Characterization of CD19.CAR⁺EVs. (A) NTA (i); AFM analysis of circulating CD19.CAR⁺EVs (ii); western blot analysis of CD63, flotillin-1, and cytochrome C in circulating CD19.CAR⁺EV samples (T lymphocytes were used as controls for cytochrome C expression) (iii); and flow cytometry evaluation of CD63 and flotillin-1 expression in circulating CD19.CAR⁺EVs (iv). CD63 and flotillin-1 expression (red histograms) were analyzed using the related fluorescence minus one (FMO) as controls (blue histograms). Mean fluorescence intensity (MFI) ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (B) Transmission electron microscopy analysis of circulating CD19.CAR⁺EVs (scale bar, 100 nm). (C) Flow cytometry evaluation of CAR expression on circulating CD19.CAR⁺EVs. CAR expression (red histogram) was analyzed using the related FMOs as controls (blue histogram). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (D) Venn diagram shows the common and uncommon proteins identified in circulating and preinfusion CD19.CAR⁺EVs. Same amount of EVs (pool of 3 individuals per condition containing 3 × 10⁶ EVs) were compared when proteomic analyses were performed to parallel circulating vs preinfusion EV cargoes. The number 4 refers to the number of identified proteins (4, corresponding to the 1.41% of the total number of identified proteins) uniquely carried by circulating EVs, whereas 275 proteins (99.97 % of identified proteins) were those shared by circulating and preinfusion CAR⁺EVs, and 4 other proteins (1.41% of identified proteins) were carried only by preinfusion CAR⁺EVs. (E) NTA (i) and AFM analysis (ii) of preinfusion CD19.CAR⁺EVs; western blot analysis of CD63, flotillin-1, and cytochrome C in preinfusion CD19.CAR⁺EV samples (T lymphocytes were used as controls for cytochrome C expression) (iii); and flow cytometry evaluation of CD63 and flotillin-1 expression in preinfusion CD19.CAR⁺EVs (iv). CD63 and flotillin-1 expression (red histograms) were analyzed using the related FMOs as controls (blue histograms). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments.