Fig. 3

RGS10-null naïve CD4+ T cells displayed intact mitogen-mediated activation and polarization into Th1 or Th17. a CD4+ T cells were isolated from spleens of WT and RGS10-null mice. Cells were treated with PMA (20 ng/ml)/ionomycin (1 μM) or anti-CD3/CD28 (5 μg/ml) in vitro for 72 h. Proliferation was measured by MTS incorporation assay and (b) cytokine production was measured by multiplexed immunoassays (MSD). *, p < 0.05, ***, p < 0.001, two-way ANOVA. c Splenic naive T cells (CD4+ CD25−) were isolated from WT and RGS10-null mice. Cells were differentiated in vitro under Th1 (top panels) or Th17 (bottom panels) polarization conditions as described in the “Methods” section. PMA/ionomycin and protein transport inhibitors were added for the last 5 h of the culture period. Cells were then stained with mAbs to surface markers and intracellular cytokines and analyzed by flow cytometry. CD4+ T lymphocytes were evaluated for IFNγ and IL-17A expression. Representative FACS plots indicate the percentage of CD4+ T cells that stained positive for IFNγ or IL-17A. Two independent experiments with similar results were performed (n = 2–3 mice in each experiment)