AID deficiency leads to an abnormal accumulation of GC B cells in the spleen. (A) WT and AID^−/− mice were immunized with NP-CGG; and 11 days later, cells were harvested to analyze GC formation via flow cytometry. B220⁺ GC cells were gated based on expression of Fas and GL7 on the cell surface. Absolute numbers of GC B cells were determined before and after immunization. (B) Splenic tissue was sectioned and stained for immunofluorescent microscopy. GCs were identified as IgD^low and PNA^high (shown in separate panels) at original magnification ×10 using a Leica confocal fluorescent microscope, and individual images were spliced together to form a complete topography of the spleen. Average GC areas and frequency of formation (identified as PNA^highIgD^low clusters located within IgD⁺ follicles) were determined and illustrated using Volocity software (Improvision) and Photoshop 7.0 (Adobe). Statistics represent comparisons between WT unimmunized (UI) versus AID^−/− UI and WT immunized (Imm) versus AID^−/− Imm. Data are mean ± SD of n = 5 to 8 mice per genotype. *P ≤ .05. ***P ≤ .001.