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1
Groves, Tim C. "Pre-TCR and TCR-Ãß signaling during T cell development." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ27657.pdf.
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Currie, James. "Stochastic modelling of TCR binding." Thesis, University of Leeds, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.590430.
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A fundamental process in the immune response to infection is the activation of T cells following contact with antigen presenting cells. This activation occurs after T cell receptors on the surface of T cells bind to immunogenic peptides expressed on the surface of antigen presenting cells. The binding of T cell receptors to ligands not only leads to the activation of T cells, it is also key to T cell selection in the thymus and the maintenance of a diverse T cell receptor repertoire. T cell receptor bindings are converted into a signal which activates a T cell but there is no universal theory which governs this process. There is experimental evidence to suggest that receptor-ligand bindings must be sufficiently long to elicit a T cell response. and that counting devices in the T cell work to allow signal accumulation, decoding and translation into biological responses. In view of these results, this thesis uses mathematical models to explore the timescales associated with T cell responses. A stochastic criterion that T cell responses occur after N receptor-ligand complexes have been bound for at least a dwell time, T, each, is used. The first model of receptor-ligand binding, in conjunction with the stochastic criterion, supports the affinity threshold hypothesis for thymic selection and agrees with the experimentally established ligand hierarchy for thymic negative selection. The initial model of ligand-receptor binding is then extended to include feedback responses, bivalent receptor binding and ligand diffusion through the immunological synapse. By including these mechanisms, the models agree with an array of experimental hypotheses which include: T cells exhibit a digital response to ligand. bivalent T cell receptor engagement stabilises receptor-ligand bindings and one ligand is sufficient to elicit a T cell response.
3
Lacroix, France. "T cell receptor (TCR) for antigen: A comparative study between the TCR alpha/beta and TCR gamma/delta subsets in noninfected and HIV infected individuals." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/6937.
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HIV infection is associated with characteristic quantitative changes in T-lymphocyte subsets: progressive depletion of the CD4$\sp{+}$ T-lymphocytes and an increased number of the CD8$\sp{+}$ T-lymphocyte. In this study, I report the results of a flow cytometric analysis of the expression of CD3, CD4, CD8, TCR$\alpha$/$\beta$, and TCR$\gamma$/$\delta$ antigens. I observed that CD8$\sp{+}$TCR$\alpha$/$\beta\sp{+}$ cells increased early in HIV disease (p 0.01) whereas the frequency of CD4$\sp{+}$TCR$\alpha$/$\beta\sp{+}$ cells was relatively unchanged. The frequency of TCR$\gamma$/$\delta\sp{+}$ cells remained unchanged. However, the mean fluorescence intensity (MFI), reflecting surface antigenic density, varied and allowed a clear distinction among different stages of infection. The expression of three activation markers (HLA-DR, CD38, CD57) was clearly increased in HIV infected individuals. The TCR$\alpha$/$\beta$ subset showed more substantial variation for activation markers. In the TCR$\gamma$/$\delta$ subset, the CD57 antigen seemed to be the most affected by the state of the disease and showed the greatest increase (p 0.01). (Abstract shortened by UMI.)
4
Mariathasan, Sanjeev. "TCR-mediated signaling in thymocyte selection." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63683.pdf.
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Rivera, Reyes Brenda Mariola. "Regulation of the TCR signaling pathway." Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1132588714.
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Barry, A. C. "Regulation of TCR signalling by SOCS." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479241.
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Mallaun, Michel. "Proximal TCR signaling in self tolerance /." [S.l.] : [s.n.], 2008. http://edoc.unibas.ch/diss/DissB_8729.
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Huang, Elizabeth Chi-Fang. "Organisation of, and ligand-independent signalling by, the TCR, with a special emphasis on the pre-TCR." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:0c01e3d4-2002-487c-a0b6-09ed20cb223b.
