Orientador: Ana Liz Garcia Alves / Resumo: O transplante de células tronco mesenquimais (CTMs) em doenças articulares vem se demonstrando eficaz. O objetivo desse estudo foi avaliar a resposta imune de repetidas aplicações intra-articulares de CTMs alogênicas e a imunogenicidade destas, utilizando ensaios de microcitotoxicidade para avaliar as respostas de anticorpos citotoxicos dependentes de complemento. Quinze equinos adultos foram distribuídos aleatoriamente entre três grupos. Seis animais no grupo ALO onde articulações hígidas receberam CTMs alogênicas, seis no grupo ALO LPS em que as articulações foram previamente inflamadas, pela indução experimental da sinovite com 0,5ng de LPS e três animais no grupo AUTO que foi mantido como controle negativo e receberam CTMs autólogas. Foram realizadas 2 aplicações na mesma articulação de células tronco mesenquimais derivadas de membrana sinovial com intervalo de 4 semanas. Foram colhidas amostras seriadas nos 3 dias após cada aplicação e a cada 7 dias por 8 semanas do liquido sinovial para análise e soro sanguíneo para os ensaios de microcitotoxicidade e exame ultrassonográfico para avaliar efusão articular através da distancia entre a superfície do talus e a capsula articular. Para análise estatística foi utilizado os testes Two Way Repeated Measures ANOVA e teste Turkey's. Foi evidenciada uma reação inflamatória moderada pelo aumento da celularidade do liquido sinovial após as aplicações. Também, foi observado que após a segunda aplicação alogênica, as articulações apres... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Transplantation of mesenchymal stem cells (MSCs) in joint diseases has been shown to be effective. The objective of this study was to evaluate the immune response of repeated intra-articular applications of allogeneic MSCs and their immunogenicity using microcytotoxicity assays to evaluate complement dependent cytotoxic antibody responses. Fifteen adult horses were randomly assigned to three groups. Six animals in the ALO group where healthy joints received allogeneic MSCs, six in the ALO LPS group in which the joints were previously inflamed, by the experimental induction of synovitis with 0.5ng of LPS and three animals in the AUTO group that was maintained as negative control and received Autologous MSCs. Two applications were performed in the same articulation of mesenchymal stem cells derived from synovial membrane with interval of 4 weeks. Serial samples were taken 3 days after each application and every 7 days for 8 weeks of synovial fluid for analysis and blood serum for the microcytotoxicity and ultrasound examination to evaluate joint effusion through the distance between the surface of the talus and the joint capsule. Two-way ANOVA and Turkey's test were used for statistical analysis. A moderate inflammatory reaction was evidenced by increased synovial fluid cellularity after the applications. Also, it was observed that after the second allogeneic application, the joints presented effusion until 28 days after application when compared to the autologous applications ... (Complete abstract click electronic access below) / Mestre

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12986号 / 論農博第2826号 / 新制||農||1038(附属図書館) / 学位論文||H28||N4961(農学部図書室) / 32456 / 名古屋大学大学院農学研究科生化学制御専攻 / (主査)教授 祝前 博明, 教授 今井 裕, 教授 廣岡 博之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24759号 / 生博第500号 / 新制||生||66(附属図書館) / 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 杉田 昌彦, 教授 井垣 達吏, 教授 野田 岳志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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

Blockering av NKG2A receptorn på NK - och CD8+ T celler med en anti-NKG2A antikropp, medför en aktivering av cytolytisk aktivitet, och är en lovande immunkontrollpunkt i immunterapi mot cancer. Nyligen har en TCR-liknande antikropp, EXX1, som binder till liganden för NKG2A receptorn, Qa-1b - en icke-klassisk MHC klass Ib molekyl i möss -studerats i tumör modeller in vitro. Resultat påvisar att den TCR-liknande antikroppen endast binder till Qa-1b om denna presenterar Qdm peptiden på sin yta, som erhålls från ledarsekvensen hos klassika MHC klass Ia H-2D. Detta väcker frågor kring strukturella faktorer som möjliggör antigenpresentation på Qa-1b och de exakta molekylära parametrarna som ger upphov till antikroppens specificitet. Syftet med denna studie var att bestämma och jämföra kristallstrukturerna för Qa-1b med Qdm (AMAPRTLLL) samt peptid 001 (AQAERTPEL). Den tunga peptidkedjan hos Qa-1b och beta-2-mikroglobulin producerades rekombinant i E.coli, återveckades med respektive peptid, renades med kromatografimetoder och slutligen kristalliserades genom ångdiffusionsmetoden med hängande droppar. Värmestabilitet hos MHC/peptid undersöktes med nano differential scanning fluorimetry, där Qa-1b /001 uppvisade bättre stabilitet. Kristaller för Qa-1b /Qdm och Qa-1b /001 kunde erhållas med 8% PEG4000, 10mM NiCl2, 0.1M natriumacetat vid pH 5.7, respektive 10% PEG4000, 10 mM NiCl2 och 0.1 M natriumacetat vid pH 6.0. Strukturen för Qa-1b /001 kunde bestämmas vid 2.43 Å med molekylär ersättning. Med anledning av negativt laddade sidogrupper i peptid 001 som har en ytlig konformation i bindningsfickan, kan avsaknaden av bindning till EXX1 förklaras av en skillnad i elektrostatiska interaktioner mellan Qdm och peptid 001. Ytterligare strukturella karakteriseringar av Qa-1b komplexen med antikroppen är av fortsatt stort intresse. / Blocking of the NKG2A receptor expressed on NK cells and CD8+ T cells with an anti-NKG2A antibody for elicitation of cytolytic activity, is a promising immune checkpoint in cancer immunotherapy. EXX1, a novel TCR-like antibody with specificity for the NKG2A ligand, Qa-1b - a murine non-classical MHC class Ib ortholog of HLA-E - has been assessed in tumor models in vitro. The antibody only engages with Qa-1b when it presents the dominant peptide Qdm, derived from the leader sequence of the classical MHC class Ia H-2D. This raises questions about the structural features of antigen presentation by Qa-1b, and the molecular parameters driving the specificity of the TCR-like antibody. The purpose of this study is to determine and compare the crystal structures of Qa-1b in complex with Qdm (AMAPRTLLL) and peptide 001 (AQAERTPEL). The Qa-1b heavy chain and mouse beta-2 microglobulin were recombinantly expressed in E.coli, refolded in the presence of respective peptide, purified using size exclusion chromatography and crystallized with the hanging drop vapor diffusion method. Thermal stability of the MHC/peptide complexes was assessed with nano differential scanning fluorimetry, implying a higher stability of Qa-1b/001. Crystals of the Qa-1b/Qdm and Qa-1b/001 were obtained with 8% PEG4000, 10 mM NiCl2, 0.1 M sodium acetate at pH 5.7, and 10% PEG4000, 10mM NiCl2 and 0.1 M sodium acetate at pH 6.0, respectively. The structure of Qa-1b/001 was resolved by molecular replacement at 2.43 Å, and the presence of negatively charged side chains that protrude from the binding groove, may imply that differences in electrostatic interactions between Qdm and 001 will determine antibody-binding. Further structural characterizations, of Qa-1b complexes with bound EXX1 are of great interest.

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