<p><strong>INTRODUCTION: </strong>Pre-clinical and clinical data strongly support the feasibility of employing immunotherapy as a strategy to treat cancer.</p> <p><strong>METHODS: </strong>Using the B16F10 murine melanoma model, we have been investigating mechanisms of T cell-mediated antitumor immunity following immunization with dopachrome tautomerase (DCT), a melanoma-associated antigen.</p> <p><strong>RESULTS: </strong>In <strong>Chapter 2</strong>, we uncovered an interesting dichotomy whereby DCT-specific CD4⁺ T cell-mediated tumor protection and autoimmunity are dependent on IL-4/STAT-6 and IFN-g/STAT-4, respectively. Our data also revealed that this phenomenon is extrinsic of CD4⁺ T cell polarization.</p> <p>To gain further insight into the targets recognized by CD4⁺ T cells, we conducted in <strong>Chapter 3</strong> extensive CD4⁺ T cell epitope mapping experiments using overlapping peptide libraries. Interestingly, while we were able to identify “helper” epitopes within DCT that were required for maximal CD8⁺ T cell expansion, we were unable to identify “effector” epitopes responsible for tumor rejection. Further examination of the requirements for the generation of CD4⁺ T cell effector epitopes showed that post-translational modifications of the protein were involved.</p> <p>In <strong>Chapter 4</strong>, we investigated the modest efficacy afforded by DCT immunization in the context of established B16F10 melanomas. Using intratumoral transcriptional analysis, we demonstrated that the vaccine rapidly promoted an IFN-g-dependent immunosuppressive state inside the tumor. Concurrent treatment with the immunomodulatory antibodies anti-4-1BB and anti-PD-1 effectively counteracted this tumor immunosuppression, resulting in complete regression of tumors and long-term survival in 70% of the mice.</p> <p><strong>CONCLUSIONS: </strong>The research described in this thesis sheds new light into the mechanisms by which vaccine-mediated CD4⁺ T cell responses participate to tumor rejection and autoimmunity. Moreover, our findings indicate that cancer vaccine-induced tumor immunosuppression significantly limits tumor regression, emphasizing the requirement of combinatorial approaches for successful cancer immunotherapy. Overall, our research offers new insight for future vaccine development.</p> / Doctor of Philosophy (Medical Science)

<p>Cancer vaccine therapy aims at harnessing effective antigen specific immune responses to treat tumor. In particular, CD8+ T cells have the unique capacity to recognize and destroy tumor cell throughout the body. One potential approach to elicit high numbers of effector CD8+ T cells to control tumor growth is through repeated vaccination, a strategy called prime-boost vaccination. However, booster immunization is relatively inefficient during primary immune response because pre-activated effector T cells tend to impair robust antigen presentation. This phenomenon has been interpreted as a negative feedback mechanism where recently activated CD8+ T cells clear the antigen-bearing dendritic cells (DCs) and prevent memory T cells from the access of the boosting antigen. Interestingly, however, using in vitro activated B cell as a viral vector delivery system, we can boost T cell responses with the minimum viral input at a very short interval between immunizations. This B cell carrier is capable of delivering different viral vectors expressing different antigens, displaying a potential for broad application. The mechanisms behind B cell carrier-mediated efficient secondary responses are three fold: 1. Without the engagement of MHC molecules and antigen presentation, B cell carrying viral vector can bypass the killing by pre-existing effector T cells 2. B cells can delivery viruses to B cell follicles, a place separated from effector T cells, and mediate memory T cell expansion. 3. B cells can deliver antigen to both spleen and lymph node and induce antigen specific T cell expansion in multiple lymphoid organs. Our studies provide a novel boosting platform to accelerate CTL responses that has important clinical implications.</p> / Master of Science (MSc)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

<p>IL-15 is a cytokine that has effects on both innate and adaptive immune cells, including NK and CD8 T cells. The involvement of these cell types in tumor immunosurveillance and eradication has led to interest in IL-15 as an immunotherapy. Its role in spontaneous solid cancers has not been studied thoroughly. Here, we have shown for the first time that IL-15 overexpression in a spontaneous breast cancer model, MMTV-polyoma Middle T antigen (MT), leads to increased survival, tumor destruction, and decreased metastasis (IL-15 TG/MT). In contrast, lack of IL-15 led to decreased survival (IL-15 KO/MT) and increased metastasis. Protection in IL-15 TG/MT mice was dependent upon the presence of highly activated NK1.1+ cells, but not dependent upon CD8 T cells. The cytokine environment in IL-15 TG/MT tumors was capable of activating human NK cells to kill human triple negative breast cancer cells. In a model of metastasis, we found that overexpression of IL-15 protected from metastasis in a NK cell dependent manner. Lack of IL-15 promoted the polarization of CD4 T cells to a Th2 phenotype and they influenced polarization of macrophages to an M2 phenotype. M2 macrophages help establish tumors at the metastatic site. Here we found that M1 polarized macrophages could prevent engrafted breast tumor formation, whereas M2 macrophages promoted it. Overall, IL-15 is an extremely promising immunotherapy that has more anti-tumor effects on the immune system than were previously appreciated. Additionally, our data argues that it could generate immune responses against both primary breast cancer and metastasis.</p> / Doctor of Philosophy (Medical Science)

