Hematologic abnormalities of Aire^–/– spleens. (A) Extramedullary hematopoiesis of spleens from aged Aire KO and WT mice (×20/1.0 NA objective magnification); sections were stained with H&E. (B) Representative immunohistochemical analysis of 6-week-old (left panel) and 15-month-old (right panel) Aire WT and KO mice. At least 4 serial sections from each mouse were stained for MOMA-1⁺ (brown) metallophilic macrophages and CD1d^hi marginal zone B cells (blue). Objective magnification: ×10/1.0 NA for young mice, ×4/1.0 NA for old mice. The arrows in the left panels outline the thickness of the metallophilic macrophages, and the double arrow in the bottom right panel outlines the extent of the CD1d-positive marginal zone. (C) The follicular versus marginal zone phenotype of the splenic B cells was measured by FACS. The dot plots are gated on CD19⁺ lymphocytes; the histogram on the right shows the expression of CD21 on IgD^loCD1d^hi marginal zone B cells in WT (▪) vs KO (gray line). n = 6 WT, 5 KO. Two representative mice are shown. (D) One-hour incorporation of BrdU in CD19⁺CD1d^hi splenic marginal zone B cells of a WT versus a KO mouse with MZL. (E) PCR of splenic DNA at the immunoglobulin heavy-chain gene D-J rearrangement junction with fluorescent primer, analyzed with capillary electrophoresis. Length of the PCR product in base pairs is indicated on the top scale. n = 3 WT, 9 KO. Two representative mice are shown.