TNF-α production indicates monocytes as primary interacting cells with EVs, and blockade of CD40 or CD40L accessory molecules decreases EV-induced T-cell augmentation response. (A) Intracellular staining of PBMCs for TNF-α and cell surface staining for monocytes (**P < .01), B cells (*P < .05), and T cells. Data are representative of 4 independent experiments, 6 hours post-stimulation of PBMCs with EVs. (B) Left: The absolute counts of exosomes and microvesicles were measured by TruCount beads (n = 3). Right: Three PBMC sets were stimulated with exosome or microvesicle fractions derived from a unique EV sample, and intracellular TNF-α was measured after 6 hours. Stimulation with exosomes but not microvesicles led to production of TNF-α in monocytes (*P < .05). (C-D) PBMCs were stained with CFSE. Anti-CD40 or anti-CD40L blocking antibodies were added to the culture in the presence of PHA+EV. Cells were harvested at day 7 and stained for CD3, CD4, and CD8 cell surface markers. Addition of blocking antibodies reduced augmentation of CD4⁺ and CD8⁺ T cells (*P < .05). Data are representative of 4 independent experiments.