Figure S3.
High-affinity BCR failed to rescue PC differentiation and involvement of UNG and TET2 for IRF4 upregulation in GC B cells. (A) In vivo generation of IRF4hiNP+ cells from B1-8hiWT and B1-8hiAID cells. Left: Gating strategy to detect NP-specific (surface staining) IRF4hi cells by flow cytometry. Right: Quantification, P < 0.01, t test (n = 5, two experiments). (B) Schematics of in vivo experimental setup and gating strategy to analyze PC differentiation capacity of B1-8hiAIDR112H (green), AIDR112H (red), and CD45.1 WT (black) cells. Representative FACS plots for GC B cells and GL7lo/−CD95+ B cells. (C) Quantification of CD138+, IRF4+, NP+CD138+, and NP+IRF4+ cells in GC and GL7lo/−CD95+ cells. CD45.1 WT versus CD45.2 AIDR112H: P < 0.0001 for all groups; CD45.1 WT versus CD45.2 B1-8hiAIDR112H: P < 0.0001 for all groups, one-way ANOVA (n = 9, two experiments). Value and error bar: mean ± SD. (D) Reduced Ig class switching to IgG1 upon UGI-IRES-GFP retroviral transfection of iGB cells. Upper: Representative FACS plots for control plasmid and UGI-expressing plasmid transfection and Ig switching. Lower: Quantification of Ig switching to IgG1 among the control plasmid and UGI plasmid–transfected and nontransfected cells. (E) iGB cell proliferation rate upon UNG inhibition by UGI. Upper: Histogram overlay for nontransfected (gray) versus transfected (green) iGB cells (left: control plasmid; right: UGI plasmid). Lower: Quantification of the percentage of cells in each division in the transfected and nontransfected cells for both WT and AIDR112H iGB. Data are from one representative experiment with replicates. (F) CpG DNA methylation status at the Irf4 locus in naive B cells from WT and AIDR112H mice. Top: Schematics for the Irf4 gene locus and the region examined for DNA methylation using bisulfite sequencing. DNA methylation level at CpG sites, number above the graph shows the CpG position relative to first exon. For each CpG site, 15–30 clones were analyzed; data are from two independent experiments. (G) Ig class switching of WT and TET2 KO iGB cells on day 3 and day 6. Upper: representative FACS plots; lower: quantification, nonsignificant, ANOVA (n = 3, one experiment). **P < 0.01, ****P < 0.0001. Refer to the image caption for details. Panel A: The left side shows a flow cytometry gating strategy to detect NP-specific IRF4 high cells (nitrophenyl antigen-specific Interferon Regulatory Factor 4 high expressing cells). The right side presents a bar graph quantifying the percentage of IRF4 high cells among B1-8 high wild-type (B1-8hiWT) and B1-8 high AID mutant (B1-8hiAID) cells, with a significant difference indicated (p less than 0.01). Panel B: A schematic illustrates the experimental setup for analyzing plasma cell differentiation. Flow cytometry plots show the gating strategy for germinal center B cells (GC B cells) and GL7 low or negative CD95 positive B cells. Panel C: Bar graphs quantify CD138 positive (plasma cell marker expressing), IRF4 positive (Interferon Regulatory Factor 4 expressing), NP positive CD138 positive (nitrophenyl-specific plasma cells), and NP positive IRF4 positive cells in GC and GL7 low or negative CD95 positive cells, with significant differences noted (p less than 0.0001). Panel D: Flow cytometry plots compare immunoglobulin class switching to IgG1 (Immunoglobulin G1) upon UGI-IRES-GFP retroviral transfection (Uracil Glycosylase Inhibitor linked via Internal Ribosome Entry Site to Green Fluorescent Protein). A bar graph quantifies immunoglobulin switching to IgG1 among control and UGI plasmid-transfected cells. Panel E: Histograms overlay non-transfected and transfected iGB cells (in vitro generated germinal center B cells), showing cell proliferation rates. A line graph quantifies the percentage of cells in each division for WT (wild-type) and AIDR112H iGB cells. Panel F: A schematic of the Irf4 gene locus and a scatter plot show CpG DNA methylation status (cytosine–phosphate–guanine dinucleotide methylation) at the Irf4 locus in naive B cells from WT and AIDR112H mice. Panel G: Flow cytometry plots and a bar graph compare immunoglobulin class switching of WT (wild-type) and TET2 knockout (TET2KO, Ten-Eleven Translocation methylcytosine dioxygenase 2 deficient) iGB cells on day 3 and day 6, with no significant difference noted (analysis of variance, ANOVA).

High-affinity BCR failed to rescue PC differentiation and involvement of UNG and TET2 for IRF4 upregulation in GC B cells. (A) In vivo generation of IRF4hiNP+ cells from B1-8hiWT and B1-8hiAID cells. Left: Gating strategy to detect NP-specific (surface staining) IRF4hi cells by flow cytometry. Right: Quantification, P < 0.01, t test (n = 5, two experiments). (B) Schematics of in vivo experimental setup and gating strategy to analyze PC differentiation capacity of B1-8hiAIDR112H (green), AIDR112H (red), and CD45.1 WT (black) cells. Representative FACS plots for GC B cells and GL7lo/−CD95+ B cells. (C) Quantification of CD138+, IRF4+, NP+CD138+, and NP+IRF4+ cells in GC and GL7lo/−CD95+ cells. CD45.1 WT versus CD45.2 AIDR112H: P < 0.0001 for all groups; CD45.1 WT versus CD45.2 B1-8hiAIDR112H: P < 0.0001 for all groups, one-way ANOVA (n = 9, two experiments). Value and error bar: mean ± SD. (D) Reduced Ig class switching to IgG1 upon UGI-IRES-GFP retroviral transfection of iGB cells. Upper: Representative FACS plots for control plasmid and UGI-expressing plasmid transfection and Ig switching. Lower: Quantification of Ig switching to IgG1 among the control plasmid and UGI plasmid–transfected and nontransfected cells. (E) iGB cell proliferation rate upon UNG inhibition by UGI. Upper: Histogram overlay for nontransfected (gray) versus transfected (green) iGB cells (left: control plasmid; right: UGI plasmid). Lower: Quantification of the percentage of cells in each division in the transfected and nontransfected cells for both WT and AIDR112H iGB. Data are from one representative experiment with replicates. (F) CpG DNA methylation status at the Irf4 locus in naive B cells from WT and AIDR112H mice. Top: Schematics for the Irf4 gene locus and the region examined for DNA methylation using bisulfite sequencing. DNA methylation level at CpG sites, number above the graph shows the CpG position relative to first exon. For each CpG site, 15–30 clones were analyzed; data are from two independent experiments. (G) Ig class switching of WT and TET2 KO iGB cells on day 3 and day 6. Upper: representative FACS plots; lower: quantification, nonsignificant, ANOVA (n = 3, one experiment). **P < 0.01, ****P < 0.0001.

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