miR-146b negatively regulates CD4 T-cell proliferation. (A) TCR-mediated expansion of CD4 and CD8 T cells from 146b transgenic mice and littermate controls on WT and tPTEN^−/− backgrounds. Total T cells were stimulated with anti-CD3/CD28. Triplicate T-cell samples were collected at each time point and total live T-cell numbers were determined by Trypan blue exclusion. CD4 and CD8 numbers were calculated based on flow cytometric analysis at each time point. Data are representative of 4 independent experiments. (B) TCR-mediated expansion of CD4 T cells from 146a8 transgenic mice and littermate controls on WT and tPTEN^−/− backgrounds. The assay was performed as described in (A). Data are representative of 2 independent experiments, one for each founder line. (C) Quantification of flow cytometric analysis of Annexin V⁺ T cells stimulated as in (A) for 72 hours. Data are representative of 2 independent experiments. (D) Flow cytometric analysis of CFSE dilution over time by T cells labeled with 1 μM CFSE before stimulation as described in (A). Histograms are representative of duplicate samples and 2 independent experiments. All mice used were littermate pairs between 6 and 10 weeks old (C57BL/6). Data in (A-C) are presented as mean ± SD and statistics were calculated by Student t test (***P < .001, n.s. not significant).