FIGURE 5.

(A) Single vesicle imaging indicates that WT Jurkat T cells produce several distinct subsets of EVs. The CD47⁺ subset is highly enriched in α4β1 and the tetraspanin CD81. The tetraspanins CD9 and CD63 are in a distinct subset of EVs that lacks CD47, some of which contain other β1 integrins. Loss of CD47 by mutation in these cells results in increased release of EVs bearing one or more of the three tetraspanins. CD81 remains preferentially associated with α4β1 integrin, but more CD81⁺ EVs are present that lack α4β1 integrin. (B) PC3 prostate carcinoma cells do not express α4β1 integrin. Therefore, although CD81⁺ EVs are more abundant than in Jurkat cells, fewer colocalize with CD47 and β1 integrins. PC3 cells also produce more abundant CD9⁺ and CD63⁺ EVs, which do not colocalize with CD47. In contrast to Jurkat cells, CRISPR/Cas9 deletion of CD47 in PC3 cells decreases the abundance of CD81⁺ and CD63⁺ EVs, and more of the CD9⁺ EVs colocalize with β1 integrins