Fig. 2

CD19⁺ Foxp3⁺ B cells exist in spleens of collagen-induced arthritis (CIA) mice. CIA was induced in 6- to 8-week-old male DBA/1J mice by intradermal immunization in the base of the tail with Cll/CFA emulsion on day 0, followed by a booster injection on day 21, as described in the “Methods” section. a Purified B cells isolated from the spleen of CIA mice were cultured with 10 µg/ml LPS or 10 µg/ml anti-IgM. Following 72 h of activation, Foxp3 mRNA (left) and protein (right) levels were determined by RT-PCR and Western blot, respectively. The optical densities of Foxp3 bands were normalized to those of β-actin. b Spleens of DBA/1J or CIA mice (5 weeks after primary CII immunization) were stained with CD19-FITC, CD4-PerCP cy5.5, and Foxp3-PE mAbs and analyzed by confocal laser microscopy. The numbers in the graphs indicate the cell count of positive cells counted microscopically. c Kinetic analysis of Foxp3-expressing B cells in the spleens of CIA mice. Original magnification, ×400. d The expression of CD19 and Foxp3 in CIA mice splenocytes. The data represent one of three similar experiments. Bar graphs show mean ± SD. Statistical significance was defined at *p < 0.05 compared with 3 weeks after immunization, **p < 0.01, ***p < 0.001 compared with untreated cells