京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19147号 / 生博第330号 / 新制||生||44(附属図書館) / 32098 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 稲葉 ｶﾖ, 教授 米原 伸, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Green open access: Authors can share their research in a variety of different ways and Elsevier has a number of green open access options available. We recommend authors see our green open access page for further information (http://elsevier.com/greenopenaccess). Authors can also self-archive their manuscripts immediately and enable public access from their institution's repository after an embargo period. This is the version that has been accepted for publication and which typically includes author-incorporated changes suggested during submission, peer review and in editor-author communications. Embargo period: For subscription articles, an appropriate amount of time is needed for journals to deliver value to subscribing customers before an article becomes freely available to the public. This is the embargo period and it begins from the date the article is formally published online in its final and fully citable form. This journal has an embargo period of 12 months. 詳細は以下のアドレスを参照https://www.elsevier.com/journals/immunology-letters/0165-2478?generatepdf=true / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19870号 / 生博第351号 / 新制||生||46(附属図書館) / 32906 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 稲葉 ｶﾖ, 教授 米原 伸, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

No description available.

Sphingolipids are found in essentially all animals, plants and fungi, and some prokaryotic organisms and viruses. Sphingolipids function as structural components of membranes, lipoproteins, and as cell signaling modulators and mediators. To complicate matters further, sphingolipids often vary in type in different regions of tissues, and even in single cells, the subcellular localization of sphingolipids and their metabolic enzymes, transport proteins and targets may influence their functions. It is important to study sphingolipids spatial distribution within living organisms to understand how sphingolipids are involved in complex biochemical processes. As part of this thesis, procedures were optimized for the use of matrix assisted laser desorption/ionization (MALDI) tissue mass spectrometry (TIMS) to visualize the location of several types of lipids including sulfatides (ST), gangliosides and phosphoglycerolipids in brains from a mouse model for Tay-Sachs/Sandhoff disease. MALDI-TIMS was next applied to human ovarian carcinoma tissue to detect sulfatide location and established that ST are associated specifically with the regions of the ovarian tissue that bear the carcinoma. Electrospray ionization tandem mass spectrometry (ESI-MS-MS) was also used to confirm that ST and galactosylceramide (GalCer) are elevated in ovarian cancer. Gene expression data using tumor cells collected using laser capture microdissection revealed greater expression of mRNAs for GalCer synthase, GalCer sulfotransferase (Gal3ST1) and other enzymes of ST biosynthesis in epithelial ovarian carcinoma cells. This is a unique combination of two complementary, profiling technologies--mass spectrometry (metabolomic approach) with analysis of gene expression to study complex cancer pathology. The next study focused on the subcellular location of sphingolipids. In comparison with wild type Hek293 cells, a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) was found to have elevated amounts of all subspecies of ceramide (Cer), but produces disproportionately higher amounts of C18-Cer and GalCer. Since Cer is known to inhibit protein ER/Golgi trafficking, these studies found that the higher production of Cer caused impairment of ER/Golgi trafficking of Ceramide synthase 1 (CerS1), thus increased C18-Cer. In addition, since GalCer is only synthesized in the lumen of the ER, this impairement of ER/Golgi trafficking also gave GalCer synthase access to its substrate and increased GalCer biosynthesis. These studies illustrate the complexity of sphingolipid biology and the usefulness of multiple tools to understand sphingolipid complex biological processes.

Glycosphingolipids (GSLs), composed of a polar saccharide head and a lipophilic ceramide tail, are ubiquitous components of the plasma membrane of eukaryotic cells. They serve in many regulatory capacities and have antigenic properties towards natural killer T (NKT) cells of the innate immune system. Critical to the recognition of glycosylceramides by NKT cells are antigen presenting cells (APC), such as dendritic cells, which are responsible for binding, processing, and delivery of ligands to these lymphocytes. This event is mediated by CD1d, a major histocompatibility complex-like protein expressed on the surface of APCs, which binds GSL antigens by the ceramide moiety and presents the polar group to the T cell receptors of CD1d-restricted cells. The subsequent immune response involves NKT cell proliferation and emission of numerous cytokines, such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), resulting in the stimulation of the innate and adaptive immune systems through maturation of APCs, activation of T cells, and secretion of antibodies by B cells. To understand the structure-activity relationship between GSLs and NKT cell activity and the requirements for intracellular processing of antigens, analogs of the model compound alphaGalCer (KRN-7000) have been synthesized. These include fluorophore-appended 6”-amino-α-galactosylceramides and N-alkenoyl GSLs, such as PBS-57, a potent alphaGalCer surrogate useful in NKT cell stimulation studies. A nonantigenic beta-C-galactosylceramide has also been prepared as an inhibitor of these innate lymphocytes. To probe the potential for using NKT cells to bias the immune system between the proinflammatory TH1 response or the immunomodulatory TH2 mode, versions of alphaGalCer with shortened ceramides have been created. One of these truncated analogs, PBS-25, has successfully been cocrystallized with CD1d and the binary complex structure solved by X-ray crystallography. Synthetic glycosphingolipids derived from Novosphingobium capsulatum and Sphingomonas paucimobilis have also been made. In assays with classical Valpha14i/Valpha24i NKT cell lines, these Gram-negative bacterial antigens were recognized directly and specifically by host immune systems through CD1d-restriction, unlike GSL-deficient microbes (e.g., Salmonella typhimurium). A search for other GSL-bearing alpha-proteobacteria led to the discovery of another natural glycosphingolipid, an N-alkenoylphytosphingoid-alpha-galactoside, isolated from the outer membrane of Ehrlichia muris.

The MHC class I status of tumour cells during immunotherapy is often underestimated. It represents one of important tumour escape mechanisms and thus can contribute to the failure of most of the cancer clinical trials that are usually based on the induction of cytotoxic T cell responses. Epigenetic changes in the promoters of genes involved in the MHC class I Ag presentation can result in decreased expression of the cell surface MHC molecules on tumour cells. Thus, epigenetic modifiers can restore an expression of the MHC class I molecules and make tumours visible to the CD8+ effector cells. Besides the epigenetic changes on the tumour cells, epigenetic modulators affect cells of the immune system such as dendritic cells (DC). Tumour cells can escape from the immune response not only by changes in the cancer cells, but also by influencing, expanding and/or activating immunoregulatory cell populations, such as regulatory T cells (Treg). This thesis focuses on the potential of the DC-based vaccines against HPV-16-associated tumours with a different MHC class I expression, on the combination of cancer immunotherapy with the treatment using epigenetic modifiers, with special attention paid to their effects on DC, and, finally, on the impacts of the anti-CD25 antibody (used for Treg elimination) on Treg and NKT...