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Understanding how immune signalling is initiated and regulated spatiotemporally is likely to be helped by investigations of receptor stoichiometry. The pre-TCR expressed by thymocytes shares similarities in structure and signalling components with the mature TCR but, in contrast to the TCR, it has no known ligands. This thesis has therefore analysed the stoichiometry of the pre-TCR using mutagenesis- and imaging-based approaches, and explored how ligand-independent signalling might be initiated by both the TCR and pre-TCR. The mutational analysis, which required considerable optimisation because the pre-TCR is very weakly expressed in transfected cells, suggested that only one contiguous surface on pre-Tα needs to be buried in the pre-TCR complex in order for it to reach the cell surface. This comprised the surface buried at the interface with the constant region of TCRβ and therefore was incompatible with previous suggestions that the pre-TCR assembles into a 'head-to-tail' dimer. Unexpectedly, super-resolution imaging experiments combined with cluster analysis, and the method of single-molecule cross-colour coincidence detection were suggestive that, rather than dimers, the pre-TCR forms higher-order oligomers in transfected cells and possibly also in thymocytes. Similar analyses showed that the organisation of the TCR was heterogeneous, depending on the level of expression of the receptor. Overall, technical limitations that emerged in the course of the study highlighted some of the difficulties in studying native receptor stoichiometry on resting cells generally. The final part of the thesis investigated ligand-independent signalling by the TCR, using a novel assay based on the binding of a superagonistic antibody to a non-signalling form of the receptor CD28. Using CRISPR/Cas-mediated gene editing, it was shown that ligand-independent signalling by the mature TCR and pre-TCR was dependent on Lck and Zap70, indicating that it is reliant on the triggering of conventional signalling pathways. Ways in which the pre-TCR might initiate signalling, that could be tolerant of complexes exhibiting a range of stabilities, are also discussed.
9
Bunse, Mario. "RNAi-mediated knockdown of the endogenous TCR improves safety of immunotherapy with TCR gene-modified T cells." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17155.
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Durch den Transfer der Gene des heterodimeren T-Zellrezeptors (TZR) mithilfe viraler Vektoren können T-Zellen programmiert werden, ein ausgewähltes Antigen spezifisch zu erkennen. In klinischen Studien wurden solche T-Zellen bereits mit Erfolg zur Immuntherapie von Krebs und viralen Infektionen eingesetzt. Genmodifizierte T-Zellen unterscheiden sich jedoch von normalen T-Zellen, weil sie neben den beiden zelleigenen auch die zwei übertragenen TZR-Gene exprimieren. Diese Situation erlaubt die Bildung vier verschiedener TZR-Heterodimere: der zelleigene TZR, der übertragene TZR und zwei gemischte TZR, bestehend aus je einer übertragenen und einer zelleigenen TZR-Kette. Gemischte TZR bergen das Risiko von Nebenwirkungen, weil sie durch Zufall gesundes Körpergewebe erkennen und so Autoimmunität auslösen könnten. In dieser Arbeit wurden deshalb virale Vektoren entwickelt, die gleichzeitig mit der Übertragung von neuen TZR-Genen den zelleigenen TZR durch RNA Interferenz (RNAi) unterdrücken. Mikro-RNA (miRNA), die in den Vektor MP71 eingefügt wurden, reduzierten den zelleigenen TZR in Maus-T-Zellen um mehr als 85%. Dies hatte zur Folge, dass beide Ketten des übertragenen P14-TZR in gleicher Menge auf der Zelloberfläche exprimiert wurden und die Bildung von gemischten TZR reduziert wurde. In einem Mausmodell der adoptiven T-Zelltherapie verhinderte die Unterdrückung des zelleigenen TZR die Entstehung von Autoimmunität, die andernfalls durch gemischte TZR verursacht wurde. Im Gegensatz dazu führte die Anwendung von gentechnisch optimierten P14-TZR-Genen weder zur angeglichenen Oberflächenexpression der P14-TZR Ketten noch zu weniger Autoimmunität im Mausmodell. Ein anderes Tierexperiment zeigte, dass die miRNA die Funktion der genmodifizierten T-Zellen nicht beeinträchtigte. Schließlich wurde ein viraler Vektor entwickelt und getestet, der die Expression des zelleigenen TZR in menschlichen T-Zellen effektiv unterdrückte und die Bildung von gemischten TZR reduzieren konnte.