Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC typedistinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other welldescribed exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and doserelated manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine. © 2004 Wiley-Liss, Inc. Key words: exosomes; membrane vesicles; hMUC1; Hsc70; cell-free tumor vaccine Exosomes were initially described as small membrane vesicles (40 -90 nm in diameter), which are released from reticulocytes in order to eliminate unnecessary proteins, such as transferrin receptor (TfR) or acetylcholine esterase, during the process of their final maturation into red blood cells. [1][2][3][4] These extracellular vesicles are also produced by various kinds of hematopoietic cells, including mast cells, platelets, T lymphocytes, B lymphocytes and dendritic cells (DCs), as well as by intestinal epithelial cells. [5][6][7][8][9][10][11][12][13] Exosomes are known to originate from the inward budding of limiting membrane of multivesicular bodies (MVBs), and these internal vesicles of MVBs are released into the extracellular space by fusion of MVBs with the plasma membrane. 14 Proteomic analysis of DC-or B-cell-derived exosomes revealed selective enrichment of a subset of cellular proteins, including antigen-presenting proteins such as MHC class I and II molecules, heat shock proteins (HSPs), targeting-related MFG-E8 and tetraspanins such as CD9, CD63, CD81 and CD82. [15][16][17][18] In addition, a recent study by Hegman et al. has demonstrated that exosomes secreted by human mesothelioma cells contain a discrete set of proteins associated with antigen presentation, signal transduction, migration and adhesion. 19 The function(s) of exosomes are not well defined but have been reflected by their protein composition and cellular origins. 14,20 Although exosomes are known to discard membrane proteins such as transferrin receptors in reticulocytes, several lines of studies have sug...

Human B£-1 is an anti-apoptotic Bcl-2 family member. Here, we found that B£-1 was converted into a potent death-promoting protein by green £uorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-B£-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-B£-1. Apoptosis induced by GFP-B£-1 was signi¢cantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-£uoro-methyl ketone, but was not blocked by either Bcl-xL or B£-1. Our ¢ndings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins. ß