Plasma membrane of T cells is abundant in diverse receptors and other molecules orchestrating immune responses. Numerous studies demonstrate that the localisation of proteins in the cell is non-random and that mislocalisation either in the context of plasma membrane at nanoscale or with respect to the cell interior can lead to the protein malfunction and subsequent aberrant T- cell response. In my first Ph.D. project we focused mainly on the role of the transmembrane domain length and amino acid composition, proximal sequences and the presence or absence of palmitoylation on the localisation of transmembrane adaptor proteins LAT, PAG and NTAL in T cells. We showed that plasma membrane localisation of PAG and NTAL is controlled by the amino acid composition of their TMD and is palmitoylation independent. We propose that NTAL localisation to the plasma membrane is, despite its suboptimal length, facilitated by the electrochemical asymmetry of its TMD. Among transmembrane adaptor proteins, LAT was the most interesting one. Dependency of LAT plasma membrane localisation on palmitoylation in combination with unusual amino acid composition of its TMD led us to investigate it in a separate project. My first author Ph.D. project was thus to elucidate the role of highly conserved helix-breaking amino acids,...

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12957号 / 論医博第2099号 / 新制||医||1011(附属図書館) / 32356 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 岩井 一宏, 教授 椛島 健治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38173/abstract;jsessionid=DA29F0C067C89EC1147E79EE7380D21A.f01t04?systemMessage=Wiley+Online+Library+will+be+disrupted+on+24th+October+2015+at+10%3A00-10%3A30+BST+%2F+05%3A00-05%3A30+EDT+%2F+17%3A00-17%3A30++SGT++for+essential+maintenance.++Apologies+for+the+inconvenience / 京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13003号 / 論医科博第3号 / 新制||医科||5(附属図書館) / 32931 / (主査)教授 杉田 昌彦, 教授 生田 宏一, 教授 三森 経世 / 学位規則第4条第2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20084号 / 医博第4177号 / 新制||医||1018(附属図書館) / 33200 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 山田 亮, 教授 椛島 健治 / 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21636号 / 医博第4442号 / 新制||医||1034(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 生田 宏一, 教授 杉田 昌彦, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Vγ9Vδ2 T cells are the main circulating γδ T cells in human adult blood. They are known for their T cell receptor (TCR)-dependent recognition of microbe and endogenous-derived non-peptide pyrophosphate antigens (phosphoantigens, PAg). With the intrinsically biased type 1 immune responses, Vγ9Vδ2 T cells are an important force in the defense of infections and tumors. However, the immune responses of Vγ9Vδ2 T cells in early life infections and in immunotherapies are not clear yet. In this thesis, we explored Vγ9Vδ2 T cell immune responses in both aspects. Vγ9Vδ2 T cells are abundant in human fetal peripheral blood, but compared to their adult counterparts they have a distinct developmental origin, are hyporesponsive towards in vitro phosphoantigen exposure and they do not possess a cytotoxic effector phenotype. In order to obtain insight into the role of Vγ9Vδ2 T cells in the human fetus, we investigated in the first part of this thesis their responses upon in utero infection with the phosphoantigen-producing parasite Toxoplasma gondii (T. gondii). Most congenital infections are caused by viruses, T. gondii is one of the exceptions. The organelle apicoplast present in T. gondii can generate the most potent Vγ9Vδ2 T cell activator. Thus infection in utero with T. gondii makes it a good model to observe Vγ9Vδ2 T cell immune responses in early life. By comparing to age-matched controls, we found that fetal Vγ9Vδ2 T cells were highly expanded in congenital T. gondii infected newborns, and these expanded cells were highly differentiated towards potent cytotoxic effector cells. While the impact of congenital infection on Vγ9Vδ2 T cell expansion and function waned after birth, the Vγ9Vδ2 TCR repertoire of infected infants possessed a clear fetal footprint with public clonotypes, reflecting the Vγ9Vδ2 T cell response in utero. Indeed, verification of the antigen recognition related complementarity-determining region 3 (CDR3) of the TCR for γ and δ chain by high-throughput sequencing revealed the enrichment of three Vδ2 sequences in congenitally-infected infants that are already generated at 8 weeks of gestation. Vγ9Vδ2 T cells possess several characteristics, including MHC-independent recognition of tumor cells and potent killing potential, that make them attractive candidates for cancer immunotherapeutic approaches. In the second part