Regulation of the immune response is mediated, in part, by the action of suppressor T cells (Ts). One intriguing aspect of these cells is the description of T cell suppressor factor (TsF): a soluble analog of the cell that shares many of its properties, such as the ability to bind free antigen (Ag) and suppress an Ag-specific immune response. The exact molecular nature of TsF and the relationship of TsF to Ts are unknown. The immune response to the small, bacterial protein, ferredoxin (Fd), was used as a model system to study TsF. A Fd-specific suppressor cell network has been described in mice that are genetically nonresponsive to this Ag. Previously, a soluble mediator, known as Fd11F, was found in the culture supernatant (SN) of the Ts hybridoma, Fd11. Fd11F possessed both Ag-binding activity and the ability to suppress the anti-Fd Ab response in mice. The TsF-specific monoclonal antibody, B16G, was used for both the recovery of Fd11F-enriched material from SN and its detection by the enzyme-linked immunosorbent assay. ' Further immunochemical, biological, and biochemical characterization of Fd11F was done with emphasis on describing the Ag-binding properties of Fd11F. It was found that Fd11F bound to solid- and liquid-phase Fd, and demonstrated preferential binding to the carrier determinant of the Ag. A spleen cell culture assay was devised which showed that Fd11F suppressed Ab production in a concentration-dependent manner. Additional experiments suggested that the suppressive effect was Ag-specific. The identification of the Ag-binding molecule was attempted by the fractionation of Fd11F-enriched material using high performance gel filtration or preparative SDS-PAGE (run under non-reducing conditions). Using SDS-PAGE, a unique, single polypeptide of about 30k relative molecular mass (Mr) was identified as the Ag-binding moiety of Fd11F. The possible relationship of this moiety to other identified materials is discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

T cell surface antigens are thought to play significant roles in immunological functions. They are involved in cellular interactions and T cell activation and proliferation. Characterization of T cell antigens is important in understanding the molecular machanisms underlying immune responses. The subject of this thesis is to characterize a novel murine T cell surface antigen called YE1/48. YE1/48, defined by two rat monoclonal antibodies YE1/48.10.6 and YE1/32.8.5, is a dimeric glycoprotein with molecular size and charge resembling the murine T cell antigen receptor α/β. It was initially detected at high levels on two T cell lymphomas, EL-4 and MBL-2. In my thesis studies, the YE1/48 antigen was characterized biochemically, a cDNA clone was isolated, and its expression in lymphoid cell populations was determined. The YE1/48 antigen was found to be distinct from the T cell receptor based on direct comparisons of their primary sequences as well as immunological analyses. It is likely a homodimer with similar or identical subunits. No homology with any known proteins could be detected, including the human T cell activation antigen CD28 (T44) which also has a similar dimeric structure as YE1/48. No function of the YE1/48 antigen could be derived from its primary sequence or with the use of the two monoclonal antibodies because the antibodies do not appear to bind to the surface of intact normal T lymphocytes. Some intriguing characteristics of the YE1/48 antigen were observed in the current studies. The YE1/48 antigen belongs to a rare group of type II membrane proteins with orientation of the amino-terminus inside the cell and the carboxy-terminus outside. The YE1/48 gene may have two alleles among different mouse strains and may belong to a multigene family. YE1/48 is expressed at low levels on a wide range of T cells with no restriction to their differentiation stages, and on spleen B cells as well as bone marrow cells. Its expression on lymphocytes is not related to activation or proliferation. However, YE1/48 expression appears to be induced at high levels by Abelson Murine Leukemia Virus-transformation of pre-B cells. Moreover, the epitopes defined by the YE1/48.10.6 and YE1.32.8.5 antibodies seem to be exposed only on three T lymphomas but not on normal T cells. It is thus tantalizing to speculate a correlation of the high level expression of YE1/48 antigen and its epitope exposure on transformed lymphocytes with cellular transformation. In summary, YE1/48 was found to be a novel T cell surface antigen which has similar dimeric structure as the murine T cell receptor α/β and human CD28 (T44). It has now been characterized biochemically, molecularly cloned, and its expression on lymphoid cells has been determined. Although the function of YE1/48 antigen remains unknown, a number of intriguing characteristics observed in the current studies have certainly called for further studies on the antigen and the determination of its function. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Les pénicillines font partie des molécules chimiques les plus fréquemment impliquées dans l’allergie médicamenteuse. Selon l’hypothèse de l’haptène, les molécules chimiques de petite taille doivent se lier aux protéines pour êtres immunogènes. Cependant, très peu est connu sur le processus d’immunisation des patients aux bioconjugués pénicilline-protéine. Notre groupe a récemment synthétisé des bioconjugués albumine sérique humaine-benzylpénicilline (HSA-BP) et a démontré l'existence de lymphocytes T CD4+ naïfs spécifiques du bioconjugué HSA-BP chez des donneurs sains. L'objectif de ce travail de thèse est d'identifier des séquences peptidiques issus de la HSA hapténisées avec la BP, impliquées dans l’activation des cellules T naïves ainsi que l’immunisation des patients allergiques et par conséquent les manifestations cliniques. Notre stratégie combine la spectrométrie de masse, la modélisation moléculaire et le criblage virtuel, la synthèse chimique orientée et la validation biologique sur des lignées de cellules T de longues durées chez les donneurs sains, et à l’aide du test de transformation lymphocytaire ainsi que les lignées de cellules T de courte durée chez les patients allergiques. Cette étude a permis: (1) l’identification des résidus lysine présents sur la HSA hapténisés par la BP par spectrométrie de masse, (2) la sélection par une approche in silico des peptides de 15-mer potentiellement immunogènes, (3) la synthèse orientée de ces peptides-BP à l’aide d’un monomère lysine-BP, (4) l’identification des épitopes reconnus par les cellules T naïves de donneurs sains, (5) la validation de deux épitopes situés sur les lysines 159 et 525 chez les patients allergiques aux pénicillines et (6) confirmation de la HSA comme un bon modèle pour l’hapténisation de la BP. / Penicillins are among the most prevalent drug-inducing allergy. According to the hapten hypothesis small chemical molecules needs to bind to proteins to be immunogenic. However, little is known on the process of patients immunization to penicillin-protein conjugates. Our group has recently synthesized benzylpenicillin-human serum albumin (BP-HSA) bioconjugate and demonstrated the existence of naïve CD4+ T lymphocytes specific to BP-HSA in healthy donors. The objective of this work was to identify peptides sequences from HSA haptenized with BP involved in naïve T-cells activation, immunization of patients and consequently the clinical manifestations. Our strategy combines mass spectrometry, molecular modeling and virtual screening, chemical oriented synthesis and biological validation using long-term T-cell lines in healthy donors and the lymphocyte transformation test as well as short-term T-cell lines in allergic patients. This study allowed: (1) the identification of lysine residues involved in the BP binding to HSA using mass spectrometry, (2) the selection of BP-peptides containing the lysine residues likely to induce immune response using an in silico approach, (3) the synthesis of the selected BP-15 mer peptide bioconjugates using a lysine-BP monomer, (4) the identification of epitopes recognized by naïve T cells from healthy donors, (5) the validation of two epitopes located on lysines 159 and 525 in allergic patients to penicillins and (6) the confirmation of HSA as a good model for BP haptenation.

Efficient delivery of genetic material to T-lymphocytes is key in gene therapy using T-lymphocytes with chimeric antigen receptors. Current procedures require the use of potentially dangerous viral vectors or large amount of input material. The diploma thesis therefore focuses on exploring new approaches for gene transfer into T-lymphocytes: use of safe virus-like particles (VLPs) derived from mouse polyomavirus in combination with the amphipathic cationic peptide LAH4. LAH4 has the potential to increase the efficiency of DNA and viral vector transport into cells. The system which combines VLPs and the LAH4 peptide was optimized for the delivery of reporter gene (encoding GFP and luciferase) to the model T-cell line Jurkat. It has been found that Jurkat cells cannot be efficiently transduced by DNA packed into VLPs. When cells were transfected only with DNA and LAH4, consistent results were not obtained, and the transfection efficiency ranged from 0.5 to 19%. The diploma thesis also analysed the effect of phosphorylation of viral structures on gene transfer. The impact of treatment of virus particles by alkaline phosphatase on the infectivity of the virus was studied and it was necessary to analyse the effect of the reaction components. Sublytic concentration of Triton-X100 in the reaction buffer...

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.

The aim of the present study was to develop a dendritic cell (DC) vaccine encoding hepatitis C virus (HCV) multiple cytotoxic T lymphocyte (CTL) epitopes that can stimulate T cell responses in vitro, and can be used for immunization in vivo. DCs were infected with recombinant replication-defective adenoviruses (Ads) expressing 2 HCV sequences fused with green fluorescent protein (GFP) and FLAG tags. One sequence (sequence 1) contained the HCV CTL epitopes, NS4B 1793-1801 and P7 774-782, as well as the HCV Th epitope, NS3 1248-1261. A second sequence (sequence 2) was the positive epitope control which contained HCV core 35-44, core 132-140 and NS3 1248-1261. The efficiency of infection was detected by flow cytometry and the expression of HCV epitopes in the DCs was confirmed by RT-PCR and western blot analysis. Ad infection significantly enhanced DC maturation and interleukin (IL)-12p70 production, resulting in T cell proliferation and increased interferon-γ secretion. The CTLs stimulated by Ad-infected DCs specifically killed Huh7.5 human hepatoma cells. The recombinant Ad-expressing multiple CTL HCV epitopes effectively infected the DCs in vitro and promoted T cell antiviral immune responses, thereby laying the foundation for the development of anti-HCV DC vaccines.

Environ, 60 % des personnes atteintes d’un cancer auront au moins une séance de radiothérapie au cours de la prise en charge thérapeutique de leur maladie. Les doses de radiothérapie sont limitées en raison du risque important de fibrose séquellaire des tissus sains. Les rayonnements ionisants (RI) peuvent induire différents types de mort cellulaire y compris l'apoptose et la sénescence. Les cellules sénescentes ont une sensibilité réduite à l'apoptose et un phénotype sécrétoire inflammatoire. De plus, les RI peuvent induire la production d’espèces réactives de l’oxygène (ERO) qui provoquent des lésions de l'ADN dans les tissus non ciblés, et des effets systémiques associés à l'inflammation. Différentes équipes ont proposé des tests prédictifs de la radiosensibilité individuelle des patients basés sur l’évaluation du taux d'apoptose radio-induite des lymphocytes T CD4+/CD8+ (LT). Cependant, l’impact des différences de sensibilité à l’apoptose/sénescence des sous-populations de LT sur le taux d’apoptose n’a pas été étudié. Notre hypothèse est que la sensibilité à l’apoptose/sénescence radio-induite des LT circulants est associée à la sur/sous-représentation de sous-populations particulières de LT CD4+ dont les fonctions sont en rapport avec la survenue de fibrose. Nos résultats chez le donneur sain montrent que les LT CCR6+Th17 pro-fibrogéniques sont moins sensibles à l’apoptose et plus sensibles à la sénescence que les LT CCR6negTh et les Treg. Cette sénescence peut être préjudiciable car les lymphocytes CCR6+Th17 situés dans les tissus irradiés peuvent sécréter de l'IL-8 et du VEGF-A. La modulation des voies ERO/MAPK ou mTOR pourrait être une cible potentielle pour la prévention de la radiotoxicité induite par les CCR6+Th17 sénescents. Enfin, le ratio de cellules circulantes H2A.J+CCR6+Th17 sénescentes / CCR6+Treg pourrait être utilisé comme marqueur potentiel de la radiosensibilité individuelle. / On average, 60% of cancer patients have at least one radiation session during their care throughout the history of their disease. The doses of radiotherapy are limited because of the high risk of fibrosis-type side effects of healthy tissues. Ionizing radiation can induce a variety of cell death responses including apoptosis, but also senescence. Senescent cells have reduced sensitivity to apoptosis, and a pro-inflammatory secretory phenotype. In addition, ionizing radiations can induce the production of reactive oxygen species (ROS) that cause DNA damage in non-target tissues, and systemic effects associated with inflammation. In order to improve the personalization of radiotherapy, different teams proposed predictive tests of the individual radiosensitivity of patients by establishing a relationship between a low rate of radio-induced apoptosis of CD4+/CD8+ T lymphocytes (LT) and a high risk of secondary fibrosis. However, the impact of the differences in individual cell sensitivity to radiation-induced senescence on the ratio between LT cell subpopulations has not been studied. Our results on healthy donors show that pro-fibrogenic CCR6+ Th17 cells are less sensitive to apoptosis and more susceptible to senescence compared to CCR6neg LT. This senescence can be detrimental as irradiated CCR6+Th17 lymphocytes located in the irradiated tissue can secrete IL-8 and VEGF-A. Modulation of ROS/MAPK or mTOR signaling pathways could be potential targets for the prevention of this CCR6+Th17-induced radiotoxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CCR6+Treg cells may be used as a potential marker of individual radiosensitivity.