Panel A: Flow cytometry plots and a bar graph show plasma cell differentiation capacity of switched (IgM negative, Immunoglobulin M non-expressing) and non-switched (IgM positive, Immunoglobulin M expressing) germinal center (GC) B cells. The bar graph compares the percentage of plasmablasts (PB, proliferating antibody-secreting precursor cells) in IgM positive and IgM negative GC B cells from wild-type (WT) and AIDR112H mice. Panel B: Histograms and line graphs depict B cell receptor (BCR) signaling in WT and AIDR112H GC B cells at different time points upon anti-BCR challenge. The line graphs quantify the levels of phosphorylated Syk (Spleen Tyrosine Kinase), Btk (Bruton’s Tyrosine Kinase), and Erk (Extracellular signal-Regulated Kinase). Panel C: Histograms and a bar graph show c-MYC (cellular Myelocytomatosis oncogene) mean fluorescence intensity (MFI, average fluorescence signal per cell) in GC B cells from WT and AIDR112H mice. Panel D: Histograms and a bar graph illustrate the up-regulation of c-MYC (cellular Myelocytomatosis oncogene) upon stimulation for 2 hours in GC B cells. The bar graph quantifies c-MYC MFI under different stimulation conditions. Panel E: Flow cytometry plots and a bar graph demonstrate the rescue of plasmablast differentiation by expressing wild-type AID protein (Activation-Induced Cytidine Deaminase) in AIDR112H iGB cells (in vitro generated germinal center B cells). The bar graph quantifies the percentage of IRF4 positive PAX5 low cells (Interferon Regulatory Factor 4 positive, Paired Box Protein 5 low; indicative of plasma cell differentiation). Panel F: Flow cytometry plots and a bar graph show the rescue of plasmablast differentiation upon treatment with 5-fluorouracil (5-FU, a thymidylate synthase inhibitor chemotherapeutic) and pemetrexed (PEM, antifolate chemotherapeutic) in iGB culture. The bar graph quantifies the percentage of IRF4 positive PAX5 low cells. Panel G: Flow cytometry plots and a bar graph illustrate the inhibition of plasmablast differentiation by expressing Ugi peptide (Uracil DNA glycosylase inhibitor peptide) in WT iGB cells. The bar graph quantifies the percentage of IRF4 positive PAX5 low cells.
PC differentiation requires WT AID protein. (A) PC differentiation capacity of switched (IgM−) and nonswitched (IgM+) GC B cells. Left: Representative FACS plots. Right: Quantification, WT versus AIDR112H, PB in IgM+, *P = 0.0102; IgM+ versus IgM− PB in WT GC, nonsignificant, unpaired t test (n = 3–5, one experiment). (B) BCR signaling in WT and AIDR112H GC B cells. Left: FACS plot histogram overlay at different time points upon anti-BCR challenge. Right: Quantification, WT versus AIDR112H, nonsignificant, unpaired t test (n = 4, two experiments). (C) c-MYC MFI in GC B cells. Right: Histogram overlays to show WT (black, solid and dotted line) and AIDR112H (red, solid and dotted line) cells, t test (n = 9, three experiments). (D) Upregulation of c-MYC upon stimulation for 2 h in GC B cells. Left: Histogram overlay of unstimulated and stimulated GC B cells. Right: Quantification of c-MYC MFI, anti-BCR versus unstimulated, nonsignificant; anti-CD40 versus unstimulated, ***P < 0.001 for both WT and AIDR112H; anti-CD40+anti-BCR versus unstimulated, ****P < 0.0001 for both WT and AIDR112H; nonsignificant between WT and AIDR112H for corresponding stimulation conditions. Two-way ANOVA (n = 4 mice per condition, two experiments). (E) Rescue of PB differentiation by expressing WT AID protein in AIDR112H iGB cells. FACS plots and quantification, Ctrl-IRES-GFP: no changes between GFP+ versus GFP− in WT and AIDR112H iGB cells; AID-IRES-GFP: nonsignificant between WT GFP− versus WT GFP+; **P = 0.0085 between AIDR112H GFP− versus AIDR112H GFP+, two-way ANOVA (one representative experiment from three). (F) Rescue of PB differentiation upon 5-FU and PEM treatment in iGB culture. FACS plots and quantification, ****P < 0.0001, WT versus AIDR112H; *P = 0.0133, AIDR112H versus AIDR112H+5-FU; *P = 0.012, AIDR112H versus AIDR112H + PEM, one-way ANOVA (n = 6, three experiments). (G) Inhibition of PB differentiation by expressing Ugi peptide in WT iGB cells. FACS plots and quantification, Ctrl-IRES-GFP: no changes between WT GFP− versus WT GFP+; Ugi-IRES-GFP: P < 0.0001 between WT GFP− versus WT GFP+, unpaired t test (n = 18, two experiments).