付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22121号 / 医科博第106号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 河本 宏, 教授 生田 宏一 / 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22371号 / 医博第4612号 / 新制||医||1043(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 文彦, 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

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Rag1-/- mutant zebrafish lack lymphocytes and were used to study the basis of acquired protective immunity in the absence of lymphocytes to the intracellular bacterium Edwardsiella ictaluri. This study morphologically identified and quantified lymphocyte like cells (LLCs) present in the liver, kidney and spleen of these fish. LLCs included Natural Killer (NK) cells and non-specific cytotoxic cells (NCCs) and were discriminated by size, and by the presence of cytoplasmic granules. The antibodies anti-NITR9, anti-NCCRP-1 (5C6) and anti-MPEG-1 were used to evaluate these cell populations by flow cytometry. Gene expression profiles in these tissues were evaluated after the Rag1-/- mutants were intra coelomically injected with the toll like receptor (TLR)-2 ligand, β glucan, TLR3 ligand, Poly I:C, or TLR 7/8 ligand, R848. The genes interferon y (infγ), expressed by activated NK cells and macrophages, tumor necrosis factor α (tnfα), expressed by activated macrophages, myxovirus resistance (mx) expressed by cells induced by IFNα, T-cell transcription factor (t-bet) expressed by NK cells and novel immune type-receptor 9 (nitr-9) expressed by NK cells were evaluated. The TLR ligands induced different patterns of expression and stimulated both macrophages and NK cells. Then fish were vaccinated with an attenuated mutant of E. ictaluri (RE33®) with or without the TLR ligands then challenged with WT E. ictaluri to evaluate protection. RE33® alone and each TLR ligand alone provided protection. Coministration of β glucan and RE33® or R848 and RE33® resulted in survival higher than RE33® alone showing an adjuvant effect. Tissue specific gene expression of ifnγ, t-bet, nitr9, NK cell lysin a (nkla), nklb, nklc and nkld were correlated to protection. The final component of this study was the development of tools to discriminate NK cell populations and evaluate the contribution of macrophages. Rag1-/- zebrafish were modified to express cherry red in lymphocyte like cells using the Lymphocyte specific tyrosine kinase (lck) promotor. Also, rag1-/- zebrafish were modified so that the gene encoding the proto-oncogene serine/threonine-protein kinase that is involved in macrophage training (raf1) is disrupted. This study indicated that the acquired protection in the absence of lymphocytes likely involves NK cells with possible contribution by macrophages.

T-cell lymphomas (TCLs) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas derived from post-thymic mature T- and NK-cells broadly classified peripheral (PTCL) or cutaneous (CTCL), indicating either a nodal or skin-homing disease, respectively. One of the main clinical challenges contributing to dismal outcomes for TCL patients is both the lack of curative treatment strategies and the high rate of relapse for currently approved therapies, underscoring the need for identification of novel targeted therapies for the treatment of TCL. While combination therapeutic strategies have been proposed and show great promise in pre-clinical and clinical trials for PTCL and CTCL, none are yet approved. Additional contributing factors toward the difficulty in studying TCL and the high rate of therapeutic failure is the highly heterogeneous genetic and molecular mechanisms driving TCL as well as the poorly understood role of non-tumor microenvironment cells in the pathogenesis of TCL. Indeed, while several studies have suggested that tumor associated macrophages play both a significant functional role in supporting tumor maintenance and are therapeutically targetable, less is known about potential tumor supporting roles of other cell microenvironment populations. Here, I used an unbiased and high-throughput approach to discover novel drug combinations in CTCL and to characterize at the single-cell level relevant molecular mechanisms driving T-cell lymphomagenesis. First, I demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an inhibitor of the epidermal growth factor receptor (EGFR) family, produces strongly synergistic antitumor effects in CTCL models, both in vitro and in vivo, using mechanisms of action that involve down-regulation of the JAK-STAT signaling pathway. This result suggests a potential therapeutic role for the combination of HDAC inhibitors with afatinib in the treatment of CTCL that had not been previously recognized. Second, we developed single-nuclei analysis on a cohort of 30 TCL (PTCL-NOS, AITL, epstein barr virus positive PTCL) and 6 normal patient samples to identify and deconvolute genomic and functional mechanisms contributing to T-cell lymphomagenesis. Here, I implemented a comprehensive bioinformatics pipeline for the analysis of sparse single-nuclei transcriptomic data and characterized heterogeneous molecular mechanisms driving T-cell lymphomagenesis, such as the upregulation of the PI3K-AKT-mTOR and WNT signaling pathways in a subset of 5/9 PTCL-NOS and 7/19 AITL samples, respectively. Additionally, I identified the enrichment of both the macrophage compartment in PTCL-NOS and AITL, and the specific enrichment of CD8+ T cells in AITL. These results suggest a correlation between patient-specific characteristics, such as mutational status, and possibly therapeutically targetable molecular mechanisms driving neoplastic cell growth that warrants further investigation.

Natural killer cells are innate immune effectors that kill virally infected or malignant cells. Natural killer cell deficiency (NKD) occurs when NK cell development or function are impaired, and individuals with NKD are susceptible to severe and recurrent viral infections. Several gene deficiencies result in NKD, including variants in MCM4, GINS1, MCM10 and GINS4, which are components of the CDC45-MCM-GINS (CMG) helicase. The CMG helicase unwinds DNA during replication and is expressed in any actively proliferating cell. NK cells are more strongly impacted by mutational deficiencies in helicase proteins than other lymphocytes, though the mechanisms underlying this susceptibility are not completely understood. NK cells from individuals with NKD as a result of helicase deficiency have increased DNA damage, cell cycle arrest, and replication stress. We found that activated NK cells undergo apoptosis and autophagy in response to this stress, unlike activated T cells. We also identified a patient with a damaging variant in CDC45 to further support these findings of the effects of replication stress on NK cells. This individual, due to broader involvement of the immune system, requires a wider definition of natural killer cell disease, termed NK IEI. However, this CDC45-deficient individual’s cells display disrupted cell cycle, increased DNA damage and replication stress, with upregulation of apoptosis genes in NK cells. These findings show that sensitivity to replication stress affects human NK cell survival and function and can contribute to NK cell deficiency and human disease.

In the field of cancer immunotherapy, NK cells are recognized for their ability to provide a form of innate immunity against tumor cells. However, the average abundance of NK cells in the blood can be as low as 5% of the total lymphocyte population. As a result, it has been a focus to find novel therapies to expand NK cells in vitro while subsequently enhancing the cytotoxicity of these cells. Previously-defined methods include the minimal expansion of NK cells with high levels of cytokines such as IL-2 and IL-15, as well as co-culturing NK cells with feeder cell populations that are genetically modified to express NK-stimulating factors. Another method involves the use of artificially-derived plasma membrane nanoparticles (PM21) that express membrane-bound IL-21 (mb21) to successfully expand NK cells by a factor of 103 in 14 days. Exosomes, which are cell-derived vesicles naturally secreted by cancer cells, may reveal a novel way to expand NK cells and enhance their cytotoxicity by taking advantage of the exchange of genetic information within the tumor microenvironment. To test this hypothesis, NK cells have been cultured with varying concentrations of exosomes derived from modified K562-mb21-41BBl (a chronic myelogenous leukemia cell line) and shown to achieve 200-fold expansion of NK cells from other PBMCs in 14 days, a growth comparable to that of PM-21 particles. In vitro assays as well as co-culturing with various tumor cell lines will determine the cytotoxicity of these expanded cells. Potentially, exosomes may be applied as an in vivo therapy for NK cell expansion.

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