MiR-146a inhibits CD4⁺T-cell proliferation induced by TLR7-preactivated CAL-1 cells. (A) Flow cytometric analysis of T-cell proliferation induced by CAL-1 cells transduced with hTR control RNA or with miR-146a–expressing vectors that were preactivated for 48 hours with or without TLR7 agonist R848 (10 μg/mL). Preactivated CAL-1 cells were cocultured together with freshly isolated allogeneic CD4⁺ T cells (ratio CAL-1:CD4⁺ T cells = 1:1) after labeling with the CellTrace Violet membrane dye. After 6 days, T cells were analyzed for expression of CellTrace Violet and 7-AAD in CD3⁺ T cells. Percentages of CellTrace-Violet^lo7-AAD⁻CD3⁺ cells represent T cells that proliferated and are alive (lower left quadrant). CD4⁺ T cells activated with anti-CD3/CD28 beads are shown as a positive control for proliferation (gray histogram) as compared with CD4⁺ T cells cultured only with medium (white histogram). Shown is 1 representative experiment of 3. Numbers in plots represent percentages of cells that fall within the indicated quadrant. (B) Cells were analyzed as in panel A. Statistical analysis of CD4⁺ T-cell proliferation of 3 independent experiments. Only the mean percentages ± SD of CellTrace-Violet^lo7-AAD⁻CD3⁺ T cells are depicted. *P < .05. (C) After 6 days of coculture, CD4⁺ T cells were restimulated with PMA/ionomycin for 6 hours in the presence of Brefeldin A and analyzed by flow cytometry for IFN-γ expression. Shown are the mean percentages of IFN-γ⁺ T cells of 1 representative of 3 independent experiments. Error bars indicate SD values of measurements done in triplicate.