Exosomes from HeLa cells induce apoptosis in EBV-specific CD4⁺ T cells only when they contain galectin-9. (A) Purified exosomes released by transfected HeLa cells expressing either GFP or galectin-9 (l-isoform) were analyzed by Western blot. The first 2 lanes on the left contain total extracts from cells transfected with GFP and galectin-9 (Cell prot). Exosome proteins are analyzed in the next 2 lanes (Exo prot). CD63, which is barely detectable in total cell extracts, is much more abundant in purified exosomes. In contrast to CD63, galectin-9 is detected in exosomes only when these are derived from cells transfected with the galectin-9 gene. (B,C) There is no induction of apoptosis in CD4⁺ T cells (EBNA1 and JM1H2 clones) treated with control HeLa exosomes (Exo Hela-GFP). In contrast, treatment with galectin-9–positive exosomes induces apoptosis in a large majority of the EBNA1-specific CD4⁺ cells (Exo Hela Gal-9). For JM1H2 cells (specific for gp350), the rate of apoptosis induced by Hela galectin-9 exosomes amount to 60% of the rate observed with C17 exosomes at the same concentration. It is entirely prevented by preincubation of target cells with the anti–Tim-3 antibody. Flow cytometry graphs representative of some experiments summarized in panel B are displayed in panel C.