Pten is the primary target of miR-19b in regulating CD4⁺ T cell effector functions. (A) Schematic illustration of the predicted targeting sites for miR-19b and miR-17 within the 3′UTR of pten mRNA. (B) The full-length 3′UTR of pten (Pten-WT) or 3′UTR with mutations at the 2 miR-19b target sites (Pten-MU) were cloned downstream of a luciferase reporter and transfected into NIH3T3 cell lines stably expressing the indicated miRNAs. The luciferase activity was measured 72 hours after transfection. Bar graphs show the means ± SD of 3 independent experiments. (C) 5C.C7 T cells transduced with mock, mir-17-92, or mir-19b were sorted by FACS, and total RNA and protein were extracted for qPCR and Western blot. (D) Relative expression of Pten mRNA in CD4⁺CD25⁻ T cells from LNs and spleens of WT, f/+, and KO mice was measured by qPCR. T cells activated with plate-bound anti–CD3/CD28 Abs for 72 hours were lysed for protein quantification. qPCR data were normalized to SDHA and are shown as relative to mock or WT. Bar graph shows the means ± SEM for 3 independent experiments. (E) miR-19b–mediated regulation on PI3K signaling on antigen engagement was visualized by fluorescence video microscopy. As described previously,²⁷  the PH domain of Akt kinase was fused with GFP as an imaging probe to monitor the production of PIP3 through PI3K activation. miR-19b or mock vectors were expressed simultaneously with this PH-GFP–imaging probe in 5C.C7 T cells, which were challenged by CH27 APCs preloaded with MCC agonist peptide. Live-cell imaging was performed to monitor the initial signaling strength and the duration of PI3K activation. T cells with PH-GFP expression and stimulated by contact with a single APC were monitored for > 1 hour in a 3D manner, and the best focus plane was used for data analysis. The activity of PI3K was represented by measuring the ratio of the average probe fluorescent intensity in the synaptic region versus the average intensity in the rest cell area. Top panels: representative montages from the DIC channel; bottom panels: representative montages from the GFP channel. (F) The highest level of probe synaptic accumulation within the first 10 minutes of T:APC contact was used as the mark for the maximal activity of PI3K activation in the initiation stage (before the formation of mature immunologic synapse) of TCR signaling. (G) A similar measurement was performed at 1 hour after the initiation of TCR signaling. *P < .05; **P < .01; and ***P < .001.