published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The search for anticancer drugs that are tumor specific and cause minimal side effects and the development of effective cancer vaccines are focal points of cancer therapy today. Dendritic cells (DC) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness in abrogating established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors.The studies in this dissertation describe a chemo-immunotherapeutic strategy, which combines a Vitamin E analog, a-tocopheryl succinate (a-TOS) that is selectively toxic to tumor cells with non-antigen pulsed, non-matured dendritic cells (nmDC) to treat established murine lung and breast tumors. The results demonstrate that a-TOS synergizes with nmDC to inhibit the growth of established tumors and significantly reduce residual lung metastasis when therapy is initiated after surgical removal of primary tumors. This outcome was correlated with increased IFN-g and IL-4 production by splenic and draining lymph node lymphocytes. In trying to understand the mechanism of action of the combination treatment we observed that a-TOS treated tumor cells factors cause DC maturation in vitro. This effect is mediated in part by heat shock proteins 60, 70 and 90 induced during a-TOS-mediated killing of tumor cells. This study demonstrates the potential usefulness of a-tocopheryl succinate, an agent non-toxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating cancer.

Cancer immunotherapy aims to use and manipulate the host’s immune system to fight against cancer. The objective of this strategy is to induce specific and persistent immune responses leading to tumor eradication. Heat shock proteins (HSP) purified from cancer tissues have been identified as unique mediators of specific anti-tumor immunity. In our laboratory, we have developed an original vaccine, termed CRCL (Chaperone-Rich Cell Lysate) that consists of multiple HSP complexes enriched from tumor lysates. CRCL immunization leads to an efficient protection against a wide variety of murine cancers by inducing a strong, long-lasting, and specific T and NK-cell dependent immune responses against the tumor from which it has been generated. Tumor-derived CRCL has been shown to be more efficient in triggering DC activation than individual purified HSP or tumor lysates. The immunostimulatory effects of CRCL arise from its superior ability to provide a wide variety of tumor antigens to the immune system and by providing potent adjuvant effects. However, CD4⁺CD25⁺ regulatory T lymphocytes (Treg) critically contribute to the mechanisms of cancer-induced suppression. Data from independent groups including ours suggests they may also restrain the function of antigen presenting cells. The current study was designed to elucidate the molecular signaling events triggered by the tumor-derived CRCL vaccine in antigen presenting cells and evaluate whether CRCL may overcome the inhibitory effects of Treg modulation of DC and macrophage activation. Our results indicate CRCL activates DC and macrophages by inducing proinflammatory cytokine chemokine secretion. CRCL induces iNOS expression and NO production in macrophages. CRCL activation of DC and macrophages results in transcription factor NF-κB activation in vitro and in vivo, and this includes the activation of additional signaling molecules upstream of NF-κB. Following CRCL treatment the phenotypic maturation of DC, the production of DC and macrophage pro-inflammatory cytokines, and the activation of the transcription factor NF-κB are not affected by Treg. Additionally, CRCL induced activation of DC is not diminished by the immunosuppressive cytokine TGF-β 1. Our results indicate tumor-derived CRCL-treated DC and macrophages are refractory to Treg inhibition. These results are important for advancing CRCL-based vaccines in Phase I clinical trials.

Among the complications of current treatments for hemophilia A, the development of anti-FVIII antibodies including “FVIII inhibitors” remains the major clinical problem in treating hemophiliacs. Factor VIII inhibitors work through neutralizing the coagulation cofactor activity of the infused FVIII and preventing the restoration of normal hemostasis. This thesis explains the influence of genetic background on the generation of FVIII inhibitors, introduces a new pre-clinical approach that reduces the immunological response towards FVIII and predicts the in vivo behavior of recombinant and plasma-derived FVIII products in hemophilic patients. First, we studied the influence of the genetic background on the formation of FVIII antibodies by treating hemophilia A Balb/c and C57BL/6 mice with repetitive FVIII infusions. We observed that the C57BL/6 mice developed higher FVIII antibody titers than the Balb/c mice. Our results suggest that differences in the cytokine immune responses due to FVIII in Balb/c and C57BL/6 mice are responsible for the different FVIII antibody titers in each of these strains. Second, we investigated the use of FVIII-pulsed immature dendritic cells in inducing immune tolerance against FVIII prior to the FVIII treatment. We showed that in vivo, FVIII does not induce the activation and proliferation of hemophilic T cells. Furthermore, infusing FVIII-pulsed immature dendritic cells into hemophilic mice resulted in a long-term reduction in immune reactivity towards FVIII. Also, we have proposed methods on how to improve the tolerogenic abilities of dendritic cells. Our results indicate that the immature dendritic cells induced the formation of T regulatory cells and that these T regulatory cells were responsible for the observed reduction in immune reactivity. Finally, we were able to identify the mechanisms behind the immune system activation in mice treated with either recombinant or plasma-derived FVIII products. We showed that plasma-derived FVIII results in reduced FVIII antibody titer formation in hemophilic mice. Our results demonstrate that the differences in antibody formation in hemophilic mice treated with either recombinant or plasma- derived FVIII products are due to the distinct cytokine micro-environment induced by each product. This thesis contributes to the current knowledge on FVIII immunology and the in vivo behavior of FVIII in hemophilic mice. The results generated from this thesis can be used to modify the available FVIII treatments in order to minimize the immunological complications of FVIII and improve the quality of life of hemophilic patients. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2008-08-14 18:22:51.56

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Diss. (sammanfattning) Göteborg : Univ. , 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Melbourne, Dept. of Medical Biology, 2006. / Typescript. Includes bibliographical references (leaves 189-208).

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 71-92). Also available in electronic version.