Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.

One of the major focuses for our lab involves examining the immunosuppressive properties of various agents and receptors in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). This dissertation encompasses an investigation of cannabidiol in the EAE model, the endocannabinoid CB1 receptor in the EAE model, staphylococcal superantigens (SAgs) as immunosuppressive agents, and various aspects of the EAE model. The first chapter covers the existing literature pertinent to these studies, the second and third chapters cover the material, methods, and results from the studies, and the fourth chapter is a discussion of how those results fit into the existing body of literature. A fifth chapter has also been included which covers two additional studies designed to develop alternative EAE models for our lab; however, both studies turned out differently than expected. One of the most interesting developments from this final chapter was the discovery of an age dependent difference in the memory T cell response of older mice, which allows for more robust disease to be induced when cells from 6 month old mice are used in the passive EAE (P-EAE) model as opposed to mice 10 weeks of age.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25166号 / 医博第5052号 / 新制||医||1071(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 藤田 恭之, 教授 上野 英樹, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Staphylococcus aureus causes chronic mastitis in bovines that is difficult to treat with current therapeutics. The goal of this research is to provide information about and improve innate immune responses to infection. Infection can result in host cell apoptosis or programmed cell death. Many pathogens can inhibit apoptosis; thereby acquiring a replicative niche, a reprieve from immune responses, and an escape from treatments. We hypothesize that S. aureus inhibits apoptosis in dendritic cells (DC). To investigate our hypothesis, DC were infected with live S. aureus (LSA), γ-irradiated S. aureus (ISA), or Streptococcus agalactiae (Strep ag.) for 2 hours. Stimulations of DC included ultraviolet light (UV) and lipoteichoic acid (LTA). Results indicate that γ-irradiated S. aureus can inhibit UV-induced apoptosis by upregulating LTA. This research provides information about S. aureus infections, but further research is needed to improve responses to this type of infection. One way to improve innate immune responses to infection is by supplementing bovines with OmniGen-AF®, a probiotic that restores neutrophil function during immunosuppression. To determine the mechanism by which OmniGen-AF® functions, wildtype, MyD88 KO, and TLR4 KO mice were fed either normal chow or supplemented with OmniGen-AF® for two weeks. Mice were immunosuppressed with dexamethasone and challenged with LTA. LTA overcame immunosuppression in a TLR4-depenent manner regardless of supplementation with OmniGen-AF®. Overall this research supplies knowledge about S. aureus inhibition of apoptosis in DC and S. aureus LTA activation of PMN regardless of immunosuppression or supplementation with OmniGen-AF®. / Master of Science

Yes / T-lymphocytes (T-cell) therapy offers a treatment for cancers. Developing this technology in the future provides the opportunity to revolutionise treatment and to make cancer a chronic condition. T-cells in themselves are a type of lymphocytes (itself a type of white blood cell) that play a central role in cell mediated immunity. They can be distinguished from other lymphocytes, such as B-cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. T-cells have the capacity to destroy diseased cells, but tumours present a considerable challenge that reduces their impact. As cancer cells are frequently ‘invisible’ to the immune system, and they create an environment that suppresses T-cell activity., genetic engineering of T-cells can be used therapeutically to overcome these challenges. T-cells can be taken from the blood of cancer patients and then modified to recognise and destroy cancer-specific antigens.

Le mélanome uvéal (MU) est le cancer intraoculaire primaire le plus fréquent chez l'adulte et représente 5% des mélanomes. La moitié des patients développent des métastases et 90% de celles-ci sont localisées dans le foie. Le MU métastatique est résistant aux traitements anticancéreux incluant l'immunothérapie. Le foie est un organe où l'immunité est finement régulée grâce à l'activité immunosuppressive de cellules immunitaires et des cellules stellaires hépatiques (CSH). Ces cellules peuvent favoriser une immunosuppression locale par le biais de l'expression de protéines immuno-régulatrices. Mon projet visait à mieux comprendre les mécanismes cellulaires/moléculaires qui mènent à une immunosuppression dans le MU métastatique et comportait deux objectifs spécifiques : 1) l'analyse du microenvironnement tumoral dans des métastases hépatiques de patients à l'aide de marquages immunohistochimiques et 2) l'optimisation d'un modèle *in vitro* de cocultures avec des cellules cancéreuses du MU (CCMU), des CSH et des cellules immunitaires pour étudier les mécanismes immunosuppressifs dans la niche hépatique. D'abord, des coupes histologiques de métastases hépatiques prélevées sur 11 patients ont été analysées par des méthodes bio-informatiques à la suite de marquages immunohistochimiques pour quantifier les lymphocytes, macrophages, cellules endothéliales et CSH activées. Deux sous-groupes de métastases ont pu être identifiés selon que l'infiltrat immunitaire intra-tumoral était fort ou faible. Les métastases présentant un faible infiltrat immunitaire étaient caractérisées par une forte densité de cellules immunitaires dans la marge. Une haute densité en CSH activées était retrouvée dans la métastase et dans la marge, suggérant une implication des CSH dans l'exclusion des cellules immunitaires de la métastase. Ensuite, un modèle *in vitro* impliquant la stimulation de cellules immunitaires par des CSH préalablement activées par le milieu conditionné de CCMU a été optimisé. Des analyses de cytométrie en flux du phénotype immunosuppressif des CSH ont révélé une expression élevée de PD-L1 sur ces cellules. Ces expériences ont permis le développement de méthodes d'analyses bioinformatiques de marquages immunohistochimiques de métastases de patients, ainsi que l'optimisation de méthodes de cocultures et d'analyses par cytométrie en flux de différents types cellulaires impliqués dans la formation de métastases hépatiques du MU. / Uveal melanoma (UM) is the most frequent primary intraocular cancer in adults and represents 5% of all melanomas. Half of patients develop metastatic disease, and 90% of them are in the liver. Metastatic UM is remarkably resistant to any treatment currently available, including immunotherapy. The liver is an organ in which immunity is finely regulated thanks to the immunosuppressive activity of immune cells and hepatic stellate cells (HSteCs). These cells could induce immunosuppression through the production of immunoregulatory proteins. My project aimed to get a better understanding of the cell/molecular mechanisms leading to immunosuppression in metastatic UM, and included two specific objectives: 1) the analysis of the tumoral microenvironment in hepatic metastases from patients using immunohistochemical staining and 2) the optimization of an *in vitro* model of cocultures with UM cells, HSteCs and immune cells to study the immunosuppressive mechanisms in the hepatic niche. First, histological sections of hepatic metastases from 11 UM patients were analyzed using computational methods following immunohistochemical staining to quantify lymphocytes, macrophages, endothelial cells and activated HSteCs. Two subsets of metastases were identified based on whether the intra-tumoral immune infiltrate was strong or weak. Metastases with weak immune infiltrate had a high density of immune cells in the margin. A high density of activated HSteCs was found within the metastasis and the margin, suggesting the involvement of activated HSteCs in the exclusion of immune cells from the metastasis. Next, an *in vitro* model involving the stimulation of immune cells by HSteCs activated beforehand by the conditioned medium of UM cells was optimized. Flow cytometry analyses characterizing the immunosuppressive phenotype of activated HSteCs revealed a high expression of PD-L1 on these cells. These experiments enabled the development of bioinformatics analysis methods for immunohistochemical stainings of patients' metastases, as well as the optimization of coculture methods and flow cytometry analyses of different cell types involved in UM metastasis formation.