of this thesis we investigated Vγ9Vδ2 T cell responses towards two kinds of hemiparasite plant Viscum album L. (European mistletoe) extract drugs in vitro. Mistletoe therapy is the most used complementary cancer therapy in European countries. Mistletoe extract drugs are considered to benefit for increasing the quality of life of cancer patients and modulate immune cells, but the mechanism of action is not clear. Here, we investigated in-depth the in vitro response of human T cells towards mistletoe extract drugs by analyzing their functional and TCR responses using flow cytometry and high-throughput sequencing respectively. Non-fermented mistletoe-extract drugs (AbnobaViscum), but not their fermented counterparts (Iscador), induced specific expansion of Vγ9Vδ2 T cells among T cells. Furthermore, AbnobaViscum rapidly induced the release of cytotoxic granules and the production of the cytokines IFNγ and TNFα in Vγ9Vδ2 T cells. This stimulation of anti-cancer Vγ9Vδ2 T cells was mediated by the butyrophilin BTN3A, did not depend on the accumulation of endogenous phosphoantigens and involved the same Vγ9Vδ2 TCR repertoire as those of phosphoantigen-reactive Vγ9Vδ2 T cells.In summary, in the first part of this thesis we showed that the human fetus intrinsically possesses a group of Vγ9Vδ2 T cells that are responding to congenital parasite infections that provide potential protective effects to the fetus. In the second part, we provided insight into the in vitro responses of Vγ9Vδ2 T cells towards mistletoe extract drugs, indicating that Vγ9Vδ2 T cells can be an important target in mistletoe therapy. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Celiac disease (CD) is a highly prevalent autoimmune disease in which intestinal inflammation is induced by dietary gluten. The means through which gluten-specific CD4+ T cell activation culminates in intraepithelial T cell (T-IEL) mediated intestinal damage remain unclear. Here, we performed multiplexed-single cell analysis of intestinal and gluten-induced peripheral blood T cells from patients with different celiac disease states and controls. Untreated, active CD (ACD) and potential CD (PCD) were associated with an enrichment of activated intestinal T cell populations including CD4+ follicular T-helper (TFH) cells, regulatory T cells (Tregs), and Natural CD8+ αβ and γδ T-IELs. Natural CD8+ αβ and γδ T-IELs expressing activating Natural Killer Cell Receptors (NKRs) exhibited a distinct TCR repertoire in CD and persisted in patients on a gluten-free diet (GFD) without intestinal inflammation. Our data further show that NKR-expressing cytotoxic cells, which appear to mediate intestinal damage in CD, arise from a distinct NKR-expressing memory population of T-IELs. Following gluten ingestion, both αβ and γδ T cell clones from this memory population of T-IELs circulated systemically with gluten-specific CD4+ T cells and assumed a cytotoxic and activating NKR-expressing phenotype. In patient-derived organoid (PDO) model of CD, gluten exposure induced the presence of this cytotoxic, NKR-expressing population exclusively in PDOs generated from CD patients. The increased abundance of cytotoxic, NKR-expressing T-IELs following gluten exposure corresponded to histologic observations of altered organoid morphology including degenerated organoid structures and the presence of infiltrating immune cells co-localized with apoptotic epithelial cells. Collectively, these findings suggest that these cytotoxic, NKR-expressing T cells in CD are rapidly mobilized in parallel with gluten-specific CD4+ T cells following gluten ingestion to mediate the destruction of intestinal epithelial cells in CD.

The Affymetrix GeneChip® Canine Genome 2.0 microarray is re-annotated using AgBase tools, up-to-date ID mapping and GO annotations associated with publicly available gene products updated on this array. This re-annotation makes the array more useful for researchers using the canine microarray for biological discovery. We use flow cytometry to determine if liposomal clodronate (LC) is an acceptable alternative to surgical splenectomy to facilitate detection of subclinical infection with Babesia canis in potential blood donor greyhounds. Our study shows that LC is not a reliable means of exposing babesiosis in greyhounds with a recent history of infection. We evaluate the effect of depletion of antigen presenting cells on regulatory T cells (Tregs) in dogs treated with LC by multi-color flow cytometry. We demonstrate that LC promotes increases in the CD4+CD25+FOXP3+ Tregs affecting mostly the CD4+CD25lowFOXP3+ Tregs subset suggesting a role of monocytes in naïve T cell priming and differentiation into Tregs.