京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12871号 / 論医博第2087号 / 新制||医||1006(附属図書館) / 31589 / 北海道大学大学院医学研究科臨床医学コース / (主査)教授 三森 経世, 教授 長澤 丘司, 教授 河野 憲二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The immune system will maintain the integrity of the organism from harmful non-malicious recognizes and protects the body against exo- and endogenous toxic substances and together with the nervous and endocrine system are among regulatory systems of the organism. Recently the evidence has supported the emerging concept of different B cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B cell homeostasis. This work is focused on the study of peripheral CD27+ B lymphocytes (one of the least explored human B lymphocytes) in healthy controls and patients with various immunopathologies, in this case we present representative results for patients with inflammatory bowel disease. Using polychromatic flow cytometry we examined 31 of peripheral blood samples, including 14 controls, 7 patients with Crohn's disease (CD) and 5 with ulcerative colitis (UC). In 6 patients with CD, we were able to perform immunophenotyping also 2 hours after intravenous administration of infliximab, and in one patient 14 days after drug...

Chronic Lymphocytic Leukemia (CLL) is a type of cancer that affects the B cells of the immune system causing problems in the process of producing antibodies. It can be sorted into mutated and unmutated CLL based on the percentage of somatic mutations in the Immunoglobulin Heavy chain Variable region (IgHV). The B cells of healthy individuals can be sorted into three groups; CD27dull memory B cells (MBCs), CD27bright MBCs and naïve B cells. The hypothesis for the project was that the unmutated CLL subset originates from CD27dull MBCs and the mutated CLL subset originates from CD27bright MBCs. RNA-sequencing data from healthy individuals were acquired from a collaboration partner in Rome and CLL-patients were collected from public datasets available online. Several bioinformatic tools were used to analyze the data. First, the quality of the data files was checked, then adapter sequence from the sequencing process and low-quality bases were removed (trimming). Good quality of the files was confirmed after the trimming. Secondly, these files were mapped against the human reference genome (GRCh38/hg38) for alignment, then the resulted data was used to check for genes that showed differential expression between the different groups. Results were analyzed and visualized using Venn diagrams, Principal Component Analysis (PCA) and heatmap plots and random forest. A list of 85 genes was generated based on the different comparisons and was used in one PCA plot that showed clear separation between the different groups. The SWAP70 gene was analyzed for single nucleotide polymorphisms (SNPs). The study concluded five genes that could be used as biomarkers for CLL and the diagnosis of its subtypes where some of them were discussed in previous studies. Also, the mutated CLL subset showed a similar behavior to the healthy individuals and this could validate the original hypothesis and justifies the better disease prognosis for this subtype.

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Gedächtnis-B-Zellen und Plasmazellen sind essentielle Komponenten der protektiven Immunität. Die Mechanismen ihrer Induktion, ihres Überlebens und der Gedächtnis-B-Zellreaktivierung sind allerding bisher nur unvollständig verstanden. Um unser Wissen diesbezüglich zu erweitern, wurden in der vorliegenden Arbeit zum einen Charakteristika von Primär- und Sekundärimmunantworten nach Immunisierung mit Keyhole Limpet Hemocyanin (KLH) untersucht und zum anderen das Vorkommen sowie der Phänotyp humaner Gedächtnis-B-Zellen in verschiedenen lymphatischen Geweben analysiert. Die primäre parenterale KLH Immunisierung führte auf serologischer und zellulärer Ebene zu einer Reihe unerwarteter Ergebnisse, welche u. a. das Auftreten von IgA Antikörpern und die gleichzeitige Präsenz von hoch- und wenig mutierten primären Plasmablasten beinhalteten. Die Untersuchung der Gedächtnis-B-Zellverteilung in verschiedenen lymphatischen Geweben ergab den größten Gedächtnis-B-Zellpool in der Milz. Blut-, Tonsillen-, Knochenmarks- und Milz-Gedächtnis-B-Zellen wiesen nur wenige phänotypische Unterschiede auf. Einer davon war die CD69 Expression auf tonsillären ruhenden Gedächtnis-B-Zellen, was darauf hindeutet, dass tonsilläre Gedächtnis-B-Zellen tatsächlich sessil sein könnten. Die hier erhaltenen Ergebnisse stellen neue Erkenntnisse über bisher unbeschriebene Mechanismen bei parenteralen Immunantworten wie zum Beispiel die Induktion von IgA Antikörpern sowie die potentielle Rekrutierung kreuzreaktiver Gedächtnis-B-Zellen dar. Es bleibt zu klären, wie die Reaktivierung solcher Gedächtnis-B-Zellen reguliert ist. Derartiges Wissen wäre insbesondere für Therapien von Erkrankungen des Immunsystems, wie Autoimmunität, von Bedeutung. Das potentiell patrouillierende Verhalten der Gedächtnis-B-Zellen ist ein markanter Unterschied zu den nischenabhängigen sessilen Plasmazellen und deutet weitestgehend darauf hin, dass Gedächtnis-B-Zellen nicht auf derartige Nischen angewiesen sind. / Memory B cells (mBC) and antibodies are major mediators of protective immune responses yet the mechanisms of their induction, maintenance and mBC reactivation are poorly understood. Therefore, to enhance knowledge in this regard this study comprehensively characterized a human primary and secondary B cell immune response to Keyhole Limpet Hemocyanin (KLH). Secondly, mBC maintenance was investigated by a systematic analysis of mBC presence, frequency and phenotype within different lymphoid organs. Parenteral primary KLH immunization yielded unexpected results on the serological and B cellular level, including KLH-specific IgA antibody induction, the simultaneous presence of low and highly mutated circulating KLH-specific primary plasmablasts and only little clonal overlap between the primary, memory and secondary KLH-specific B cell repertoires. With respect to the organ distribution of human mBC, the spleen was identified as a major mBC reservoir. Splenic, tonsillar, bone marrow and blood mBC pools exhibited a largely comparable phenotype. Yet, we found tonsillar mBC to express CD69. Due to their resting state tonsillar mBC could therefore constitute a tissue resident cell population. The observations described allow insights into hitherto unknown potential mechanisms behind primary immune responses, i.e. prominent IgA induction by parenteral challenge and inclusion of cross-reactive mBC. The so far unclear regulatory players involved deserve future investigation, as such knowledge may be crucial for therapeutic interventions in immune system disorders. Furthermore, strikingly different to the resident plasma cells in the bone marrow, mBC appear to distribute between lymphoid organs and continuously recirculate in peripheral blood indicative of their potential permanent screening activities, suggesting that human mBC do not require one dedicated niche for their principle survival.