CD4⁺ T cells exposed to autologous EVs from CD40/IL-4–stimulated CLL cells exhibit enhanced immunological synapse signaling and proliferation. (A) Confocal images of T-cell F-actin immune synapse formation (F-actin rhodamine phalloidin, red) interactions with autologous tumor CLL cells (CMAC-labeled, blue) from a representative patient. CD4⁺ T cells (negatively selected) were treated with purified 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 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. 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. Quantitative image analysis measured the total area (μm²) of F-actin polymerization (F-actin rhodamine phalloidin, red) at CD4⁺ T-cell contact sites and synapses with CLL cells within the experimental T-cell population. The dot plot shows the mean synapse/interaction area (μm²) ± SEM from a representative patient (from 6 independent CLL samples) (Mann-Whitney t test, **P < .01). (B) Intercellular CD4⁺ T cell: tumor CLL conjugates formed between CD40/IL-4 CLL-EVs or CLL-EVs treated CD4⁺ T cells and sAg-pulsed autologous CLL tumor cells were scored using a confocal microscope (scale bar, 5 μm) and image analysis software. Bar chart shows the mean percentage CD4⁺ T-cell:tumor CLL conjugates ± SEM from 6 independent CLL samples with 100 random T cells analyzed per experiment (Mann-Whitney t test, *P < .05). (C) CD4⁺ T-cell:tumor CLL conjugates formed during 20 minutes were fixed, permeabilized, and stained with anti-Lck (green). Tumor cells were labeled with CellTracker blue dye, CMAC. T cells are denoted by white dashed lines. Quantitative image analysis (relative recruitment index [RRI]) of Lck accumulation at the immunological synapse is shown, representative evaluation of 20 conjugates from 3 independent experiments, with the mean value shown as a blue bar ± SEM. Scale bar, 5 μm. (D) MFI was calculated for CD4⁺ T-cell CD69 expression levels (Alexa 488, green fluorescent channel) following treatment with CD40/IL-4 CLL-EVs or CLL-EVs T cells are denoted by white dashed lines. Tumor cells were labeled with CellTracker blue dye, CMAC. Dot plot chart shows the CD69 MFI ± SEM for a representative patient from 6 CLL samples (Mann-Whitney t test, **P < .01). Original magnification, ×60. Scale bar, 5 μm. (E) Nuclear Ki-67 expression, a marker for cellular proliferation, was measured in autologous patient CD4⁺ T cells exposed to CLL-EVs (CD40/IL-4 CLL-EVs or CLL-EVs) and with and without anti-CD3/-CD28 crosslinking stimulation for 72 hours (-CD3/-CD28 and unstim., respectively). Bar chart shows mean Ki-67 ± SEM for 3 independent CLL patient experiments (1-way ANOVA, **P < .01). (F) The confocal images show increased nuclear (DAPI, blue) Ki-67 expression (Alexa 647 denoted using green with analysis software) in CD4⁺ T cells (F-actin, red) treated with CD40/IL-4 CLL-EVs compared with coculture with control CLL-EVs. Scale bar, 5 μm. (G) Z-stack confocal analysis (3-dimensional central T-cell region analysis) reveals increased nuclear (DAPI, blue) Ki-67 expression (Alexa 647 denoted using green with analysis software) in CD4⁺ T cells (F-actin, red) treated with CD40/IL-4 CLL-EVs compared with control CLL-EVs. Scale bar, 5 μm. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole.