Molecular Modifications and Functional Conditioning of Dendritic Cells (DC) for DC-Based Tumor Vaccines

This dissertation, "Molecular Modifications and Functional Conditioning of Dendritic Cells (DC) for DC-based Tumor Vaccines" by Kar-wing, To, 杜嘉詠, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Molecular Modifications and Functional Conditioning of Dendritic Cells (DC) for DC-based Tumor Vaccines Submitted by Kar-wing To for the degree of Doctor of Philosophy The University of Hong Kong February 2007 Dendritic cell (DC)-based tumor vaccine is a novel therapeutic approach for cancer treatment developed during the last decade. Unlike conventional cancer therapies, it aims to induce specific immunity against cancer cells. Despite limited success has been achieved so far clinically, it remains as a promising immunological approach in the battle against cancer. However, inconsistency over the effectiveness, clinical outcome and ways of DC generation points to a need for new strategies to enhance its general efficacy. DCs are the most potent antigen presenting cells (APCs) important for the activation of T cells, especially naive T cells. However, DCs are heterogenous cell populations which can be either immunogenic or tolerogenic, depending on their cellular transducing signals exerted on T cells. Abnormalities in DC functions may induce tolerance rather than immunity in cancer patients, therefore, the types or functional conditions, hence the immunogenic 'quality', of the DC employed are critical for developing effective DC-based vaccines. By molecular modifications and functional conditioning of DCs, the main objective of this study was to develop immunologically programmable DC vaccines with high efficacy. Two principal ways of DC modulation targeting both positive and negative arms of immune regulatory mechanisms, including over-expression of functional molecules important for DC immunogenecity and blockage of immunosuppressive factors, were evaluated. The roles of DC-associated cell surface functional molecules were first studied. DCs over-expressing CD40, CD80 and CD86 showed enhanced immunogenecity in vitro, but their in vivo anti-tumor effects were marginal as determined in a melanoma mouse model. Further analysis revealed that after tumor antigens loading, DCs changed their functional phenotype producing enhanced levels of IL-10 that suppressed IL-12 expression. IL-12 is a Th1 cytokine important for anti-tumor immunity. Subsequent experiments were designed for functional modulation of DCs by over-expressing IL-12, and by using an immunomodulator (Bergapten) best known for its anti-microbial activity. Again, despite enhanced immunogenic activities in vitro, modifications of DCs either by over-expressing IL-12 or treating with Bergapten failed to boost significant anti-tumor activity in vivo. Similarly, a marked reduction in IL-12 was accompanied by increased IL-10 expression of these cells after loading with tumor antigens. Subsequent experiments focused therefore on the negative regulator of DC functions, IL-10. Two molecular approaches were used to block endogenous IL-10 expression at the APC level, including DCs generated from IL-10 knockout (KO) mice and selective IL-10 gene knock-down (KD) by siRNA. The siRNA-treated DCs showed about 50% drop in IL-10 production, up-regulation of MHC class II and CD86, and enhanced allogenic T cell stimulating capacities. The level of IL-10 reduction however had limited effect on the in vivo anti-tumor activity. In contrast, DCs generated from the IL-10 KO mice showed markedly increased immunogenecity both in vitro