The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

Cancer is a disease that is caused by mutations in somatic cells. Metastasis is the major cause of death from cancer and often complicates treatment. Malignant tumours secrete degradative enzymes such as cathepsin L which degrade the extracellular matrix to facilitate tumour invasion and metastasis. The immune system does not normally recognize and eradicate tumours because they arise from self tissues to which the immune system is tolerant. Self antigens are poorly immunogenic because they lack T cell help. In this study, a foreign glucosidase was conjugated to self rabbit cathepsin L using glutaraldehyde to specifically provide T helper cell epitopes. The conjugate was used to immunise two male rabbits. A second pair of rabbits (male and female), was primed with sheep cathepsin L (to induce T helper cell activation) and received rabbit cathepsin L boosters. A third pair of rabbits which served as a control was immunised with sheep cathepsin L. The two pairs of test rabbits made high avidity antibodies against rabbit cathepsin L, showing a similar response to control rabbits when antibodies were tested in an ELISA. Western blot analysis showed that these anti-cathepsin L autoantibodies were specific for rabbit cathepsin L. Rabbits which were immunised with the conjugate were · inoculated with sheep cathepsin L nine weeks after the final inoculation with the conjugate. Analysis of antibodies in an ELISA showed that antibody responses against rabbit cathepsin L were augmented in a manner that is characteristic of memory responses. Low titre antibodies against sheep cathepsin L were also produced. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Current treatment methods for Acute Leukemia (AL) only provide temporary therapeutic efficacy as most patients will experience relapse within 2 years following first remission. Our lab has determined that vaccination with autologous cells infected with oncolytic virus MG1 can provide durable cures in a pre-clinical mouse model of AL. However, the mechanism(s) by which the infected cell vaccine (ICV) stimulates T cell dependent anti-tumour immunity and provides protection against tumour growth is unknown. This thesis was aimed to determine 1) what antigen presenting cell populations are activated post ICV immunization and 2) what T cell subsets are important in developing anti-tumour immunity during ICV immunization. My thesis has demonstrated that ICV immunization is more effective at inducing in vivo dendritic cell activation compared to irradiated L1210 cells alone and this activation may be a reason as to why we see improved anti-tumour efficacy in our ICV model. In addition, we have determined that CD4 T cells play an essential anti-leukemic role during ICV immunization and that neutralizing antibody production is a CD4 T cell dependent mechanism. Our data also demonstrates that both CD4 and CD8 T cell populations from ICV immunized mice provide a leukemia-specific anti-tumour immune response. Taken together, this data suggests that CD4 T cells may be acting as helper T cells to aid in the robust activation of leukemia-specific anti-tumour CD8 T cells. Our pre-clinical data characterizing the immune response has improved our understanding of the mechanism(s) which contribute to the efficacy of the ICV and will help provide a rationale framework with which to begin translating this treatment to clinical trials.

Submitted in part fulfillment for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2003. / Evidence suggests that the induction of tissue kallikrein, and the subsequently formed kinins, enhances proliferation of tumour cells because of their mitogenic property. Additionally, the kinin peptides are believed to promote the invasion of normal tissue by tumour cells. TGF-l is a potent inhibitor of the growth of renal epithelial cells, and is a classical anti-mitogen, which is central to many of its antiproliferative effects. No studies thus far have been performed, as to whether the proposed anti-mitogenesis ofTGF-1 has a regulatory effect on the cell proliferative action of kinins on renal epithelial and carcinoma cells. / M

Adoptive T cell immunotherapy is emerging as a powerful approach to treat diseases ranging from cancer to autoimmunity. T cell therapy involves isolation, modification, and reintroduction of T cells as “living drugs” to induce a durable response. A key capability to fully realize the potential of T cell therapies is effective manipulation of ex vivo T cell activation, with the aim of increasing T cell production and promoting specific phenotypes. While initial efforts to modulate T cell activation have heavily focused on mimicking biochemical signaling and ligand-receptor interactions between T cells and antigen presenting cells (APCs), there is increasing appreciation for understanding the role of mechanics at this interface and utilizing these insights to improve T cell activation systems. The aims of this dissertation is to contribute to this understanding by elucidating how mechanical properties of an activating surface regulate T cell activation, and apply these insights to generate biomaterial based systems to enhance activation from leukemia patient derived T cells. We first use a hydrogel system to investigate patterns T cell activation to substrate stiffness, discovering a biphasic response of T cell activation to stiffness that is synergist with ligand density. We then generate electrospun fiber scaffolds as an alternative platform to improve T cell expansion; we discover that 3D geometry in the form of fiber diameter and span lengths affects T cell activation. Lastly, we characterize the starting makeup of T cell populations from leukemia patients to study patterns of T cell exhaustion, utilizing the developed electrospun fiber scaffold system to enhance expansion of exhausted T cells from leukemia patients, and demonstrate patient-specific responses to different scaffold formulations. This approach allows for engineering of biomaterial designs that can leverage T cell mechanobiology to enhance T cell activation, with potential to be tailored to patient-specific expansion conditions and increasing the availability of T cell therapy to a wider range of patients.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22347号 / 医博第4588号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 江藤 浩之, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cancer immunotherapy, including immune checkpoint blocking antibodies are important components for treatment of patients with various types of cancer as they enhance the ability of the immune system to fight tumours. However, tumor cells have the ability to develop resistance to a variety of transitional therapies such as chemotherapy. In this study, in vitro Tumour-Immune co-culture system (TICS) has been developed to evaluate the impact on the antitumor activity of the primary human lymphocytes and response to PD-1 (nivolumab) and PD-L1 (durvalumab) checkpoint blocking antibodies against acquired chemotherapy resistance cancer cell lines. Using paired ovarian and neuroblastoma cancer cell lines obtained prior to chemotherapy (naïve) and after chemotherapy resistance, the results show that resistant ovarian cancer cells have differential effect on activation of lymphocytes and respond poorly to nivolumab and durvalumab, compared to chemotherapy naïve cells. On the other hand, chemotherapy neuroblastoma resistance cells show to respond to PD-1/L1 blockade therapy in TICS. Furthermore, blocking important molecular interactions between cancer cells and human lymphocytes such as HLA-ABC, HLA-DR and IFN-γ receptor compromises response to immune checkpoint blockade. In accordance, deletion of programmed death ligand 1 (PD-L1) on cancer cells by the CRISPR/Cas9 system significantly increases antitumor activity of immune cells in TICS. Moreover, deletion of beta-2-microglobulin (B2M) on human cancer cells resulted in substantial downregulation of HLA-ABC, which influenced immune activation induced by PD-1 blockade. Together, these findings demonstrate that chemotherapy resistance in human cancer cells could limit efficient response of PD-1/L1 blockade and thus immune checkpoint therapy could be more effective in early stage cancers.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21637号 / 医博第4443号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 松田 道行, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM