IL-21 induces activation of STAT1, STAT3, and STAT5 in human naive and memory B cells. Human naive, IgM memory and isotype-switched memory, or total memory, B cells were sort-purified from normal donor spleens. (A) These B cell subsets were cultured for ∼18 h with anti-Ig, rested, and then cultured in the absence (red histograms) or presence (blue histograms) of IL-21 for 30 min. Phosphorylation of STAT1, STAT3, STAT4, STAT5, and STAT6 was determined by intracellular staining. Histograms on the left show representative staining in naive and memory B cells. Right panels plot increase in mean fluorescence intensity of pSTATs in naive, IgM memory, and isotype-switched memory B cells cultured with IL-21; response of unstimulated cells were normalized to a value of 1.0. These values represent the mean ± SEM of two independent experiments using B cells from different donor spleens. Identical results were obtained when the B cell subsets were prestimulated with CD40L/anti-Ig. (B–D) Human B cell subsets were cultured for ∼18 h with anti-Ig, rested, and then left unstimulated or stimulated with IL-21 or anti-Ig for 15–30 min. Cells lysates were prepared and subjected to SDS-PAGE and Western blotting to detect phosphorylated or total STAT3 (B), phosphorylated or total ERK (C), or phosphorylated AKT or 14.3.3 as a loading control (D). B–D are representative of three to four similar experiments.