IL-21 induces IL-10 production during the differentiation of B10 cells via activation of p-STAT3.

<p>(<b>A</b>) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 for the indicated times and stimulated with PIB for the final 5 hours. STAT3 mRNA expression was detected by real-time RT-PCR. (<b>B</b>) The expression of p-STAT3 and STAT3 in sorted CD19⁺CD5⁺CD1d^high B cells from MRL/lpr mice and B6 mice was analyzed by Western blot. (<b>C</b>) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 for the indicated times. Then, p-STAT3 proteins were analyzed by Western blot. (<b>D</b>) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 or AG490 for the indicated times, and the levels of p-STAT3 and STAT3 proteins were determined by Western blot. (<b>E</b>) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 or AG490 for the indicated times and stimulated with PI for the final 5 hours. IL-10 in supernatants was detected by ELISA. (<b>F</b>) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without the indicated concentrations of IL-21 or AG490 for 48 hours and stimulated with PI for the final 5 hours. IL-10 in supernatants was detected by ELISA. (<b>G</b>) Sorted CD19⁺CD5⁺CD1d^high B cells from MRL/lpr mice were cultured in the presence of LPS and 10 ng/ml IL-21 with or without AG490 for 48 hours, and IL-10 in supernatants was detected by ELISA. (<b>H</b>) Naïve B cells sorted from B6 mice were cultured with or without 10 ng/ml IL-21 or SPI for 15 minutes, p-STAT3 proteins were then analyzed by Western blot. (<b>I</b>) Naïve B cells sorted from B6 mice were cultured in the presence of LPS with or without 10 ng/ml IL-21 or SPI for the indicated times and stimulated with PI for the final 5 hours, IL-10 in supernatants was then detected by ELISA. (<b>J</b>) CFSE-labeled CD4⁺CD25^- T cells from MRL/lpr mice were cultured for 3 days with anti-CD3 and anti-CD28 antibodies in combination with 20% of the supernatants from IL-21-induced B10 cell cultures (from MRL/lpr mice) with or without neutralization of IL-10. The proliferation of these cells was determined by flow cytometry. All the above results shown are representative of at least three independent experiments.