B cells infiltrate liver and thymus of aged Aire-deficient mice. (A) Number of lymphocytic infiltrates in liver sections. n = 11 WT, 11 KO. Each symbol corresponds to a different animal, and median is indicated by horizontal lines. P value was calculated with Mann-Whitney test. (B) Immunofluorescent stainings for CD3, B220 (×20/1.0 NA objective magnification) and CD9 (×10/1.0 NA objective magnification) were performed on cryostat sections of livers from 3 aged Aire KO mice. Negative control stainings were performed on adjacent sections by adding secondary antibody in the absence of primary antibody. In addition, the negatively staining anti-CD3 antibody is an isotype control of the anti-B220 and anti-CD9 antibodies. Arrows point at the site of lymphocytic infiltration. (C) The follicular versus marginal zone phenotype of the liver B-cell infiltrates was measured by FACS. Leukocytes were gated on forward scatter/side scatter (FSC/SSC) by comparison with a spleen, and then CD19⁺ cells were further gated. n = 6 WT, 5 KO. Two representative mice are shown. (D) Aire WT or KO thymi were FACSed for B220 expression (1 KO and 1 WT shown). Histograms shown are gated on lymphocytes from FSC/SSC. In an additional experiment, Aire WT or KO thymi were investigated by FACS for CD19 expression. Histograms shown are gated on CD3^– lymphocytes.