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Abstract

Vaccination has been regarded to as the most attractive immunotherapy for cancer because of its ability to instruct immune cells to recognize and kill tumor cells. Exosomes are one of the most recent discoveries translated from basic biology into experimental vaccine therapy of cancer. Exosomes are 30-150 nm membrane vesicles released by many cell types. In particular, dendritic cell (DC)-derived exosomes (Dexo) inherit the molecules that make DCs able to activate antigen-specific immunity. As a consequence, Dexo have been tested in clinical trials for the vaccination of melanoma and lung carcinoma patients, however resulting in poor therapeutic outcomes due to weak immunogenicity. We therefore developed a protocol for the production of Dexo with improved immunogenic properties by exploiting the physiological ability of DCs to mature into immunostimulatory cells in the presence of antigens and immunogenic signals. We cultured Dexo-producing DCs with a murine melanoma cell lysate, that provided tumor antigens, and poly(I:C), a synthetic nucleic acid analog that provided immunogenic signals. The result was a Dexo vaccine capable of eliciting robust tumor-specific immunity in melanoma-bearing mice, leading to significantly reduced tumor growth and enhanced survival of experimental mice. These data indicate our strategy as a valuable improved alternative to currently available protocols for the production of potent tumor-tailored Dexo vaccines. Healthy tissues are spared from immune aggression via the establishment of immune tolerance to self antigens. The liver has recently been attributed an important role in peripheral tolerogenesis to circulating extracellular antigens, mainly due to the antigen scavenging properties of liver sinusoid endothelial cells. However, even though hepatocytes are the most abundant cell type of the liver and possess enzymatic activities necessary for processing of blood-borne molecules, their ability to take up and induce tolerance to extracellular antigens has yet to be explored. In this thesis we show for the first time in vitro and in vivo that hepatocytes efficiently take up, process and present extracellular antigens on MHC-I complexes inducing CD8+ T cell tolerance. We also show that the negative regulatory PD-1/PD-L1 pathway is involved in hepatocyte-dependent tolerogenesis. Therefore, we propose that hepatocytes are instrumental in the establishment and maintenance of liver-mediated peripheral tolerance towards extracellular antigens reaching the liver through the bloodstream. In addition, this thesis also provides evidence that hepatocytes are capable of directly expressing antigens specific of other peripheral tissues, a process known as promiscuous gene expression. Promiscuous gene expression allows to broaden the pool of self antigens presented to T lymphocytes in central and peripheral lymphoid organs, thus increasing the likelihood of inducing T cell tolerance to self antigens. We describe here for the first time that Deaf1, a transcription factor involved in promiscuous gene expression, is significantly expressed in murine hepatocytes, together with other well characterized peripheral tissue-restricted antigen-coding genes. Even though preliminary, these data further suggest a central role of hepatocytes in peripheral tolerogenesis and confirm the relevance of hepatocyte-targeted approaches as valuable immunotherapy candidates for the treatment of autoimmunity and hepatic chronic infections.

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