Ultraviolet radiation (UVR) stimulates melanogenesis in melanocytes, primarily via release of alpha-melanocyte stimulating hormone from keratinocytes. UVR also induced an inflammatory response in the skin in which Interferon-gamma (IFNγ) cytokine plays an important orchestrating role. Here we report that recombinant IFNγ induces a temporal increase of melanogenesis in mouse melanoma cells. IFNγ elevates expression of microphthalmia-associated transcription factor (Mitf), which is the master regulator of melanogenesis by initiating transcription of melanogenic enzymes, tyrosinase (Tyr), tyrosinese-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct). Interestingly, tyrosinase protein, but not mRNA expression, accumulated in response to IFNγ treatment and was consistent with tyrosinase activity. In addition, glycosidase digestion showed that IFNγ induced ER-resistant, fully mature tyrosinase via post-transcriptional mechanisms, rather than increased de novo synthesis or early processing in the ER. Most strikingly, IFNγ mediated alkalization of melanosomes by elevating Oca2 expression, which leads to facilitate melanosome maturation and sequential accumulation of mature tyrosianse. Both Jak1/Jak2 inhibitor Ruxolitinib and knockout of Stat1 mediated by CRISPR-CAS9 blocked the IFNγ-induced Mitf, tyrosinase, Oca2 expressions and melanin biosynthesis. Our data reveals that IFNγ-Jak1/2-Stat1 axis regulates melanogenesis by inducing maturation of melanosomes and accumulation of mature tyrosinase via post-translational mechanisms. CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-gamma (IFNγ) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNγ activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence (GAS) site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNγ -response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. We also show that ectopic expression of Ctla4 in mouse melanoma cells promotes tumor growth in immunocompetent mice. Ctla4-enhanced melanomagenesis is blocked in immunodeficient NSG mice. In addition, ligation of CD86 (one of Ctla4 ligands) in T cells inhibits CD8 T cells proliferation in vitro. Expression of Ctla4 in melanoma cells are resistant to CD8 T cell cytoxicity in vitro. Our data demonstrates and highlights the novel and unrecognized functions of CTLA4 in melanoma cells that aids their survival, immunoevasion and tumorigenic capabilities. Taken together, these findings have potential implications for the conventional and prototypical roles of the IFNγ signaling pathway and CTLA4 in tumor immunosurveillance and tumor immunoevasion. More importantly, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses. / Biomedical Sciences

T lymphocytes engineered with chimeric antigen receptors (CARs) have shown remarkable success in the treatment of leukemias. Conventional CARs seek to recapitulate TCR and costimulatory signals through fusion of T cell signaling elements into a single receptor. The robust anti-tumor activity of CAR T cells is often accompanied by debilitating toxicities due to excessive T cell activation and cytokine production following infusion. Our lab has generated a novel chimeric receptor termed T cell antigen coupler (TAC), which is designed to engage native T cell signaling domains for cellular activation. In a murine xenograft model, we previously found that TAC T cells mediated rapid tumour regression in the absence of toxicities. Comparatively, CAR T cells elicited significant lethal toxicities to the mice due to reactivity against an unspecific antigen that resulted in excessive proliferation and cytokine production in vivo. Here, we report that TAC and CAR T cells have fundamentally different biology, both at rest, and during activation. TAC T cells were more sensitive to the context of stimulation compared to CAR T cells. Whereas TAC T cells can discriminate between antigen bound to a bead, or antigen present on a cell, CAR T cells do not make the same distinction and responds equally well to both. Compared to several different CAR constructs, TAC T cells are less prone to tonic signaling and T cell differentiation in the absence of antigen. These findings support that TAC T cells may pose a safety benefit as a cancer immunotherapy, due to its distinct biology from CAR T cells that enables them to require more stringent contexts for activation. / Thesis / Master of Science (MSc) / Cytotoxic T cells are also known as “resident killer” cells of the immune system, as they can seek and eliminate diseased or infected tissue, including cancer cells. However, cancer cells can evade elimination by T cells over time. Genetic engineering of T cells allows us to re-arm T cells against cancer cells. T cells isolated from a patient are genetically modified to recognize cancer cells specifically. So far, these modified T cells have been successful against several leukemias. However, the side effects of this treatment can be substantial and life-threatening, due to the massive reaction of the T cells against the cancer cells following infusion. We explore the biology of two different types of engineered T cells to better understand the interaction between T cell and tumour cell. Our results aim towards mitigating the side effects of T cell treatment, while investigating how we can improve its effectiveness for the future.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23603号 / 医博第4790号 / 新制||医||1055(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 上野 英樹, 教授 金子 新, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23837号 / 薬科博第152号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23845号 / 薬博第852号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM