Early growth response (Egr) gene 2 and 3 are genes encoding transcription factors important for maintaining immune homeostasis. Here we define a fundamental role of Egr2 and 3 to control T cell proliferation and differentiation of effector T cells. Egr2 and Egr3 deficiency in T cells resulted in impaired T cell proliferation, but hyper-activation and excessive differentiation of T cells in response to viral infection, while, conversely, sustained Egr2 expression enhanced proliferation, but severely impaired effector differentiation in to T helper (Th) subsets, such as, Th1 and Th17 subtypes. T-bet is important for differentiation of effector T cells in response to pathogen and in particular it is a master regulator for modulating the T helper 1 lineage specific differentiation programme. Although T-bet has been extensively studied in T cells, the regulation of T-bet function is less well known. We have now discovered that Egr2 and 3 are potent inhibitors for Tbet function in CD4 and CD8 effector T cells. Together with Egr2 and 3, T-bet is induced in naïve T cells by antigen stimulation, but the expression was reciprocally regulated by IFNγ, which inhibited Egr2 and 3, but promoted Tbet expression. The expression of Egr2 and 3 in CD4 T cells under TH2 and TH17 condition was essential to suppress TH1 differentiation in vitro. In response to viral infection, sustained Egr2 expression in T cells profoundly inhibited differentiation of effector cells, while Egr2 and 3 deficient T cells produced excessive levels of IFNγ. We found that both Egr2 and 3 can directly interact with the Tbox domain of T-bet, block its DNA binding and inhibit T-bet mediated production of IFNγ. Thus, Egr2 and 3 are antagonists for T-bet function in effector T cells and essential for the control of T cell differentiation and immune pathology.

CD8+ T cells are essential for host protection against intracellular pathogens and tumors. During antigen-driven responses, CD8+ T cell fate is governed by transcriptional and epigenetic processes that allow naïve CD8+ T cells to develop into a wide range of effector and conventional memory cell subsets. Over the last decades, novel techniques and major efforts led to a better understanding of the origin, nature, and short- and long-term effects of these processes on individual CD8+ T cells. Under certain conditions, naïve CD8+ T cells can acquire memory phenotype and functions in an antigen-independent manner. Although homeostatic cytokines and initial activation pathways that drive the development of these unconventional memory cells had been identified, the ensuing transcriptional profile of these cells and their degree of similarity with conventional memory cells remained ill-defined. The epigenetic events that accompany unconventional memory formation were also not known.Here, we show that innate memory cells, a type of thymic unconventional memory cells, are transcriptionally close to conventional memory cells but only partially epigenetically programmed toward the full memory fate. We also show that the sole overexpression of the transcription factor Eomesodermin (EOMES), a master regulator of effector and conventional memory cells, is able to drive many of the phenotypical, functional, transcriptional, and epigenetic features of innate memory cells, and to induce the recruitment of BRG1, a member of chromatin remodeling complexes, to innate memory gene regulatory regions. We further show that the in vivo interleukine-4-dependent development of innate memory cells is largely dependent on BRG1. We bring to light that, in innate memory cells, EOMES is recruited in many instances to genomic regions previously bound by the transcription factor RUNX3. Overall, we provide insights into the mechanisms that allow memory cell formation and T cell receptor stimulation to be uncoupled. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

5 Regulatory T cells (Tregs) are essential for the maintenance of peripheral self-tolerance and prevention of autoimmunity by suppressing the response of self-reactive CD8+ and CD4+ T cells. However, while interactions of Tregs with CD4+ T cells have been extensively studied, their effect on the self-tolerance of CD8+ T cells has not been explored in detail. The main aim of this diploma project was to provide evidence whether and how Tregs prevent autoimmunity induced by CD8+ T cells. We used an experimental mouse model of autoimmune diabetes allowing us to acutely deplete Tregs and titrate the number of self-reactive T cells, self- antigen affinity, and self-antigen doses. We found out that Tregs play an important role in the prevention of CD8+ T-cell mediated autoimmunity. Moreover, we revealed that Tregs suppress both high-affinity T cells that escape negative selection and relatively weakly self-reactive, but numerous, positively selected T cells. Tregs do so by increasing requirement for the number of self-reactive CD8+ T cells required for the autoimmunity induction. Intriguingly, presence of Tregs does not impact threshold for self-antigen. Moreover, for the first time, we showed that Tregs can suppress CD8+ T-cell-mediated autoimmunity in the absence of conventional CD4+ T cells. This means that...

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Chlamydia spp. infect epithelial cells of the respiratory, intestinal, and reproductive tracts. Chlamydial infections in women may lead to pelvic inflammatory disease, ectopic pregnancy, chronic pelvic pain, and infertility. In addition to infecting infection the female reproductive tract (FRT), Chlamydia also infects the gastrointestinal tract (GIT) of animals and humans. In mice Chlamydia muridarum disseminates from the FRT to the GIT via internal routes and in a stepwise manner. Initially Chlamydia spreads from the FRT to infect the FRT-draining iliac lymph nodes (ILNs), then the spleen, and then the GIT. The first step of this dissemination (FRT to ILN) is mediated by tissue CD11c+ DCs. Chlamydia transport from ILN to the spleen is dependent on cell transport and is mediated by sphingosine 1-phosphate (S1P) signaling. The third step of Chlamydia transport from the spleen to the GIT is significantly hindered in splenectomized mice. However, which cells mediate this transportation of the second and the third step remain unknown. Using mouse-specific C. muridarum as a model pathogen we show that following depletion of CD8+ T cells or monocytes, Chlamydia dissemination to the spleen and the GIT is significantly hindered. Furthermore, this study reveals that Chlamydia may infect various cell types which then mediate its dissemination internally. It remains to be determined what role systemic dissemination may have in Chlamydia pathogenesis.

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TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy.

Interleukin-7 is an essential cytokine that plays a major role in the development and homeostatic maintenance of T-cells. The presence of soluble forms of various cytokine receptors have been proposed to be involved in the endogenous regulation of cytokine activity. Due to the natural ability of soluble CD127 (sCD127) to bind to IL-7, there is an interest in its potential application as an immunotherapeutic agent in diseases, where IL-7 has been found to be relevant, including HIV infection. In this study, I hypothesize that by administering sCD127 to healthy mice, IL-7 activity should be enhanced, thus enhancing T cell proliferation in vivo. The work presented here focuses on three main objectives: 1) evaluating the effect of IL-7 with or without sCD127 on T cell proliferation in healthy mice; 2) validating a mouse model of T cell depletion using anti-CD4 and CD8 antibodies; and 3) determining the effect of sCD127 treatment with or without IL-7 on T cell reconstitution and proliferation in the T cell depletion model. To assess the effect of administering exogenous sCD127, IL-7 or the combination on T cell proliferation, peripheral blood mononuclear cells and spleen were isolated, and stained to characterize T cell number, proliferation, and surface CD127 expression by flow cytometry. For the T cell depletion model, wild type C57BL/6 mice were injected intra-peritoneally with 150 μg single dose of anti-CD4 and anti-CD8 depleting antibodies. Consequently, mice were bled weekly to demonstrate the kinetics of T cell reconstitution following depletion (from d7 to d63). Our results demonstrated that in healthy mice daily treatment with murine IL-7 significantly stimulated T cell proliferation and consequently increased cell number. This observation was further boosted by pre-complexing IL-7 with sCD127. For T cell depletion experiments, the kinetics of T-cell reconstitution was different between the CD4+ and CD8+ T cells. CD4+ T cell reconstitution was almost complete 6 weeks following T cell depletion, while CD8+ T cells were only partially reconstituted at this time point. Treatment with IL-7 or combined therapy had a transient and significant effect on T cell proliferation and reconstitution, and this influence was abrogated after treatment discontinuation. Interestingly, CD8+ T cells exert greater responses to our treatments in that a more pronounced proliferation and significant increase in cell number was observed relative to the effect seen on CD4+ T cells in both healthy and depleted mice. In conclusion, antibody-mediated T cell depletion is a potentially valuable tool to investigate lymphopenia-induced proliferation and potential therapies thereof. This study suggests that combining sCD127 and IL-7 therapies enhances IL-7-mediated T cell proliferation, and provides important information for the potential therapeutic use of sCD127 and its impact on IL-7 function.

