Increased numbers of MZ and B1 B cells in AID^−/−µS^−/− mice compared with those of AID^−/−, μS^−/−, and WT mice. The B-cell populations of 8- to 12-week-old age-matched WT C57Bl/6, AID^−/− (AID), μS^−/− (μS), and AID^−/−µS^−/− mice were analyzed by flow cytometry and confocal microscopy. (A) Proportions of CD21^loCD23^hi follicular, CD21^hiCD23^lo MZ, and CD21^loCD23^lo (B1 and activated B cells) among splenic mature CD93⁻ B cells are shown. (B) Absolute numbers of mature follicular (FO), MZ, and CD21^loCD23^lo B cells were determined based on the gates defined in A. (C) Cells obtained by peritoneal lavage were counted and total numbers of B1 cells identified as IgM⁺Mac-1⁺ by FACS analysis were calculated, as shown in supplemental Figure 1B. (D) Confocal images of spleen sections show the structure of the white pulp with the T zone, stained with CD3ε (green), the follicles containing the follicular B cells, in pink, as result of the co-staining with IgD (red) and IgM (blue) and the MZ B cells forming a blue IgM⁺IgD⁻ outer ring around the follicles. Representative dot plots and confocal images are shown. Numbers in dot plots indicate the percentage of each population; results are representative of 3 independent experiments; *P ≤ .05, **P ≤ .01, ***P ≤ .005. White bars on confocal images represent 200 μm.