Mechanism of EV-induced immune augmentation. T cells were stained with CFSE and cultured alone or after restoration of monocytes for 7 days in PHA or PHA+EV conditions. Cells were stained for CD3, CD4, and CD8 cell surface markers. (A) Stimulation of purified T cells with low (0.5 µg/mL) or high (5.0 µg/mL) PHA concentration. (B) Addition of purified monocytes to purified T cells prior to addition of low concentration of PHA+/−EVs. (C) Schematic experimental design for testing the effect of EVs on purified T cells or purified monocytes. (D-E) Incubation of EVs with purified CD4⁺ (D) or CD8⁺ (E) T cells or purified monocytes for 1 hour followed by washing and mixing with purified monocytes or T cells, respectively, and as outlined in diagram (C). Data are representative of 3 independent experiments (*P < .05, **P < .01).