Fig. 3

In vitro generation of regulatory B cells with suppressor activity by transfection of Foxp3. Splenic CD19⁺ B cells from DBA/1J mice were transfected with Foxp3 shRNA, a Foxp3 over-expression construct, or control plasmid DNA, and then stimulated with 10 µg/ml LPS, or 10 µg/ml anti-IgM for 72 h. a Relative Foxp3 mRNA levels in Foxp3 shRNA or Foxp3 over-expressing cells were determined by RT-PCR using primers specific for Foxp3 or β-actin. The relative quantity of Foxp3 was normalized to that of β-actin in each sample. b Cell viability of CD19⁺ B cells by MTT assay 24 h after plasmid transfection. c Suppression of the proliferation of responder CD4⁺CD25⁻ T cell stimulation with anti-CD3 by B cells. CD4⁺ CD25⁻ T cells isolated from the spleens of CIA mice were cultured with Foxp3 shRNA or Foxp3-transfected CD19⁺ B cells in the absence (left panel) or presence (right panel) of Cll. Proliferation was determined after 3 days of culture by ³H-thymidine incorporation. Cultures were harvested and cpm were determined. Values are the mean ± SD from triplicate cultures. Data represent one of the two independent mice