No description available.

<p>Linker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLC&#947;1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLC&#947;1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ &#945;&#946; T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLC&#947;1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.</p><p>First, we investigated the importance of the LAT-PLC&#947;1 interaction in &#947;&#948; T cells by crossing LATY136F mice with TCR&#946;-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial &#947;&#948; T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ &#947;&#948; T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by &#945;&#946; T cells in LATY136F mice. Development of these hyperproliferative &#947;&#948; T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ &#947;&#948; T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLC&#947;1 signaling may function differently in various subsets of &#947;&#948; T cells. </p><p>In addition to examining &#947;&#948; and &#945;&#946; T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ &#945;&#946; T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ &#945;&#946; LATY136F T cells demonstrated that NF&#954;B, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLC&#947;1 signaling in regulating IL6 production by T cells during autoimmunity. </p><p>Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLC&#947;1 signaling in modulating Tfh cell differentiation and BCL6 expression.</p> / Dissertation

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23110号 / 医科博第121号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 竹内 理, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is crucial in linking TCR engagement to downstream signaling events, such as calcium flux and Ras-MAPK pathway. Following TCR engagement, LAT is phosphorylated at its membrane-distal tyrosine residues, which mediates the binding of Grb2/Sos, PLC-&#61543;1, and GADS/SLP-76 complexes. This multi-protein signaling complex initiates signaling cascades eventually leading to the activation of transcription factors that regulate the genes required for T cell proliferation and effector functions. The indispensable role of LAT in thymocyte development has been evidenced as LAT-deficient mice completely lack peripheral T cells. To study the function of LAT in mature T cells, our lab previously generated a conditional knock-in mouse line in which the lat gene can be deleted by Cre recombinase. Deletion of LAT in mature T cells revealed the critical role of LAT in T cell activation. Here, we used this inducible LAT deletion mouse line crossed with the OT-I transgenic mice to study the role of LAT in mature CD8 T cells. </p><p>To analyze the contribution of LAT in CD8 T cells during the course of pathogen infection, we infected mice with Listeria monocytogenes-expressing Ova to elicit activation of antigen-specific CD8 T cells, and then inducibly deleted LAT in these cells at different stages of infection under the control of tamoxifen treatment. We show that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction. In addition, memory CD8 T cell can persist in the absence of LAT, suggesting that LAT-signaling is not necessary for memory maintenance. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1lowIL-7RhighCD62Lhigh memory T cells. Together, these data demonstrate that, while it is dispensable for contraction and memory maintenance, LAT-signaling regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.</p><p>The fundamental activity of CD8 T cells is to elicit cytotoxicity toward target cells that express foreign antigens, and this is mediated through granule-dependent and Fas ligand-dependent mechanisms. The signaling events that regulate these processes remain unclear. We showed that LAT-deficient cytotoxic T cells (CTLs) failed to upregulate FasL and produce IFN-&#61543; after engagement with target cells. Moreover, they displayed reduced granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT-deficiency led to altered synapse formation, subsequently causing unstable T cell:APC conjugates. MTOC polarization and granule reorientation were also impaired by LAT-deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and PLC activity. This revealed an unexpected finding that CTL function is not entirely dependent on LAT. Collectively, these data indicate that the signaling circuits governing CTLs are programmed to adopt multiple pathways, allowing CTLs to effectively eliminate various pathogens during adaptive immune responses.</p><p>&#8195;</p> / Dissertation

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20971号 / 医博第4317号 / 新制||医||1026(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 河本 宏, 教授 濵﨑 洋子, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Maintaining balance in the human immune system is critical for the body’s ability to discriminate between foreign and self-antigens. This balance is achieved, in part, by a subpopulation of T cells known as induced regulatory T cells (iTregs). Dysregulation of this population may contribute to the onset and progression of cancer, chronic inflammation and autoimmune diseases. Therefore, manipulation of iTreg development holds promising therapeutic potential; however, studying this vital population has proven difficult due to low numbers, heterogeneous cell populations, substantial phenotypic differences between mouse and human cells, and the high plasticity seen in iTregs. These current limitations have prevented a full understanding of