Transfer of CLL-EVs containing miR-363 to CD4⁺ T cells downregulates the expression of the immunomodulatory receptor CD69. (A) Potential binding sites for miR-363 in the 3′ UTR of human CD69 identified by TargetScan v7.0. (B) Diagram showing the position of the 3 miR-363–binding sites (labeled 1, 2, and 3) (TargetScan v7.0) in the CD69 3′ UTR. The 3′ UTR has a total length of 995 bp and site 1 is at 758 bp, site 2 at 870 bp, and site 3 at 907 bp. (C) Plots demonstrating conservation of miR-363–binding sites (horizontal black lines). Sequences from 6 species (human, mouse, dog, cat, cow, and rabbit) were compared (weblogo.berkely.edu). The overall height of the stack indicates the sequence conservation at that position, whereas the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position. (D) Luciferase activity, in HEK293T cells, following cotransfection of reporter constructs with either miR-363 mimic or scrambled control miRNA expressed as percentage of luciferase activity following transfection of the reporter construct alone. Five reporter constructs were tested: wild-type (WT), construct-bearing mutations of site 1 (labeled 1), construct-bearing mutations of site 2 (labeled 2), construct-bearing mutations of site 3 (labeled 3), and construct-bearing mutations of sites 1, 2, and 3 (labeled 1, 2, 3). The miR-363 mimic caused repression of luciferase activity compared with the scrambled control unless all 3 miR-363 sites were mutated (n = 4). Mean ± SEM. The 2-tailed Student t test was used (***P < .001, **P < .01, *P < .05). (E) Flow cytometry histograms showing repression of CD69 expression in the human Jurkat T cells following transfection with miR-363 mimic. T cells were either transfected with scrambled control miRNA (solid blue line) or miR-363 mimic (solid red line) and stimulated with PMA/ionomycin. FACS histograms for control unstimulated cells are shown as red and blue dotted lines. Bar chart shows that miR-363 mimic caused a significant (Student t test, *P < .05) reduction in MFI (n = 4). (F) Incubation of primary CD4⁺ T cells with autologous CLL-EVs following CD40/IL-4 stimulation increased intracellular levels (relative change) of T-cell miR-363. Mean ± SEM from 3 separate experiments are shown (Student t test, **P < .01). (G) Flow cytometry analysis revealing that incubation of primary CD4⁺ T cells with autologous CLL-EVs following CD40/IL-4 stimulation (red line) led to significant suppression of CD69 expression following crosslinking of T cells with anti-CD3/CD28 compared with the treatment of T cells with control CLL-EVs from unstimulated CLL cells (blue line). Dotted lines are the respective isotype control experiments. The bar chart shows changes to MFI expression of CD69 following treatment with CLL-EVs (Student t test, *P < .05) (n = 4). FACS, fluorescence-activated cell sorter; MFI, mean fluorescence intensity; n.s., not significant; PE, phycoerythrin; Scr, scrambled; SEM, standard error of the mean.