Exosomes : immunomodulators in cancer and therapy

Abstract: Exosomes are nano-sized membrane vesicles derived from the late endosomal compartment. They are capable of transferring proteins, lipids and RNA between cells. B cell and dendritic cell (DC)-derived exosomes express major histocompatibility complex (MHC) class I and II, as well as costimulatory molecules (CD80/86) and can initiate T cell responses. Several clinical trials have shown DC-derived exosome-based cancer immune therapy to be safe but limited in inducing antigen-specific T cells. In contrast, tumour cell-derived exosomes can express immune inhibitory molecules and play an important role in spreading oncogenic activity by carrying tumour antigens, inducing angiogenesis at distant sites and preparing tissues for metastasis. This thesis aimed at I) analysing how to enhance the immunogenicity of exosomes for therapy, II) investigating whether MHC complexes on exosomes are needed to induce an anti-tumour immune response, III) comparing microvesicles and exosomes side by side for their immunogenic capacity, IV) understanding the metastatic process induced by tumour-derived exosomes from bladder cancer patients and whether certain exosomal proteins can be used as markers for diagnosis and prognosis. Study I reveals that exosomes loaded with the NKT cell ligand alpha-galactosylceramide (αGC) and the model antigen ovalbumin (OVA) activate NKT cells, induce strong NK and γδ T cell innate immune responses, and induce OVA-specific T and B cell responses far better than only OVA-loaded exosomes. Exosomes loaded with αGC/OVA decreased tumour growth and increased median survival compared to exosomes loaded with OVA only or soluble αGC + OVA alone in a B16 melanoma model. This study demonstrates how to increase the immunogenicity of DC-derived exosomes for cancer treatment. Study II demonstrates that exosomal MHC class I is dispensable for the induction of antigen-specific T cell responses if whole OVA is present. We show that OVA-loaded DC-derived exosomes from MHCI-/- mice induce antigen-specific T cells to the same extent as wild type exosomes. Even exosomes with MHC class I and II mismatch induced tumour-infiltrating CD8+ T cells and increase survival in a B16 melanoma model. This study provides new opportunities for the design of allogeneic exosome-based vaccines and therapies. Study III compares microvesicles (MV) and exosomes from OVA-exposed DCs side by side for their capacity to induce OVA-specific immune responses in vivo. MV and exosomes express similar surface markers but only exosomes induced OVA-specific CD8+ T cells and OVA-specific IgG antibodies. In contrast, MV induced a higher number of plasma cells. Finally, we found that exosomes contain more OVA compared to MV. We conclude that exosomes from DCs are superior in inducing antigen-specific immune responses in vivo compared to MVs, while MVs might activate the immune system unspecifically. Study IV evaluates the proteomic profile of exosomes from tumour tissue explants and urine from urinary bladder cancer patients. We show that exosomes from malignant or benign tissue can be distinguished by the proteomic profile and are involved in platelet, metabolic and immune signalling networks. We show that, even if no tumour is left, exosomes can express a metastatic memory phenotype which might be involved in cancer progression. In summary, this thesis gives new insights into how to design vesicle-based cancer vaccines and provide new opportunities for the use of allogeneic DC-derived exosomes in patients. In addition, we demonstrate that exosomes isolated from the urine of urinary bladder cancer patients express specific markers for malignancy, which provides new possibilities for diagnostic strategies.