Fig. 1.

FO B cells in spleen of K5-TSLP mice showed activated phenotype. (A) Alteration of cell surface phenotype of K5-TSLP FO B cells. After 3 weeks of 1 mg ml⁻¹ dox treatment, single cell was isolated from normal littermate control mice (NLC) or K5-TSLP mice spleens and analyzed with flow cytometry. Cells were gated on FO B cells (B220⁺CD21/CD35^lowCD23⁺). Data (left) represent one of more than seven independent experiments; right, summary of mean fluorescence intensity relative to NLC. *P < 0.005. (B) Proliferative response of spleen B cells from K5-TSLP or NLC mice. B cells purified from spleens were cultured with indicated stimulations for 24 h. Cells were pulse-labeled with [³H]-thymidine during the last 8 h. Error bars, SD of three replicates. Data represent one of three independent experiments. (C) In vitro BrdU incorporation assay of splenic B cells from K5-TSLP or NLC mice. B cells purified from spleens were cultured with 40 μM of BrdU and indicated stimulations for 24 h. Cells were washed, fixed with ethanol, stained with anti-BrdU antibodies and propidium iodide and analyzed. Numbers near gated areas indicate percent of the population. Data represent one of two independent experiments. (D) In vivo BrdU incorporation in immature (B220⁺CD21/CD35⁻CD23⁻) and FO B cells in NLC and K5-TSLP mice. Mice were given i.p. inoculations of 0.8 mg of BrdU in PBS at 12-h interval during the last 3 days of 3-week dox treatment. Splenocytes were stained with antibodies for surface antigens, fixed and permeabilized. Following DNase treatment, cells were stained with anti-BrdU antibodies and analyzed. Data represent one of two independent experiments.