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

Over the past decades, information has accumulated concerning the mechanism how an exterior signal induced by ligand on neighboring cells is transmitted to the nucleus through the Notch receptor and the cellular effects of Notch signaling on the regulation of differentiation, proliferation and apoptosis in many cell types. However, the function and the mechanism of Notch signaling in peripheral T cells still remains to be addressed. Therefore, we asked whether Notch1 is involved in CD8+ cytolytic effector T cell (CTLs) maturation and effector functions and how Notch1 exerts its cellular function in the nucleus and in the cytoplasm. The maturation of naïve CD8+ T cells into CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator, Eomesodermin (EOMES), which is thought to regulate the expression of two key effector molecules, perforin and granzyme B. In addition, the data from previous studies in our lab showed that Notch signaling results in the activation of NF-κB, IFN-γ secretion and cell proliferation both in CD4+ and CD8+ T cells. Therefore, we hypothesized that Notch1 may be involved in CD8+ T cell maturation and effector function. We observed that Notch1 regulates the expression of EOMES, perforin and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL development through direct regulation of EOMES, perforin and granzyme B. We further investigated the molecular steps leading to the formation of intracellular Notch1 (N1ICD)/CSL (also known as CBF1/RBP-Jκ in mammals; Suppressor of Hairless in Drosophila; and Lag-1 in C. elegans) with other co-factors in target promoters of Notch1 signaling. We proposed that the association of two nuclear complexes with N1ICD controls the transcription of genes, allowing the development of effector CTL in the immune system. Recent studies proposed a model where Notch1 colocalizes with CD4, a component of the immune synapse, upon T cell stimulation and directly associates with p56Lck and CD28, as well as PI3K. However, the link between Notch and the TCR signalosome needed further investigation. We found that Notch1 functions as a scaffold, associated with the cytosolic components, Carma1, Bcl10, PKCθ and the IKK complex upon TCR stimulation, leading to the activation of NF-κB and IL-2 production. We further showed that the N-terminal region of N1ICD is essential for interaction with Carma1 and that deficiency of Notch1 abolishes the nuclear binding of NF-κB on the il- 2 promoter, leading to reduced IL-2 production.

Advanced liver fibrosis in chronic hepatitis C infection (HCV) is associated with a generalized impaired immune system. Many immune cells are affected in chronic liver disease, including CD8+ T cells. The Crawley lab reported CD8+ T cell hyperfunction in cirrhotic HCV-infected individuals that persisted after effective antiviral therapy. To evaluate the link between CD8+ T cell dysfunction in advanced fibrosis, we adapted a hepatotoxic carbon tetrachloride (CCl4) murine model. We consistently observed severe fibrosis in CCl4-treated mice resembling fibrosis in chronic HCV infected individuals. After stimulation of PBMC, the proportion of granzyme B+, and IFN-γ+ CD8+ T cells in fibrotic mice was significantly higher than the controls, particularly naïve and central memory CD8+ T cells. This state of hyperfunction was sustained after liver insult removal and significant fibrosis regression to near normal tissue integrity. Sex differences were also studied in this model and were apparent after prolonged exposure to CCl4 and in the capacity to repair liver fibrosis. Following an ectopic challenge with cancer cells, tumor growth was significantly greater in fibrotic mice. Moreover, the response to immunotherapy was significantly delayed in CCl4-treated mice. In summary, we reported for the first time that circulating CD8+ T cells are hyperfunctional in a murine model of advanced liver fibrosis in response to a hepatotoxin. In this context, affected mice failed to control the growth of a tumor whose growth is known to be controlled by a robust CD8+ T cell response. In addition, the reduced responses to immunotherapeutic effects suggest deficiencies in antigen-specific CD8+ T cell responses. Therefore, this animal model might be useful to identify mechanistic targets with translational potential for immune restoring treatments in human chronic liver diseases with advanced liver fibrosis.