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...

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Extensive interest in cancer immunotherapy is reported according to the clinical importance of CTLA-4 and (PD-1/PD-L1) [programmed death (PD) and programmed death-ligand (PD-L1)] in immune checkpoint therapies. AXL is a receptor tyrosine kinase expressed in different types of cancer and in relation to resistance against various anticancer therapeutics due to poor clinical prognosis. AXL and its ligand, i.e., growth arrest-specific 6 (GAS6) proteins, are expressed on many cancer cells, and the GAS6/AXL pathway is reported to promote cancer cell proliferation, survival, migration, invasion, angiogenesis, and immune evasion. AXL is an attractive and novel therapeutic target for impairing tumor progression from immune cell contracts in the tumor microenvironment. The GAS6/AXL pathway is also of interest immunologically because it targets fewer antitumor immune responses. In effect, several targeted therapies are selective and nonselective for AXL, which are in preclinical and clinical development in multiple cancer types. Therefore, this review focuses on the role of the GAS6/AXL signaling pathway in triggering the immunosuppressive tumor microenvironment as immune evasion. This includes regulating its composition and activating T-cell exclusion with the immune-suppressive activity of regulatory T cells, which is related to one of the hallmarks of cancer survival. Finally, this article discusses the GAS6/AXL signaling pathway in the context of several immune responses such as NK cell activation, apoptosis, and tumor-specific immunity, especially PD-1/PDL-1 signaling.

Tumor immunotherapy, capable of inducing both cellular and humoral immune responses, is an attractive treatment strategy for cancer. It has been reported that the inactivation of cell-mediated immunity by hyper-activation of humoral immunity-referred to as immune deviation-does not inhibit tumor growth. We investigated the ability of several adjuvants to elicit Thomsen-Friedenreich (T/Tn)-specific humoral immunity while avoiding immune deviation and conferring protection against tumorigenesis. T/Tn (9:1) antigen was purified from blood type O erythrocytes donated by healthy Korean volunteers. Immunization was performed using T/Tn only, T/Tn mixed with Freund's adjuvant (T/Tn+FA), keyhole limpet hemocyanin (KLH)-conjugated T/Tn mixed with FA (KLH-T/Tn+FA), or oxidized mannan-conjugated T/ Tn mixed with FA (ox-M-T/Tn+FA). Anti-T/Tn antibodies were generated in the T/Tn+FA, KLH-T/Tn+FA, and ox-M-T/ Tn+FA groups. The antibody level was highest in the KLH-T/Tn+FA group. Mice immunized with ox-M-T/Tn+FA showed specific complement-dependent cytotoxicity, and were protected against T/Tn-positive mammary adenocarcinoma cell challenge, although anti-T/Tn antibody levels were the highest in the KLH-T/Tn+FA group. These results demonstrate that an ox-M-conjugated T/Tn vaccine mixed with FA can promote cellular immunity while moderating the humoral immune response, thereby effectively inhibiting tumor growth.

Inducing cancer-specific cellular immune responses has become an attractive strategy in cancer treatment. In this study, we investigated the role of several adjuvants in eliciting T/Tn-specific cellular immunity and protection against T/Tn expressing tumor challenge. T/Tn (9:1) antigen was purified from blood type “O” erythrocytes donated from healthy Korean volunteers. Immunization was performed using: T/Tn only, T/Tn mixed with Freund’s adjuvant (T/Tn + FA), keyhole limpet hemocyanin (KLH)-conjugated T/Tn mixed with FA (KLH-T/Tn + FA), and oxidized mannan-conjugated T/Tn mixed with FA (ox-M-T/Tn + FA). Mice immunized with ox-M-T/Tn + FA generated T/Tn-specific CD3, helper T (Th) cells, major histocompatibility complex (MHC) II, and MHC I; T/Tn presentation was significantly high and tolerogenic CD11b+ was the lowest among the tumor models. To verify Th type, we stained intracellular cytokines (interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and IL-10) using CD3 co-staining. Th1 (IFN-γ and GM-CSF) cytokines were highly expressed and showed high FasL/Fas ratios, cytotoxic T lymphocyte (CTL) activity, and cytotoxic T lymphocyte precursor (CTLp) activity in mice immunized with ox-M-T/Tn + FA. Lymphocyte infiltration was highest in mice immunized with ox-M-T/Tn + FA. Additionally, we monitored FasL, MHC I, CD301, and T/Tn expression levels using immunohistochemistry (IHC) on macrophage and tumor sites. The expression of all markers was highest in the ox-M-T/Tn + FA group. Furthermore, tumor retardation and survival rate were highest in the ox-M-T/Tn + FA group. These results demonstrate that a vaccine formulation of T/Tn conjugated with ox-M and mixed with FA-induced cellular immunity and sustained a humoral immune response without over-activating the immune system, thus effectively inhibiting tumor growth.