京都大学 / 新制・課程博士 / 博士(医学) / 甲第24803号 / 医博第4995号 / 新制||医||1067(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 金子 新, 教授 伊藤 能永, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Kinesin family member C1 (KIFC1) is a minus-end-directed motor protein that is critically involved in microtubule crosslinking and spindle formation. KIFC1 is essential for supernumerary centrosomes, and it is associated with the initiation and progression of cancers. In the present study, we initially reviewed the The Cancer Genome Atlas database and observed that KIFC1 is abundantly expressed in most types of tumors. We then analyzed the gene alteration profiles, protein expressions, prognoses, and immune reactivities of KIFC1 in more than 10,000 samples from several well-established databases. In addition, we conducted a gene set enrichment analysis to investigate the potential mechanisms for the roles of KIFC1 in carcinogenesis. The pan-cancer analysis of KIFC1 demonstrates significant statistical correlations of the KIFC1 expression with the clinical prognoses, the oncogenic signature gene sets, the myeloid-derived suppressor cell infiltration, the ImmunoScore, the immune checkpoints, the microsatellite instabilities, and the tumor mutational burdens across multiple tumors. These data may provide important information on the understanding of the role and mechanisms of KIFC1 in carcinogenesis and immunotherapy, as well as on the clinical progression of a variety of cancers.

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

Bladder cancer, as one of the most common cancer types and with high recurrence risk, is considered a candidate for novel immunotherapy strategies. An important aspect of the research for immunotherapy drug development for bladder cancer is to study the tumor microenvironment (TME) and it’s immune contexture. Besides tumor-infiltrating lymphocytes (TILs) as the main drivers of anti-tumor response, recent studies revealed the importance of tumor-associated tertiary lymphoid structures (TLSs) and high endothelial venules (HEVs) in the TME. Structures similar to these were found to spontaneously form in the orthotopic MB49 model used for bladder cancer research in our group. The aim of this study was to perform a deeper characterization of the TME in this model, by using immunofluorescent staining and microscopy. Specifically, the co-localization of tumor infiltrating lymphocytes (CD8+ and CD4+ T cells, CD19+ B cells), CD11c+ dendritic cells and HEVs along with CCL21 signaling were analyzed within orthotopic MB49 tumors, with and without immune stimulation. The quantification of cells expressing CD8, CD19 and CD11c immune markers, CCL21 levels, vascular density and numbers of HEVs, showed higher densities within the immune-stimulated tumors, indicating a rapid effect of immune stimulation on increasing immune cell infiltration and vascular density after only 24 hours post CpG therapy. Also, the highest frequency of TILs, CCL21 chemokine and vascular density was located in regions of the tumor border indicating that these regions should be studied further in depth as a potential target for entry of cells to the tumor with immunotherapy or as a model of the tumor microenvironment since tumor cell density is maintained high in these locations.

No description available.

Background Cancer immunotherapy has yielded many successes. Yet to some hard-to-treat brain tumors, such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), it still lacks substantial improvement. Neoantigens resulting from mutations in malignant cells are the key targets for employing adoptive cell therapies. A novel therapeutical strategy may be developed based on the identification of T cell receptors (TCRs) targeting specific neoantigens. Methods Previous work had been done to provide essential materials, including candidate neoantigen peptides, human leukocyte antigen (HLA) genotypes, and peripheral blood mononuclear cell (PBMCs) from patients and healthy donors (HDs). Autologous antigen-presenting cells (APCs) and T cells were isolated from PBMCs for in vitro assays. The activation of T cells against peptides was evaluated by the upregulation of 41BB utilizing flow cytometry (FACS). The cell populations with positive signals were sorted through FACS for TCR sequencing directly or after rapid cell expansion. Results T cells and APCs from 12 HDs were isolated. T cells from 10 HDs were analyzed after in vitro stimulation. T cells from HD30 showed reactions to several public neoantigens; while T cells from HD49 and HD53 showed reactions also to private neoantigens restricted in GBM patient C6. Conclusion The upregulation of 41BB indicated the activation of T cells and the existence of reactive TCRs against either public or private neoantigens in some HDs. Those reactive TCRs and their encoding sequences were the fundamentals of future works. Due to practical reasons, TCR sequencing cannot be done within this project. In future works, wildtype peptides will be included to further validate the results, ensuring identified TCRs recognize neoantigens specifically. Furthermore, the identified TCRs will be cloned and transferred to freshly isolated T cells to confirm their functionality. Keywords Cancer immunotherapy, brain cancer, neoantigen, MHC/HLA, TCR

