These experiments also demonstrated that PBMCs loaded with CD154+/gp350+ exosomes were constantly better lysed than PBMCs loaded with CD154+/gp350-exosomes. This prompted us to investigate whether T cells specific for gp350 accounted for this improved lysis. Therefore, we tested the cytolytic activity of these T cells against EBV-infected lymphoblastoid cells (LCLs), which are known to be targets for gp350-specific T cells [21] , and against EBV-negative B blasts [22] from an HLA-DR13+ matched healthy donor. We found that the T cells efficiently lysed allogenic LCLs, whereas they completely ignored EBV-free B blasts, indicating the presence of EBV-specific T cells in the effector population ( Figure 3 ). These data corroborate that the stimulation of PBMCs with CD154+/gp350+ carrying exosomes is an efficient method for the activation of B-CLL cells and the reactivation and Immunoblots demonstrated that vesicles carry gp350 and that these vesicles co-sediment with vesicle that carry the exosome markers hsp70, tsg101, CD63 and ganglioside (GM)1 (www.exocarta.org). Calnexin is present in cell lysates (CL), its absence from exosomes demonstrated the purity of the preparation. (B) Latex beads were coated with gp350+ exosomes and stained with a gp350-specific antibody. gp350 is accessible and thus probably located in the exosome membrane (isotype control is shown as grey tinted histogram). (C) gp350 confers B-cell tropism to exosomes. PBMCs from a healthy donor were incubated for 18 h with gp350+/gfp+ exosomes and then analyzed for GFP fluorescence by flow cytometry. Only CD19+ B-cells stained positive, whereas an interaction of gp350+ exosomes with CD3+ T cells and CD19-cells (T and NK cells, monocytes) could not be observed. An immunoblot with a CD154-specific antibody revealed that CD154 is highly expressed in transfected cell and that the protein is incorporated into exosomes. Shown are immunoblots of cell lysates (CL) and lysates from purified exosomes (EL) of normal and transfected 293 cells, incubated with CD154-(upper panel) and tsg101-specific antibodies (middle panel). An OptiPrep TM gradient revealed co-sedimentation of CD154-and gp350-carrying vesicles (see Figure 1A ). (B) Exosomes carrying gp350+ specifically bind to CD19+ B cells from a B-CLL patient. Fresh B-CLL cells were incubated with purified exosomes from 293 cells transfected with a gp350 expression plasmid and binding of exosomes was measured by flow cytometry. (C) Exosomes carrying CD154 bind only weakly to B-CLL cells, probably through interaction with CD40, which is highly expressed on these cells. Binding of CD154-carrying exosomes is drastically enhanced by the co-expression of gp350. 293 cells were co-transfected with expression plasmids for CD154 and/or gp350. Fresh B-CLL cells were co-cultivated with CD154+/gp350-and CD154+/gp350+ exosomes for two days and binding of exosomes and thus transfer of CD154 was measured by flow cytometry. 