the molecular signaling events that govern their development and function. Our lab has established a novel cell culture system that mimics in vivo human iTreg development. This system allows for the discrimination and comparison of naïve, memory and iTreg T cell populations simultaneously within a single donor. These iTregs exhibit high levels of CD25, FoxP3, CTLA4, GITR, low levels of CD127 and display strong suppressor activity. Using this innovative system, we have demonstrated a rewiring of T cell receptor (TCR) signaling in iTregs compared to conventional T cells. We found that the voltage gated K+ ion channel-Kv1.3 is not active in response to TCR engagement in iTregs, even though Ca2+ influx remains intact. Kv1.3 and the linked Src-family kinase Lck were redistributed to the highly active IL2-Receptor (IL2-R) complex. Additionally, we have shown that there is increased AKT protein expression in iTregs versus conventional T cell populations that does not correlate with the TCR-induced increase in its active (phosphorylated) form. This blockage appears to be due to an imbalance of kinase to phosphatase activity in iTregs with a specific TCR-induced inhibition of mTOR activity. We have also demonstrated that AKT accumulation in iTregs leads to its physical association with SMAD3, suggesting a novel, non-enzymatic function of AKT through transcription factor inhibition. This study sheds light on the reciprocal cross talk between the IL-2R and TCR signaling pathways and uncovers the mechanism of AKT blockade in primary human iTregs, thus opening novel avenues for therapeutic manipulation

Les lymphocytes T sont au coeur du système immunitaire adaptatif et leur dérégulation est à la base de pathologies. Les cellules T se développent dans le thymus et passent par de nombreuses étapes de maturations identifiables par l'expression des corécepteurs CD4+/CD8+ à la surface des cellules. À leur sortie du thymus, les cellules T sont divisées en deux sous-types principaux: les cellules T auxiliaires CD4+ spécifique aux antigènes présentés sur complexe majeur d'histocompatibilité (CMH) de classe II et les cellules T cytotoxiques CD8+ reconnaissant un antigène présenté sur un CMH-I. Toutes les cellules T proviennent d’un précurseur commun. Leur différenciation en cellule T CD4+ et T CD8+ est influencée par l'intensité et la durée de la signalisation du récepteur des cellules T (RCT) et des cytokines. Cette signalisation résulte en l’expression des facteurs de transcription ThPOK pour la différenciation de cellule T CD4+ et Runx3 pour les cellules T CD8+. Il a été démontré que ThPOK est à la fois nécessaire et suffisant pour le développement des lymphocytes T CD4+, puisque le gain et la perte de la fonction de ThPOK favorise le développement de cellules lymphocytes T CD4+ et CD8+, respectivement. Ma thèse vise à approfondir notre compréhension du choix de la lignée CD4+/CD8+ en explorant les mécanismes moléculaires de la voix de signalisation de ThPOK et du RCT. Dans cette étude, nous avons étudié l'impact d'un gain-de-fonction de ThPOK sur la différenciation des thymocytes, en utilisant trois lignées transgéniques exprimant des niveaux variables de ThPOK. Une analyse approfondie de ces transgènes chez des souris dont le RCT est restreint soit au CMH de classe I ou de classe II, a démontré que, comparés aux thymocytes restreints au CMH-II, les thymocytes restreints au CMH-I requéraient des niveaux plus importants de ThPOK pour se différencier en CD4+. L’introduction d’un transgène exprimant un niveau moins élevé de ThPOK comparé aux deux autres transgènes, mais un niveau plus élevé de ThPOK par rapport au niveau endogène dans les cellules CD4+ WT, n'induit qu'une réorientation partielle des cellules T CD8+ en CD4+, ce qui a mené à la génération, à la fois de lymphocytes T CD4+, DN (doubles négatifs) et CD8+ matures. L'analyse génotypique, plus précisément celle des cellules DN chez les souris porteuses du transgène ThPOK et dont le RCT est restreint au CMH-I, a révélé que l’inhibition des gènes spécifiques à la lignée CD8+ 2 nécessitait des niveaux d'expression différents de ThPOK comparés à ceux requis pour l’induction des gènes spécifiques à la lignée CD4+. En effet, cette étude nous a permis de démontrer que l’intensité du signal dérivé du RCT ainsi que sa spécificité pour un CMH donné jouent un rôle essentiel dans le choix de différentiation CD4+/CD8+ induit par ThPOK. Ainsi, la réorientation CD8+/CD4+ chez les souris exprimant le transgène ThPOK-H est significativement augmentée par l'amplification de l’intensité du signal dérivé du RCT dans les cellules spécifiques aux CMH-I. De plus, la fréquence des cellules CD4+ était plus élevée lorsqu’une quantité identique de ThPOK était exprimée dans des lymphocytes T spécifiques au CMH-II, suggérant qu’il existe un aspect qualitatif quant à la régulation de la différenciation des lymphocytes T CD4+ par la signalisation induite par le RCT. Nous avons également tenté d’étudier la voie de différenciation CD4+ en l’absence de ThPOK, à la suite de la perturbation physiologique de la voie de signalisation induite par le RCT, par rapport à la perte de fonction de ThPOK. Bien que nous ayons observé une réorientation des thymocytes spécifiques au CMH-II vers la lignée CD8+, aussi bien à la suite d'une délétion de Thpok, qu’à la perturbation de la signalisation RCT les deux modes de redirections semblent toutefois être différents. En effet, notre investigation a démontré qu’en l’absence de ThPOK, la signalisation induite par le RCT dans les