Figure 3.

FasL⁺MHCII⁺ exosome production by EBV-transformed B-LCL. Epstein-Barr virus (EBV) has been used for many years to experimentally transform human B cells into immortalized B lymphoblastoid cell lines (B-LCL). The figure is a schematic of the methods recently employed to demonstrate the killer function of B-LCL-derived exosomes. Use of the non-replicative laboratory EBV strain, B95-8, resulted in consistent induction of FasL expression by transformed B-LCL. These B-LCL packaged FasL along with MHC class II molecules into exosomes that were then spontaneously released into supernatants of the B-LCL cultures grown in exosome-free media. Multiple rounds of centrifugation at increasing speeds removed cells, debris, and larger vesicles from the culture supernatant, which was then spun at 110,000 × g to pellet the exosome fraction. Resuspended exosomes were exposed to exogenous tetanus toxoid peptide or a bacterial superantigen, washed, and then added to cultures of autologous T_H cells. The method resulted in T_H cell death that was exosome and antigen-dependent, and FasL-mediated as demonstrated by the use of FasL-neutralizing antibodies.