Loss of Bcl6 in T reg cells leads to humoral autoimmunity. (A) Histopathology analysis of SG, lung, and pancreas from WT and KO mice at 30 wk of age. Black arrows indicate the immune cell infiltrates in KO mice. Bars, 100 µm. n = 6 per group. (B) Anti-IgG immunofluorescent staining of SG and kidney. WT, n = 3; KO, n = 6 per group. Bars, 50 µm. (C) Anti-dsDNA autoantibodies in the sera of WT and KO mice were measured by ELISA. n = 9 per group. (D) Saliva flow rates were measured in WT and KO mice. n = 5–6 per group. (E) FACS staining (left), frequency quantitation (middle), and cell number (right) analysis of CXCR5⁺Bcl6⁺ Tfr cells in CD4⁺Foxp3⁺ cells in different organs from the old steady-state control and KO mice. n = 6 per group. (F) FACS staining (left), frequency quantification (middle), and cell number (right) analysis of Bcl6⁺CXCR5⁺ Tfh cells in CD4⁺Foxp3⁻ T cells from different organs of WT and KO mice. (G) FACS staining (left), frequency quantification (middle), and cell number (right) analysis of GL7⁺Fas⁺ GC B cells in B220⁺ cells from different organs of WT and KO mice. (H–J) After restimulation with PMA and ionomycin for 5 h, IFN-γ (H), IL-4 (I), and IL-17A (J) expression in CD4⁺ T cells from different organs was measured by flow cytometric analysis. All mice were 30-wk-old unmanipulated mice. n = 6 per group in F–J. SP, spleen. All experimental data were verified in at least two independent experiments. Bcl6^fl/flFoxp3Cre/Cre mice were KO, and Bcl6^fl/flFoxp3WT/WT mice from the Bcl6^fl/flFoxp3Cre/WTxBcl6^fl/flFoxp3WT breeder were used as control. Data shown are mean ± SEM; two-tailed t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significance.