miR-17 modulates CD4⁺ T cell effector responses by targeting TGFβRII and CREB1. (A) Schematic representation of the putative miR-17–binding sites in the 3′UTR of tgfbr2 and creb1. (B-C) A portion of the 3′UTR of tgfbr2 (NM009371 position 2171-2552) or creb1 (NM133828 position 3442-3792 containing site 3644 or position 6758-7144 containing site 6984) was cloned downstream of a luciferase reporter and transfected into an NIH3T3 cell line stably expressing miR-17, miR-20a, or mock control. The luciferase activity was measured 72 hours after transfection. Bar graphs show the means ± SD of 3 (B) or 6 (C) independent experiments. (D-E) CD4⁺CD25⁻ T cells from WT or KO mice were transduced with indicated retrovirus, and CD4⁺GFP⁺ T cells were FACS sorted and total RNA and protein were extracted for qPCR (D) and Western blot (E). The bar graph shows means ± SEM from 3 independent experiments. (F) T cells were transduced with indicated virus and cultured under iTreg-differentiation conditions for 5 days. CD4⁺GFP⁺ T cells were then sorted, lysed, and analyzed for Smad3 Ser423/425 phosphorylation by Western blot. (G-I) 5C.C7 T cells were primed and transduced with retrovirus containing both the CREB1-IRES-GFP expression cassette and the indicated miR-17 expression cassette, miR-17 alone with GFP marker, or GFP only. (G) Assessment of CREB1 expression in 5C.C7 T cells by qPCR. The graph shows means ± SEM from 3 independent experiments. (H) Death profile of CD4⁺GFP⁺ T cells after restimulation with anti–CD3/CD28 Abs. Left: representative FACS plot; right: bar graphs showing means ± SEM from 3 independent experiments. (I) 5C.C7 T cells were transduced as indicated and cultured under iTreg-differentiation conditions for 5 days. The percentage of CD25⁺Foxp3⁺ cells inside of the CD4⁺ T-cell populations and the MFI of the intracellular Foxp3 staining were measured. The numbers on the left corner show the means ± SEM of the percentage of iTreg cells from 3 independent experiments. *P < .05; **P < .01; and ***P < .001.