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21635号 / 医博第4441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 椛島 健治, 教授 岩田 想, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

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The gut-brain axis describes a paradigm wherein the trillions of microorganisms inhabiting the gastrointestinal tract engage in bidirectional communication with the host central nervous system. Adaptive immunity represents an important intermediate in this dynamic crosstalk; previous work in our lab has demonstrated that T-lymphocytes, a main class of immune effector cells, contribute to neurodevelopmental processes and behavioral outcomes across the lifespan. Parallels between the phenotype of T-cell deficient and germ free mice led us to hypothesize that bidirectional T-cell-microbe communication is critical for normal neurodevelopment, and that T-cell deficiency impacts the neural circuitry underpinning behavior via disruption of the gut-brain axis. The main objective of this thesis was to elucidate the mechanisms by which T-cells mediate developmental gut-brain signalling. The first installation examined the gut microbiome, gut metabolome, and neurochemical profile in wild-type and T-cell deficient mice from adolescence to adulthood, demonstrating that absence of T-cells impacts the developmental trajectory of functional microbiome output and levels of neuroactive molecules in the brain. Experiment two investigated the impact of T-cell deficiency on gut-brain communication through the lens of host gene expression in the parenchyma and the intestine. T-cell deficient mice showed significant changes in genes related to intestinal immunity and barrier function, in addition to decreases in microglia-related genes in the prefrontal cortex during early life. The final experiment transitioned into a wild-type model to measure the co-evolution of T-cell subsets in mucosal and central immune compartments with composition and diversity of the microbiota. We demonstrated a parallel diversification of the gut microbiome and the functional T-cell repertoire, whereby emergence and proliferation of specific T-cell subsets is linked to compositional shifts in dominant microbial communities across development. Together, our results demonstrate the importance of T-cells for normal development of the holo-organism, with implications for the developmental wiring of functional brain circuitry. / Thesis / Doctor of Philosophy (PhD) / Modern medicine has increasingly placed emphasis on the mind-body connection. This has been exemplified by a series of recent discoveries surrounding the importance of the gut microbiome in maintaining our physical and mental health. One of the key channels through which the microbiome communicates with the host is through the immune system, an equally complex network of cells and proteins that protect the body against invading pathogens. Indeed, these systems evolve alongside each other and engage in constant crosstalk throughout the lifespan, with downstream impacts on the developing brain. This thesis sought to further explore the role of T-cells, a key component of the adaptive immune system, in coordinating gut-microbiome-brain interactions across development. The first experiment examined the microbiome as well as small molecules in the gut and brain of normal mice and mice lacking T-cells. The second experiment built on this work to examine how T-cells influence the expression of different genes in the gut and brain. Finally, the third experiment mapped different populations of T-cells and microbiome composition from the first week of life to adulthood, to better understand how they interact at different stages of development. This work will offer insight into how T-cells talk to the microbiome and how they transmit signals from the gut to the brain, with implications for understanding neurodevelopmental disorders and how they arise.