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer and urgently requires new therapies. Oncolytic viruses (OV) are a strong contender. OVs interact with immune components of the TME, which can be altered due to specific genetic mutations. The present study evaluates the impact of specific tumour mutations on the response to carboplatin, the current standard of care, and VSV∆M51, a promising OV candidate. After a study of genetically diverse models, constitutive KRas activation enhanced VSV∆M51 replication in-vitro and sensitivity in syngeneic in-vivo models. VSV∆M51 prolonged survival in syngeneic tumour- bearing mice with KRas, Trp53 and Pten mutations, including one tumour model that did not respond to carboplatin. Response to VSV∆M51 in-vivo was associated with activation of CD4+ and CD8+ T lymphocytes in the peritoneal TME. In summary, VSV∆M51-based immunotherapy has shown promise in diverse murine models of EOC bearing clinically relevant mutations.

In addition to protecting the brain from circulating pathogens and neurotoxins, the blood-brain barrier (BBB) limits both the delivery of drugs to the brain and the migration of neurological disease biomarkers from the brain into the blood. Focused-ultrasound blood-brain barrier opening (FUS-BBBO) addresses both of these transport limitations by transiently and noninvasively opening the BBB. Although originally designed as a drug delivery method, FUS-BBBO has also been shown to be an effective neuroimmunotherapy and method of improving liquid biopsy specificity for neurological disease. Prior to the work presented herein, the mechanism of FUS-BBBO neuroimmunotherapy remained poorly characterized and FUS-BBBO liquid biopsy remained poorly optimized. Initially, we present the temporal response of brain macrophages to FUS-BBBO. Due totheir role as the main phagocyte in the brain and the well-documented association between their dysfunction and neurodegenerative disease progression, we hypothesized that FUS-BBBO affects brain macrophage population composition and phenotype. Utilizing temporal single-cell RNA sequencing, we establish that treatment remodels the immune landscape via a number of processes including microglia proliferation, disease-associated microglia population size increase, and central-nervous-system associated macrophage recruitment. To further elucidate the functional role of the brain macrophage response to FUS-BBBO, we find that their depletion is associated with significantly decelerated BBB restoration. Secondly, we compare FUS-BBBO with two other methods of focused ultrasound neuroimmunotherapy, focused ultrasound neuromodulation (FUS-N) and focused ultrasound with microbubbles without BBBO (FUS+MB). FUS-N utilizes FUS parameters that alter neuronal connectivity via a combination of mechanosensitive receptor interactions and transient hypothermia without the injection of microbubbles (MB). FUS+MB is the combination of MB and FUS below the pressure threshold for BBBO (FUS+MB). FUS+MB has been shown to trigger morphological activation of brain macrophages and has proven efficacious as a method of immunotherapy within the peripheral nervous system. Due to the findings of brain macrophage modulation in response to FUS-BBBO, we compare brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s Disease (AD) pathology. We identify FUS-BBBO as the paradigm which maximizes brain macrophage modulation including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between FUS cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to characterize the effect of FUS-BBBO on brain macrophage distribution in both wild-type and Alzheimer’s disease animals. Given their relevance within neurodegeneration and perturbation response, we emphasize the distribution of three brain macrophage populations - disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find a genotype-specific redistribution of each population, with an overall trend towards increased interaction with the brain-cerebrospinal fluid barrier after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. Finally, we investigate the role of extracellular vesicles (EVs) in both the mechanism ofFUS-BBBO neuroimmunotherapy and as a method of improving FUS-BBBO liquid biopsy. EVs are lipid vesicles that are responsible for the transport and exchange of diverse cargo between cells and have been reported to modulate the immune system. Isolation of EVs has emerged as a method of improving biomarker detection. Prior to this study, the effect of FUS-BBBO neuroimmunotherapy on EV concentration and content remained unexplored. We investigate the concentration and content of isolated EVs from the serum of mice and Alzheimer’s Disease patients prior to and after treatment with FUS-BBBO. We illustrate a 100% increase in EV concentration one hour after treatment in both mice and patients. Furthermore, we illustrate an increase in murine EV RNA that is associated with the previously reported neuroimmunotherapeutic responses to FUS-BBBO including synaptic remodeling and neurogenesis. Finally, we illustrate an increase in AD biomarker concentration within the patient EVs three days after treatment that is proportional to the volume of blood-brain barrier opening. Overall, we establish that FUS-BBBO drug-free neuroimmunotherapy triggers complex brain macrophage modulation in a manner incomparable by other FUS neuroimmunotherapy paradigms. Furthermore, we illustrate the effect of FUS-BBBO on EV concentration and content in both preclinical and clinical experiments, indicating the role of EVs in FUS-BBBO neuroimmunotherapy and their utility as a method of improving liquid biopsy specificity. The results presented herein support the potential of FUS-BBBO as both a method of neuroimmunotherapy and a method of amplifying liquid biopsy specificity in Alzheimer’s Disease.

