Panel A: Flow cytometry plots show the gating strategy for identifying plasmablasts (PB, proliferating antibody-secreting precursor cells) and plasma cells (PC, terminally differentiated antibody-secreting cells) within germinal center (GC) and GL7 low or negative CD95 positive cell populations. The plots are gated on B220 (pan B cell marker) and CD138 (plasma cell marker) markers. A bar graph quantifies the proportion of GC-derived B cells in GL7 positive CD95 positive and GL7 low or negative CD95 positive populations in the S1PR2ERT2Cre-RS-loxStoplox-tdTomato mouse model (tamoxifen-inducible Cre recombinase under S1PR2 promoter with tdTomato reporter). Panel B: A Gene Ontology (GO) term enrichment TreePlot displays the enriched biological processes in GL7 positive CD95 positive (GL7 high) and GL7 low or negative CD95 positive (GL7 low) cells. Panel C: Flow cytometry plots and a bar graph illustrate the gating strategy and quantification of GC-derived IRF4 high PAX5 low cells (Interferon Regulatory Factor 4 high, Paired Box Protein 5 low; indicative of plasma cell differentiation) in GL7 positive CD95 positive and GL7 low or negative CD95 positive populations. Panel D: Flow cytometry plots and bar graphs show the effect of BI3802 treatment (BCL6 degrader compound) on plasma cell differentiation in wild-type (WT) and AIDR112H mice, with quantifications of BCL6 low (B cell lymphoma 6 low), IRF4 high PAX5 low cells, and IRF4 high cells within the BCL6 low population. Panel E: Flow cytometry plots and a bar graph depict the impaired CXCR5 downregulation (C-X-C motif chemokine receptor 5) in AIDR112H GL7 low CD95 positive cells, with quantifications of GL7 low CD95 positive, CXCR4 high (C-X-C motif chemokine receptor 4 high), and CD83 slash 86 positive (activation markers CD83 and CD86 expressing) cells. Panel F: A flow cytometry plot and a line graph show the upregulation of IRF4 (Interferon Regulatory Factor 4) versus cell division in iGB cells (in vitro generated germinal center B cells), with data from two experiments. Panel G: Flow cytometry plots and bar graphs illustrate the overexpression of IRF4 (Interferon Regulatory Factor 4) in iGB cells by retroviral transfection, with quantifications of IRF4 high PAX5 low cells in control and IRF4-transfected cultures. Panel H: A flow cytometry plot shows increased immunoglobulin (Ig) production of IRF4 high PAX5 low cells in WT iGB cultured B cells, with IgG1 high (Immunoglobulin G1 high expressing) cells overlaid with total B220 positive iGB cells.
Fate mapping of GC-experienced B cells in WT mice and impaired IRF4 upregulation and CXCR5 downregulation of GC B cells in AID R112H mice. (A) Proportion of GC-derived B cells in GL7+CD95+ and GL7lo/−CD95+ cell populations in S1PR2ERT2Cre-RS-loxStoplox-tdTomato mouse model. Gating strategy and quantification of cells with GC identity or GC-derived (BCL6+ and tdTomato+) (n = 6, one experiment). (B) GO-term enrichment treeplot for GL7+CD95+(GL7.hi.) cells and GL7lo/−CD95+(GL7.lo.) cells. (C) GC-derived IRF4hiPAX5lo cells in GL7+CD95+ and GL7lo/−CD95+ cell populations in S1PR2ERT2Cre-RS-loxStoplox-tdTomato mouse model. Representative FACS plots and gating strategy. Quantification of tdTomato+ cells among the IRF4hiPAX5lo cells (n = 6, one experiment). (D) PC differentiation in vivo during the GC response upon BI-3802 treatment for 24 h. Left: Representative FACS plots. Right: Quantification of BCL6lo: WT versus AIDR112H, P < 0.0001; WT + BI-3802 versus AIDR112H + BI-3802: P < 0.01; AIDR112H versus AIDR112H + BI-3802: P < 0.01; IRF4hiPAX5lo cells: WT versus AIDR112H, P < 0.001; WT + BI-3802 versus AIDR112H + BI-3802: P < 0.0001; AIDR112H versus AIDR112H + BI-3802: nonsignificant; IRF4hi within the BCL6lo population: WT versus AIDR112H, P < 0.01; WT + BI-3802 versus AIDR112H + BI-3802: P < 0.01; nonsignificant difference in treated versus nontreated, two-way ANOVA (n = 4–5, one experiment). (E) Impaired CXCR5 downregulation in AIDR112H GL7loCD95+ cells. FACS plots and quantification. Same FACS plot on column 1 and column 4 in each row is displayed to show gating strategy for GC and GL7loCD95+ population, respectively. FACS plot for GC and GL7lo/−CD95+ gating used here from the same samples is shown in Fig. 3 A. GL7loCD95+: P < 0.01; CXCR4hi: P < 0.0001; CD83/86+: P < 0.05, one-way ANOVA (n = 8, two experiments). (F) Upregulation of IRF4 versus cell division in iGB cells. Representative FACS plot and quantification, IRF4 MFI versus cell division, data from two experiments. (G) Overexpression of IRF4 in iGB cells by retroviral transfection. Left: Representative FACS plots. Right: Quantification of IRF4hiPAX5lo cells in control and IRF4-transfected cultures. Ctrl-IRES-GFP: no significant changes between WT GFP− versus WT GFP+; no significant changes between AIDR112H GFP− versus AIDR112H GFP+; IRF4-IRES-GFP: P < 0.0001, WT GFP− versus WT GFP+; P < 0.0001, AIDR112H GFP− versus AIDR112H GFP+, two-way ANOVA (n = 5–6, two experiments). (H) Increased Ig production of IRF4hiPAX5lo cells in WT iGB cultured B cells. Representative FACS plot, IgG1hi cells overlaid with total B220+ iGB cells. MFI, mean fluorescence intensity. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.