SHP-1 negatively regulates IL-21–mediated Th17 development. (A) Representative IL-21 dose-response assay. CD4⁺ T cells from +/+ and me/+ C57BL/6 mice that were cultured under Th17-inducing with the indicated IL-21 concentrations followed by intracellular staining for IL-17A/F. Profiles show CD4⁺-gated population. (B) Relative Th17 T-cell generation in IL-21 dose-response assays comparing +/+ and me/+ T cells (maximum % Th17 cells: 23% +/+, 30% me/+). EC₅₀: +/+ cells 10 ng/mL IL-21 (n = 6); and me/+ cells 5 ng/mL IL-21 (n = 6; P = .01). (C) Representative graph of absolute numbers of IL-17⁺CD4⁺ T cells generated in IL-21 dose-response assays, as shown in panel A. (D) IL-21R surface expression of splenic CD4⁺ T cells. (E) CD4⁺ T cells (0.7 × 10⁶) purified from +/+, me/+, and me/me C57BL/6 mice were stimulated with IL-21 for the indicated times and assessed for STAT3 phosphorylation (Tyr705), STAT3, and β-actin protein expression by immunoblotting. Numbers represent relative band densities in arbitrary units. (F) Kinetics of IL-21–induced STAT3 phosphorylation presented in panel E. pSTAT3 band densities normalized to β-actin (left panel) and pSTAT3 band densities normalized to STAT3 (right panel). Data represent 2 or 3 independent experiments with multiple mice per genotype.