Immune checkpoint blockade (ICB) therapies have transformed the treatment landscape for advanced melanoma, extending patient survival and improving quality of life for numerous patients with a disease that was once considered to be universally fatal. However, despite the success of ICB for many patients, over half are either resistant to initial therapy, or develop resistance over time after an initial response. The mechanisms underlying this therapy resistance remain unclear for the majority of patients. We have recently identified loss of the co-stimulatory and adhesion molecule CD58 on melanoma cells as a driver for cancer immune evasion and ICB resistance. In this thesis, we use in vitro co-culture models of patient-derived melanoma cells and tumor infiltrating lymphocytes as well as in vivo patient-derived xenograft models to demonstrate the necessity of CD58 interactions with its ligand CD2 on T cells for T cell activation, tumor infiltration, and effector cytotoxicity. Furthermore, using genome-wide genetic and protein screening approaches, we identify CMTM6 as a positive regulator of CD58, and uncover its role in mediating CD58’s regulation of inhibitory PD-L1 signaling by binding to both proteins and preventing their lysosomal degradation. Thus, CMTM6 co-regulates these co- inhibitory and co-stimulatory signals such that, in the absence of CD58, CMTM6 becomes available to bind and stabilize additional PD-L1, enhancing its inhibitory signals to T cells. Finally, we identify a potential role for CD58 on T cells as a marker of effector memory T cells with enhanced effector and progenitor function. The CD58-CD2 axis therefore serves a multi-faceted, underappreciated role in melanoma cancer immunity, and may serve as a therapeutic target for enhancing anti-tumor T cell responses.

Oncolytic viruses (OVs) are promising tumor-selective treatments, and the efficacy of OV therapies has been shown to depend heavily on the successful delivery and spread of these agents within the tumor mass to generate profound immunostimulatory effects. We have previously reported the potential of vanadium-based compounds such as vanadyl sulfate (VS) as immune-stimulatory enhancers of OV immunotherapy. These compounds, in conjunction with RNA-based OVs such as oncolytic VSVΔ51, improve viral spread and oncolysis, leading to long-term antitumor immunity and prolonged survival in resistant tumor models as previously reported. This effect is associated with a virus-induced antiviral type I IFN response shifting towards a type II IFN response. Here, the systemic impact and the relevant immunological changes following VS/VSVΔ51 combination therapy were investigated to understand the immunological mechanism of action leading to improved antitumor responses. We screened for the secretion of chemokines and cytokines in vivo to understand the mechanism of action regulating the recruitment of immune cells to the tumor in the CT26WT tumor model following treatment. Additionally, the antigen-specific immune response was investigated to further identify the relevant immunological changes following treatment with the VS+VSVΔ51 combination. Our data revealed that VS+VSVΔ51 combination therapy significantly increased the levels of IFN-γ and IL-6, and other key important pro-inflammatory cytokines and chemokines. Improved tumor antigen-specific T-cell responses were observed following the combined therapy. Supported by relevant immunological changes and as a proof of concept for the design of more effective therapeutic regimens, we found that local delivery of VSVΔ51 encoded with IL-12 or with other transgenes in combination with VS further improved therapeutic outcomes in a syngeneic CT26WT colon cancer model. We found that CD8+ T cells and Natural Killer (NK) cells play significant roles in establishing the therapeutic efficacy that we observed; Furthermore, engineering new and targeted therapeutic platforms to impact the antitumor immune response further improves the therapeutic benefits of the combined therapy.