Knockdown of miR-363 in parental CLL cells prevents the ability of released CLL-EVs to confer altered functional properties to autologous CD4⁺ T cells. miR-363 was knocked down in CLL cells using LNA-based antisense oligonucleotides, prior to CD40/IL-4 stimulation and isolation of released EVs (anti-miR-363 compared with scrambled control [Con.] antisense miR oligonucleotides). CD4⁺ T cells (negatively selected) were treated with purified CLL-EVs from the transfected autologous parental CLL cells. Treated T cells were then conjugated with autologous CLL B cells (pulsed with superantigen [sAg] cocktail and labeled with CellTracker blue dye, CMAC). T-cell conjugates were analyzed by immunofluorescence and confocal microscopy as described in Figure 4. Confocal images (n = 10 per patient treatment sample) were acquired using an A1R confocal microscope (Nikon) and analyzed with NIS-Elements software (Nikon). Original magnification, ×60. Scale bar, 5 μm. Dot plot charts show (A) the mean F-actin polymerization (rhodamine phalloidin, red) synapse/interaction area (μm²) ± SEM from a representative patient (from 3 independent CLL samples), (B) the mean RRI of Lck accumulation (green) at the immunological synapse, representative analysis (±SEM) from 3 independent patient experiments and (C) the CD69 MFI ± SEM for a representative patient from 3 independent CLL patient samples. CD69 expression is denoted in white. Treatment of autologous CD4⁺ T cells with CLL-EVs derived from miR-363 knocked down cells (anti-miR-363) blocked modulation of T-cell migration rates when compared with EVs derived from control transfected tumor cells (Scrambled Con. miR). Dot plot charts show the (D) speed of migration (micromolar per minute) and (E) distance of migration (μm) for a representative patient from 3 independent CLL samples (Mann-Whitney t test, *P < .05, **P < .01).