CD4⁺ T cells exposed to autologous EVs from CD40/IL-4–stimulated CLL cells exhibit enhanced CCL19-induced migration. Primary patient CD4⁺ T cells were treated with autologous CLL-EVs for 48 hours (CLL-EVs following CD40/IL-4 stimulation [CD40/IL-4 CLL-EVs], compared with control EVs from unstimulated CLL cells [CLL-EVs]). Treated T cells were then allowed to adhere to immobilized ICAM-1 for 10 minutes following exposure to the chemokine CCL19. Video microscopy (Biostation IM; Nikon) was used to observe the migration of CD4⁺ T cells for 20 minutes before the migration of individual cells was tracked (NIS-Elements analysis software; Nikon). (A) Representative phase-contrast images (original magnification, ×20) and motility tracks (colored) of CD4⁺ T cells from CLL patients treated with experimental CLL-EVs as denoted (top microscopy images). Representative migratory tracks of individual T cells from CLL patients treated with CLL-EVs are shown in the bottom data plots (n = 20 cells per experiment). Dot plot charts show the (B) speed of migration (micromolar per minute) and (C) distance of migration (μm) for a representative patient from 6 independent CLL samples (Mann-Whitney t test, **P < .01).