Panel A: The panel shows a series of flow cytometry plots and bar graphs. The flow cytometry plots illustrate the gating strategy for identifying different stages of B cell development in the bone marrow. The bar graphs display the total cell number in the bone marrow and the percentage of cells at different developmental stages (FrA, FrB plus C, FrD plus E). Panel B: This panel includes flow cytometry plots and bar graphs showing B cell development in the spleen. The flow cytometry plots depict the gating strategy for identifying various B cell subsets, while the bar graphs quantify the total number of spleen cells, B220 plus cells, and the percentage of cells at different transitional stages (T1, T2 MZP, MZB, FoB). Panel C: The panel presents immunofluorescence images of spleen sections on day 7 post-SRBC immunization, stained for IgD, GL7, and CD21/35. A bar graph quantifies the number of germinal centers (GCs). Panel D: This panel includes flow cytometry plots and bar graphs showing the GC response on day 7 post-SRBC immunization. The flow cytometry plots illustrate the gating strategy for GC, dark zone (DZ), and light zone (LZ) cells, while the bar graphs quantify the percentage of GC, DZ, and LZ cells and the DZ/LZ ratio. Panel E: The panel shows flow cytometry plots and a bar graph quantifying active Caspase3 plus apoptotic cells in GCs. Panel F: This panel includes flow cytometry plots and a bar graph quantifying memory B cell differentiation in the spleen. Panel G: The panel presents a schematic of the experimental setup for bone marrow chimeric mice, flow cytometry plots of splenic plasma cells (PB) and plasma cells (PCs), and a bar graph quantifying the log2 ratio of AIDR112H/WT in different B cell subsets (GC, DZ, LZ, PC, MB). Panel H: This panel includes flow cytometry plots and bar graphs showing the GC response in bone marrow chimeric mice, with quantifications of GC, DZ, LZ, and the DZ slash LZ ratio.
B cell development and differentiation in AID R112H mice and bone marrow chimeric mice. (A) B cell development in bone marrow by the Hardy classification. Upper panels: Representative FACS plots for gating strategy. Lower panels: Total cell number of bone marrow, P = 0.032, WT versus AIDR112H, unpaired t test (n = 5, one experiment); % of FrA (fraction A), FrB + C (fraction B + C), FrD + E (fraction D + E), nonsignificant when comparing WT versus AIDR112H in all groups. (B) B cell development in the spleen. Upper panels: Representative FACS plots for gating. Lower panels: Quantifications, total spleen cells, nonsignificant; total B220+ cells, P = 0.044, unpaired t test (n = 5, one experiment); % of T1, T2 MZP; B220+, MZB, FoB, nonsignificant for all groups, WT versus AIDR112H, unpaired t test (n = 5, one experiment). (C) GC in spleen section on day 7 upon SRBC immunization. Blue: IgD; green: GL7; red: CD21/35. Scale bar, 200 µm. (D) GC response on day 7 upon SRBC immunization. Left panels: representative FACS plot for GC, DZ, and LZ; Right panels: quantification, WT versus AIDR112H; GC, P < 0.0001; DZ, P = 0.137; LZ, P = 0.0037. DZ/LZ ratio, P = 0.0097, unpaired t test (n = 13, three experiments). (E) Active caspase 3+ apoptotic cells in GC. FACS plots and quantification, WT versus AIDR112H, P < 0.01, t test (n = 19–21, four experiments). (F) Memory B cell differentiation in the spleen. FACS plot and quantification, P < 0.01, unpaired t test (n = 13, three experiments). (G) Antigen-dependent B cell differentiation in the spleen of bone marrow chimeric mice. Upper panel: Experiment scheme. Middle panel: FACS plot of splenic PB and PCs in the chimeric mice. Lower panel: Quantification, y axis: log2 of measured AIDR112H/WT-to-grafted AIDR112H/WT ratio; GC, DZ, and LZ: P < 0.0001; PC: P < 0.001; MB: P < 0.01, one-sample t test (n = 14–22, three experiments). (H) GC response in the bone marrow chimeric mice. Left panels: Representative FACS plot; right: quantification, CD45.1 WT versus CD45.2 AIDR112H; GC, P < 0.0001, paired t test (n = 33, four experiments); DZ, P < 0.0001; LZ, P < 0.0001, two-way ANOVA (n = 14, three experiments); DZ/LZ ratio, P < 0.0001, paired t test (n = 14, three experiments). Value and error bar: mean ± SD. MB, memory B cell; T1, transitional 1; T2 MZP, transitional 2 marginal zone progenitor; MZB, marginal zone B cells; FoB, follicular B cells. *P < 0.05, **P < 0.01, ***P < 0.001.