cellules restreintes au CHM-II induit l’activation de certains gènes, suggérant ainsi leur implication dans la voie de différenciation CD4+. Ces résultats suggèrent également que la contribution de la signalisation du RTC dans la différenciation des thymocytes restreints au CMH-II ne se limitait pas à l'induction de ThPOK. Étonnamment, seul un effet synergique limité a été observé sur la différenciation des thymocytes restreints au CMH-I, lorsque Gata3, un autre facteur de transcription également induit dans les thymocytes restreints au CMH-II, et ThPOK étaient surexprimés en même temps dans ces cellules, suggérant peu de chevauchement fonctionnel entre ces deux facteurs de transcription. L’ensemble de ces résultats indique que ThPOK et la signalisation induite par le RCT fonctionnent en synergie durant le développement des lymphocytes T CD4+. / T lymphocytes are at the core of the adaptive immune system and their dysfunction is associated with several disorders and pathologies, which are at times fatal. The two main types of T-cells in mice and man are: the major histocompatibility complex (MHC) class-II-restricted CD4+ helper T-cells, and the MHC-I-restricted CD8+ cytotoxic T-cells. Developmental stages of the two types of T-cells occurs in the thymus in multiple sequential maturation stages that are identified by cell-surface CD4+/CD8+ co-receptor expression. Differentiation of the two types of T-cells in the thymus from a common precursor is influenced by the intensity and duration of signals derived from the T-cell receptor (TCR) and cytokines secreted by the thymic stromal cells. These signals lead to the activation of ThPOK or Runx/CBF transcription factors, which control the transcriptional network regulating CD4+ and CD8+ lineage fate, respectively. Studies have demonstrated that ThPOK is both necessary and sufficient for CD4+ T-cell development as gain- and loss-of-ThPOK function redirects positively selected MHC-I- and MHC-II-restricted thymocytes into CD4+ and CD8+ T-cell lineage fate, respectively. However, the role of TCR signaling and the extent to which ThPOK expression influences CD4+ lineage choice remains to be investigated. My thesis aims to elucidate the fundamental basis the CD4+/CD8+ lineage choice by exploring the molecular mechanism of action of ThPOK and TCR signaling in CD4+ lineage fate of MHC-I- and MHC-II-specific thymocytes. In this study, we have characterized gain-of-function of ThPOK in three independent transgenic mouse lines expressing varying amounts of ThPOK. Extensive analysis of the three ThPOK transgenic lines expressing MHC-I- and MHC-II-specific monoclonal TCR indicated that MHC-I-restricted, compared to MHC-II-restricted, thymocytes required significantly more ThPOK for efficient differentiation into the CD4+ lineage. Interestingly, the founder line with the lowest transgene expression, despite expressing significantly higher amounts of ThPOK compared to the endogenous levels in WT CD4+ T cells, induced a partial CD8+ to CD4+ redirection of MHC-I-restricted cells, leading to the generation of mature CD4+, DN and CD8+ T-cells in the same mouse. Lineage specific gene expression analysis, specifically in DN mature T cells from ThPOK transgenic mice expressing MHC-I-specific TCR, showed that, compared to induction of helper program, suppression of cytotoxic program required lower amount of ThPOK. Further investigation showed that TCR signal strength and MHC specificity of 4 developing thymocytes played a critical role in determining ThPOK-induced CD4+ lineage fate. While increase in TCR signal strength augmented the efficiency of ThPOK-induced CD4+ lineage choice of MHC-I-restricted thymocytes in part via endogenous ThPOK induction, it appeared to have ThPOK independent function as well as judged by significantly different CD4+ T-cell frequencies in OTI mice expressing the same amount of ThPOK but transduced quantitatively different TCR signal. Importantly, the efficiency of CD4+ lineage choice of MHC-I-specific thymocytes with augmented TCR signal strength was still significantly lower compared to the efficiency of CD4+ lineage choice of MHC-II-restricted thymocytes expressing only the transgene-encoded ThPOK suggesting a qualitative role for TCR signaling as well in CD4+ lineage choice. We then evaluated CD4+ lineage fate decision in the absence of ThPOK induction in physiologically relevant alteration in TCR signaling versus loss of ThPOK function. While we observed CD4+ to CD8+ lineage redirection of MHC-II-specific thymocytes due to Thpok-deficiency as well as lack of ThPOK induction due to disruption of TCR signaling, the two modes of lineage redirection appeared to be due to different mechanisms. Our investigation demonstrates that TCR signaling in MHC-II-restricted thymocytes induces the expression of select genes in loss-of-function of ThPOK model suggesting potential role for these genes in establishing the CD4+ helper program. These results also suggest that the contribution of MHC-II-specific TCR signaling in driving CD4+ lineage choice is not limited to Thpok induction. Interestingly, only a limited synergistic effect was observed when both Gata3, which is also induced in MHC-II-signaled thymocytes, and ThPOK were overexpressed in MHC-I-restricted thymocytes suggesting a limited functional overlap between the two transcription factors. Collectively, these data indicate that ThPOK and TCR signaling work synergistically to promote the development of CD4+ T-cells with some ThPOK independent function for TCR signaling.

