represent two or three independent experiments. b Spleen B cells from CD19-cre-Usp22 KO and WT littermates were stimulated for IgG3 CSR (by LPS) or for IgA CSR (by LPS+ IL-4+ TGF-β+ IL-5+ anti-IgD dextran) for 2.5 days, exposed to γ-radiation, and then collected for γH2AX immunofluorescent staining. Line within micrograph represents 10 μm. The number of γH2AX foci per cell was analyzed 1 or 4 h post irradiation for Usp22-WT (black circles) and CD19-cre-Usp22-KO (white circles) (bottom panel), and representative images for IgG3-CSR group (γH2AX in green and DAPI in blue) are shown in the top panel. Approximately 30–50 cells were analyzed per sample and data represent two or three independent experiments. c Representative flow cytometry plots (left panel) and compiled cell cycle analysis (right panel) of Usp22-WT and CD19-cre-Usp22-KO spleen B cells. Cells were stimulated with LPS for 2.5 days and then incubated with EdU for 4 h, before FACS staining (n = 3 samples per group). d Purified splenic B cells from CD19-cre Usp22-KO or WT littermates was stimulated with LPS and growth of these cells was monitored over time (n = 3 samples per group). Data were tested by two-way ANOVA. e CFSE-pulsed spleen B cells from CD19-cre-Usp22 KO or WT littermates were stimulated by LPS plus IL-4 for CSR to IgG1 for 4 days. The gating (G2 to G11) is based on the intensity of CFSE and the percentage of IgG1 switch per gate is summarized (n = 3 samples per group). Data in c–e represent two independent experiments; data in a–c and e were analyzed using two-tailed unpaired Student’s t-test. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 5: CD4+ T cell depletion prior to WCV priming affects T cell-dependent isotype class switching in WCV. (A to C) P. aeruginosa-specific IgM (A) and IgG (B) serum antibody titers and IgA lung supernatant antibody titers (C) of vaccinated and challenged mice (n = 5 to 19 per group). Data represent those from six independent experiments. Kruskal-Wallis test with Dunn’s multiple-comparison test for statistical analysis was used. (D to F) Correlation of CD4+ T cells blood counts with P. aeruginosa-specific serum IgM (D) and serum IgG (E) and lung supernatant IgA titers (F) in whole-cell-vaccinated animals. Yellow filled dots represent whole-cell-vaccinated IgG2b isotype control mice (WCV mIgG2b). Yellow circles represent whole-cell-vaccinated anti-CD4-treated mice (WCV α-CD4). Each circle/dot represents values from one individual mouse. The R2 and P values for each correlation were calculated using linear regression analyses. (G) P. aeruginosa-specific serum IgG2a, IgG2b, IgG1, and IgG3 antibody titers (n = 10 per group). Mann-Whitney U test was used for comparisons. Error bars indicate SEMs. Limits of detection are shown with dashed lines. The asterisks show statistical significance as follows: *, P ≤ 0.05; **, P ≤ 0.01; and ****, P ≤ 0.0001. (H) Percentage of anti-P. aeruginosa IgG subtype response above the limit of detection in whole-cell-vaccinated anti-CD4-treated mice.