京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18129号 / 医博第3849号 / 新制||医||1001(附属図書館) / 30987 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 髙折 晃史, 教授 五十嵐 樹彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19267号 / 医博第4031号 / 新制||医||1011(附属図書館) / 32269 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 松岡 雅雄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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Advancements of antiretroviral therapies (ART) have made significant strides in reducing human immunodeficiency virus (HIV) viral loads in patients to undetectable levels. Upon interruption of ART, viral load rebounds and AIDS symptoms return. Latent reservoirs of virus are responsible for this phenomenon because they contain integrated provirus, which is transcriptionally silent, thus unaffected by ART and hidden from host immune surveillance. A commonly proposed mechanism for HIV latency is the presence of host cell transcription factors that lead to transcriptional silencing. CD4+ T cells and other immune cells, whether due to their subset phenotype, activation state, or stage in development, will vary in their battery of transcription factors. Of particular interest is Bcl11b, a critical transcription factor involved in the commitment to a T-cell fate during thymocyte development that has recently been shown to play a role in silencing HIV-1 transcription. Bcl11b is required for inhibiting the development of natural killer cell-like traits during the early development of T cells. The repressive role of this zinc-finger transcription factor has recently been shown to inhibit HIV-1 transcription in the context of microglial cells via recruitment of chromatin remodeling factors. Also, Bcl11b has been shown to interact with other HIV-1 transcriptional silencing factors such as NuRD and NCoR. Preliminary mass spectrophotometry results have pointed to a physical interaction of Bcl11b with NELF, another proven repressive factor of HIV transcription. We hypothesize that Bcl11b represses HIV transcription and is recruited to the HIV-1 long terminal repeat (LTR) through a paused RNA polymerase II complex, contributing to the establishment and maintenance of latency. Our studies confirm Bcl11b's repressive role in T cells, and investigate the mechanism with NELF. Transfection of HEK293T cells with HIV-LUC shows nearly 50% reduction in HIV transcription in the presence of Bcl11b, and analysis of viral protein output by p24 ELISA confirms this result. Furthermore, when co-transfected with NELF-B, the two transcription factors lead to nearly 90% reduction in HIV transcription. Results suggest that these factors cooperate to repress HIV transcriptional elongation. Protein and chromatin immunoprecipitations (ChIP) were also performed to see a direct interaction between the two transcription factors and the HIV LTR. Physical interaction of the two factors was not witnessed, while ChIP analysis shows enrichment of RNA polymerase II at the transcriptional start site suggesting Bcl11b increasing RNA polymerase II pausing. We conclude that Bcl11b plays a repressive role in HIV transcription through promoter-proximal pausing with a synergistic effect with NELF, but a yet to be identified factor is responsible for the coordination of the two factors. As an important T-cell commitment factor, Bcl11b may play an important role in establishing and maintaining cellular latency through transcriptional repression via a complex with NELF. Confirming Bcl11b's role as a repressive transcription factor and providing further support for a synergistic role with NELF, could highlight a new target for therapeutic strategies against the elusive latent reservoir.

<p>The most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.</p><p>Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both <italic>in vitro</italic> and <italic>in vivo</italic> using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials. </p><p>In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.</p> / Dissertation

Cancer is one of the most pervasive health problems in the world today. Despite major advances in its treatment in recent decades, conventional therapies have seen limited success. Aggressive, drug-resistant cancer cells can reemerge after treatment, resulting in relapse. Immunotherapy, a strategy that utilizes a patient's own immune system to specifically destroy cancer cells, is a potential solution to this problem. Immunotherapy, however, is limited by multiple mechanisms of cancer-induced immunosuppression. One of the most important of these mechanisms is the induction of Treg, which are capable of suppressing multiple arms of the anti-cancer immune response. In the current study, we evaluated strategies to hinder the deleterious function of Treg on cancer immunotherapy. First, we determined that imatinib mesylate could inhibit Treg function in vivo and in vitro and increase the efficacy of dendritic cell-based immunization against an imatinib-resistant lymphoma. Then, searching for further methods to inhibit Treg, we found that Th-1 cells were capable of inhibiting Treg function and synergizing with a tumor lysate vaccine to treat leukemia. This process was dependent on IFN-γ secretion by the Th-1 cells. While investigating the influence of Th-1 on Treg and the resulting immunomodulatory effects of these cells in vivo, we discovered that they were capable of promoting the non-conventional direct tumor killing function of DC. We determined that Th-1 induce the cytotoxic function of bone marrow-derived DC generated with GM-CSF and IL-4 by a mechanism dependent on IFN-γ. Finally, because our results indicate that the antigen presenting function of KDC may depend upon their cytotoxic ability, and since DC generated with IL-15 have been reported to be more efficient APC than those generated with IL-4, we evaluated their ability to also function as direct tumor cell killers. We found that while IL-15 DC can indeed kill tumor cells, only LPS and not IFN-γ was capable of inducing this capability. These findings contribute to both arms of anti-cancer immunity by impairing immunosuppression with imatinib and Th-1, and promoting anti-tumor immunity with KDC. This double-pronged approach may contribute to further strategic advances in the field of cancer immunotherapy.

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No description available.

Early during an immune response, dendritic cells (DC) interact closely with CD4+ T cells, and cross-talk between these cells can come in the form of tumour necrosis factor (TNF) superfamily ligand-receptor interactions. These signals are critical for the maturation, function and survival of DC, and thereby dictate the capacity of DC to prime a robust T cell response. Among these cues, helper T cell-expressed CD40L interaction with DC-expressed CD40 is required to fully mature DC for cross-priming of help-dependent CD8+ T cell responses. The lymphotoxin-beta receptor (LTβR) is another TNF family receptor on DC, and it’s ligands LTα1β2 and LIGHT are expressed on activated T cells. Since abrogated LTβR signaling impairs T cell immunity, we have examined whether LTαβ represents another possible helper T cell-derived cue for full DC maturation. However the LT pathway controls lymphoid tissue organization and DC homeostasis, a second possible mechanism explaining the necessity of LTβR signaling for T cell immunity. Here we dissect the role of helper T cell-expressed LTβR ligands and DC-intrinsic LTβR signaling, independent of DC homeostasis or lymphoid organization, in DC function and T cell immunity. Absence of LTα1β2 and not LIGHT on helper T cells results in compromised T cell priming by DC ex vivo, and LTβ-/- CD4+ T cell responses are impaired in vivo. Ag-specific CD4+ T cell-expressed LTα1β2 and DC-intrinsic LTβR signaling are required for an optimal cytotoxic T lymphocyte (CTL) response in vivo. While CD40 induces IL-12 and is required for CTL function, DC-intrinsic LTβR signaling is necessary for CTL activation and expansion, early up-regulation of CD86 and IFNα/β production. Our results reveal non-redundant roles for distinct TNF family receptors in enabling DC to program different features in Ag-specific CD8+ T cells.