CD8+ T Zellen unterstützen die Beseitigung von Pathogenen und sind somit entscheidend bei der Bekämpfung von Infektionen. Neben der Antigendosis und dem inflammatorischen Zytokinemilieu hat auch die Stimulation durch den TZR einen entscheidenden Einfluss auf die CD8+ T Zellantwort. Das transkriptionelle Programm, die finale Größe und Dauer der klonalen Expansion und der Start der Kontraktionsphase werden durch die TZR-Signalstärke bestimmt. Schwache TZR-Stimulation führt zu einer verminderten Expansion und vermittelt eine frühzeitige Kontraktionsphase, die eine Entwicklung von Gedächtniszellen auf den Kosten der Effektorzellen favorisiert. IRF4 wird nach TZR-Ligand-Interaktion in CD8+ T-Zellen hoch reguliert. Seine Expressionskinetik ist stark von der TZR-Signalstärke der Aktivierung abhängig, übersetzt diese und sorgt für die Umsetzung in ein entsprechendes transkriptionelles und differentielles Programm. In dieser Arbeit konnte erstmals gezeigt werden, dass die IRF4-Defizienz in CD8+ T-Zellen zu einem verfrühten Abbruch der Expansion und zu einem vorzeitigen Beginn der Kontraktion führt, die durch den FAS-vermittelten Tod-induzierenden Signalweg initiiert wird. Außerdem präsentieren IRF4-defiziente CD8+ T-Zellen vermehrt Phosphatidylserine an ihrer Oberfläche und Komplementdeposition, beides begünstigt die Erkennung und Aufnahme durch Phagozyten. Diese Ergebnisse weisen zudem stark darauf hin, dass durch die fehlende Expression von IRF4 in CD8+ T Zellen, ein schwaches TZR-Signal übermittelt wird, unabhängig von der tatsächlichen Stärke und Dauer des aktivierenden Signals, dass zu einer Verkürzung der Expansionsphase führt und eine verfrühte Kontraktionsphase der Effektorzellen auslöst. Diese Arbeit erweitert schon bekanntes Wissen um IRF4 als Schlüsselregulator für die Differenzierung und Funktionalität der ag-spezifischen CD8+ T-Zellen, da es den Beginn der Kontraktionsphase diktiert mittels Aktivierung von verschiedenen Apoptose- und Phagocytose Signalwegen. / CD8+ T cells promote pathogen clearance and play a crucial role in controlling infections. Besides antigen dose and inflammatory cytokine milieu, the TCR stimulation contributes to the programming of the CD8+ T cell response. A distinct developmental program, the final magnitude and duration of clonal expansion, as well as the timing of the onset of T cell contraction, are determined by the TCR signaling strength. Weak TCR stimulation results in a diminished magnitude of expansion and accelerates the onset of contraction, as it favors the development of memory cells at the expense of effector cells. IRF4 is a transcription factor, that is upregulated in CD8+ T cells following TCR stimulation. Furthermore, its expression kinetic is highly dependent on the TCR signaling strength, which initiated activation. Therefore, it translates the strength of the activating signal and transmits it into a proper transcriptional and developmental program. This study provides unique evidence that the absence of IRF4 expression in CD8+ T cells leads to a hasted termination of clonal expansion and a premature contraction, initiated by the FAS-mediated cell death pathway. Moreover, IRF4-deficient CD8+ T cells exposed phosphatidylserine on their cell surface and showed complement deposition, both facilitating their recognition and uptake by phagocytes. The findings of this study additionally strongly indicate that IRF4 deficiency mimics weak TCR engagement and in turn transmits every TCR signal, independent of its actually affinity and duration, into a developmental program, that give rise to an early memory formation and results in a premature onset of effector CD8+ T cell contraction. This data extend previous knowledge of IRF4 being essential for the differentiation and functionality of ag-specific effector CD8+ T cells, as it furthermore dictates the onset of CD8+ T cell contraction via the activation of several death and phagocytosis inducing pathways.