Figure 4. Panel A: Multiple flow... | Rockefeller University Press

Figure 4.

Panel A: Multiple flow cytometry plots and bar graphs depict the gating strategy and quantification of transcription factors BCL6 (B cell lymphoma 6), IRF4 (Interferon Regulatory Factor 4), and PAX5 (Paired Box Protein 5) in germinal center (GC) and GL7 low CD95 positive B cells. The flow cytometry plots show the gating strategy for identifying different cell populations based on markers such as B220 (pan B cell marker), CD38 (B cell activation marker), CD95 (Fas receptor), GL7 (germinal center marker), and IRF4 (Interferon Regulatory Factor 4). The bar graphs compare the percentage of GC B cells, GL7 low or negative CD95 positive B cells, IRF4 high PAX5 low cells (Interferon Regulatory Factor 4 high, Paired Box Protein 5 low) in GC, IRF4 high PAX5 low cells in GL7 low or negative CD95 positive cells, IRF4 high in BCL6 low (B cell lymphoma 6 low) GC cells, BCL6 high in GL7 low CD95 positive cells, and IRF4 high in BCL6 low GL7 low CD95 positive cells between WT (wild-type) and AIDR112H mice. Significant differences are indicated with p-values. Panel B: Overlaid histograms and bar graphs show the expression levels of IRF4 (Interferon Regulatory Factor 4) and PAX5 (Paired Box Protein 5) in GC B cells. The histograms compare the mean fluorescence intensity (MFI, average signal intensity per cell) of IRF4 and PAX5 between WT and AIDR112H mice. The bar graphs quantify these differences, with a significant reduction in IRF4 MFI in AIDR112H mice compared to WT, while PAX5 MFI shows no significant difference.

AID ^R112H GC B cells have reduced capacity to upregulate IRF4. (A) Gating strategy for expression of transcription factors BCL6, IRF4, and PAX5 in GC and GL7^loCD95⁺ B cells. FACS plots and quantification, WT versus AID^R112H, GC: P < 0.0001; GL7^lo/−CD95⁺, P < 0.0001; IRF4^hiPAX5^lo in GC: P < 0.0001; IRF4^hiPAX5^lo in GL7^lo/−CD95⁺, P < 0.0001; IRF4^hi in BCL6^lo (GC): P < 0.0001; BCL6^hi in GL7^loCD95⁺: P < 0.0001; IRF4^hi in BCL6^lo (GL7^loCD95⁺): P < 0.0001, unpaired t test (n = 9–10, three experiments). ***P < 0.001, ****P < 0.0001. (B) Expression level of IRF4 and PAX5 in GC B cells. Overlay of histogram of IRF4 and PAX5 acquired by flow cytometry and quantification, WT versus AID^R112H, IRF4 MFI: P = 0.0006, unpaired t test (n = 4, two experiments). Nonsignificant differences for PAX5 MFI. Value and error bar: mean ± SD. Value and error bar: mean ± SD.