NPC exosomes induce apoptosis in EBV-specific CD4⁺ T cells through galectin-9/Tim-3 interaction. (A) NPC exosomes (C17) containing galectin-9 induce apoptosis in EBV-specific CD4⁺ T cells (GB3C clone, anti-EBNA3C) without significant effects on polyclonal CD4⁺ T cells sorted directly from PBMCs (CD4⁺ PBMC). Tested cells were incubated for 5 hours with purified C17 exosomes at a final concentration of 100 μg/mL exosomal proteins (corresponding approximately to 10 ng/mL galectin-9; Exo C17). Control cells were incubated without exosomes (W/O Exo). Cell clusters visible at the top of the upper right portions of the graphs are related to nonspecific necrosis. (B,C) NPC exosomes (C17) induce apoptosis in EBV-specific CD4⁺ cells from the JM1H2 clone (anti-gp350). Apoptosis of CD4⁺ T cells is almost entirely suppressed by preincubating the T cells with a blocking anti–Tim-3 antibody or by preincubating the exosomes with the 9M1-3 antibody directed to the galectin-9 CRD2. In contrast, apoptosis induced by NPC exosomes is not prevented by a nonspecific Ig (NS IgG) or a monoclonal antibody directed to the extracellular portion of the DR-α chain (anti–DR-α, DA6.147). Flow cytometry graphs representative of some experiments summarized in panel B are displayed in panel C. PI-positive/annexin V–negative cells were consistently detected in the presence of anti–Tim-3 and anti–galectin-9 antibodies even in the absence of NPC exosomes, without satisfactory explanation. (D) NPC exosomes (C17) induce apoptosis in EBNA1-specific CD4⁺ cells. Percentages of annexin V–positive and annexin V/PI-positive cells are presented on the right side of the panel. Spontaneous cell death is more prevalent in this clone compared with GB3C and JM1H2: 3.8% annexin V and 21.42% annexin V/PI-positive cells in the absence of exosomes (W/O Exo). Nevertheless, the percentage of annexin V–positive cells is dramatically increased by the incubation with C17 exosomes, in the absence of blocking antibodies (Exo⁺ NS IgG). This effect is almost entirely reversed by anti–Tim-3 or anti–galectin-9 antibodies (Exo⁺ anti–Tim-3, Exo⁺ anti–Gal-9).