Figure 6. Panel A: The experiment scheme... | Rockefeller University Press

Figure 6.

$High-affinity BCR is unable to rescue PC differentiation in AIDR112HB cells. (A) PC differentiation of B1-8hiWT and B1-8hiAIDR112H cells in vivo. Left: Experiment scheme. Middle: gating strategy to analyze GC response and PC differentiation from adoptively transferred B1-8hiWT and B1-8hiAIDR112H cells. Right: Quantification, IRF4hiCTVlo in the adoptive transferred WT and AIDR112H cells: **P = 0.005; B1-8hiAIDR112H/B1-8hiWT ratio in GC: ***P = 0.0009; paired t test (n = 6, two experiments). (B) NP binding capacity of B1-8hi GC B cells. Upper: Gating strategy to detect the NP binding capacity of B1-8hi B cells in GC by FACS. Middle: Left, surface NP-PE MFI in GC and GL7loCD95+, B1-8hiWT versus B1-8hiAID cells: *P < 0.05, one-way ANOVA (n = 5, two experiments). Right: Fraction of GC B cells that were NP-PE+ and NP-PE− among the host B, B1-8hiWT and B1-8hiAID B cells; fraction of BCR (IgMa)-expressing GC B cells that were NP-PE+ and NP-PE− among B1-8hiWT and B1-8hiAID B cells. Lower: Left, quantification of IgM+ B1-8hiWT and B1-8hiAID cells that lost NP binding in GC and GL7loCD95+ cells, ****P < 0.0001 for GC, nonsignificant for GL7loCD95+, one-way ANOVA (n = 5, two experiments). Right: Fraction of GL7loCD95+ B cells that were NP-PE+ and NP-PE− among the host B, B1-8hiWT, and B1-8hiAID B cells; fraction of BCR (IgM)-expressing GL7loCD95+ B cells that were NP-PE+ and NP-PE− among B1-8hiWT and B1-8hiAID B cells. Refer to the image caption for details.$ Panel A: The experiment scheme shows the adoptive transfer of B1-8hiWT and B1-8hiAIDR112H cells into a host, followed by analysis after immunization. The middle section displays flow cytometry plots for gating strategies to analyze germinal center (GC) response and plasma cell differentiation. The right section quantifies IRF4hiCTVlo cells in adoptively transferred WT and AIDR112H cells, showing significant differences. Panel B: The flowchart shows B cells adoptive transfer and the gating strategies to detect NP binding capacity of B1-8hi B cells in GC by flow cytometry plots. The bar graphs compares surface NP-PE MFI in GC and GL7loCD95 positive cells, and the fraction of NP-PE positive and NP-PE- GC B cells among host B, B1-8hiWT, and B1-8hiAID B cells. The bar graph also quantifies IgM plus B1-8hiWT and B1-8hiAID cells that lost NP-binding in GC and GL7loCD95 positive cells, and the fraction of NP-PE positive and NP-PE- GL7loCD95 positive B cells among host B, B1-8hiWT, and B1-8hiAID B cells.

High-affinity BCR is unable to rescue PC differentiation in AID ^R112H B cells. (A) PC differentiation of B1-8^hiWT and B1-8^hiAID^R112H cells in vivo. Left: Experiment scheme. Middle: gating strategy to analyze GC response and PC differentiation from adoptively transferred B1-8^hiWT and B1-8^hiAID^R112H cells. Right: Quantification, IRF4^hiCTV^lo in the adoptive transferred WT and AID^R112H cells: **P = 0.005; B1-8^hiAID^R112H/B1-8^hiWT ratio in GC: ***P = 0.0009; paired t test (n = 6, two experiments). (B) NP binding capacity of B1-8^hi GC B cells. Upper: Gating strategy to detect the NP binding capacity of B1-8^hi B cells in GC by FACS. Middle: Left, surface NP-PE MFI in GC and GL7^loCD95⁺, B1-8^hiWT versus B1-8^hiAID cells: *P < 0.05, one-way ANOVA (n = 5, two experiments). Right: Fraction of GC B cells that were NP-PE⁺ and NP-PE⁻ among the host B, B1-8^hiWT and B1-8^hiAID B cells; fraction of BCR (IgMa)-expressing GC B cells that were NP-PE⁺ and NP-PE⁻ among B1-8^hiWT and B1-8^hiAID B cells. Lower: Left, quantification of IgM⁺ B1-8^hiWT and B1-8^hiAID cells that lost NP binding in GC and GL7^loCD95⁺ cells, ****P < 0.0001 for GC, nonsignificant for GL7^loCD95⁺, one-way ANOVA (n = 5, two experiments). Right: Fraction of GL7^loCD95⁺ B cells that were NP-PE⁺ and NP-PE⁻ among the host B, B1-8^hiWT, and B1-8^hiAID B cells; fraction of BCR (IgM)-expressing GL7^loCD95⁺ B cells that were NP-PE⁺ and NP-PE⁻ among B1-8^hiWT and B1-8^hiAID B cells.