EVs from CD40/IL-4–stimulated CLL cells rapidly enter autologous CD4⁺ T cells. CD4⁺ T cells from CLL patients were incubated with fluorescently PKH67-labeled CD40/IL-4 CLL-EVs for the indicated times before washing and fixation (0 time point, 24 and 48 hours). EV transfer studies were followed by confocal imaging. (A) Quantification of EV internalization using ImageJ software. MFI of PKH67-EVs ± SEM (n = 3). **P < .01. (B) The confocal images show maximal uptake of PKH67-labeled CLL-EVs into autologous CD4⁺ T cells by 24 hours (CD4⁺ T-cell F-actin, rhodamine phalloidin [red], PKH67-labeled CD40/IL-4 CLL-EVs [green], CD4⁺ T-cell nuclei [blue], and a merged image [bottom right of image panels]). Scale bar, 20 μm. (C) Confocal images showing serial sections of confocal micrographs (CD4⁺ T-cell F-actin, rhodamine phalloidin [red] and PKH67-labeled CD40/IL-4 CLL-EVs [green]) showing internalization of CLL-EVs within CD4⁺ T cells by 24 hours. Scale bar, 5 μm.