<br>T cells can be genetically modified using viral vectors. The transfer of genes encoding both chains of the heterodimeric T cell receptor (TCR) programs T cells to specifically react towards an antigen of choice. Such TCR gene-modified T cells were already successfully applied in clinical studies to treat cancer and viral infections. However, in contrast to nonmanipulated T cells these cells express the transferred TCR in addition to the endogenous TCR and this situation allows the assembly of four different TCR heterodimers: the endogenous TCR, the transferred TCR, and two mixed TCR dimers, composed of one endogenous and one transferred TCR chain. The formation of mixed TCR dimers represents a safety issue because they may by chance recognize self-antigens and thereby cause autoimmune side effects. To overcome this problem, an RNAi-TCR replacement vector was developed that simultaneously silences the endogenous TCR and expresses an RNAi-resistant therapeutic TCR. The expression of miRNA encoded by a retroviral MP71 vector in transduced mouse T cells reduced the surface levels of the endogenous TCR by more than 85%. The knockdown of the endogenous TCR in turn resulted in equal surface expression levels of both transferred P14 TCR chains and prevented the formation of mixed TCR dimers. Accordingly, the development of lethal mixed TCR dimer-dependent autoimmunity (TI-GVHD) in a mouse model of adoptive T cell therapy was dramatically reduced by the knockdown of the endogenous TCR. In contrast, the usage of genetically optimized TCR genes neither resulted in equal surface levels of both P14 TCR chains nor in reduced autoimmunity. A second mouse model demonstrated that the in vivo functionality of the transduced T cells was not negatively influenced by the expression of the miRNA. Finally, an RNAi-TCR replacement vector for human T cells was developed that effectively reduced the expression of the endogenous TCR and prevented the formation of mixed TCR dimers.
10
Sommermeyer, Daniel. "Generation of dual T cell receptor (TCR) T cells by TCR gene transfer for adoptive T cell therapy." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16051.
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Die Herstellung von T-Zellen mit definierten Spezifitäten durch den Transfer von T-Zellrezeptor (TCR) Genen ist eine effiziente Methode, um Zellen für eine Immuntherapie bereitzustellen. Eine besondere Herausforderung ist dabei, ein ausreichend hohes Expressionsniveau des therapeutischen TCR zu erreichen. Da T-Zellen mit einem zusätzlichen TCR ausgestattet werden, entsteht eine Konkurrenzsituation zwischen dem therapeutischen und dem endogenen TCR. Bevor diese Arbeit begonnen wurde war nicht bekannt, welche TCR nach einem Gen-Transfer exprimiert werden. Daher haben wir Modelle etabliert, in denen TCR Gene in Maus und humane T-Zellen mit definierten endogenen TCR transferiert wurden. Die Expression beider TCR wurde mithilfe von Antikörpern und MHC-Multimeren analysiert. Diese Modelle haben gezeigt, dass bestimmte TCR andere TCR von der Zelloberfläche verdrängen können. Dies führte in einem Fall zu einer vollständigen Umkehr der Antigenspezifität. Aufgrund dieser Ergebnisse haben wir das Konzept von „starken“ (gut exprimierten) und „schwachen“ (schlecht exprimierten) TCR vorgeschlagen. Zusätzlich wurde die Verdrängung „schwacher“ und „starker“ humaner TCR durch Maus TCR beobachtet. Parallel dazu wurde berichtet, dass die konstanten (C) Regionen von Maus TCR für die erhöhte Expression auf humanen Zellen verantwortlich sind. Dies führte zu einer Strategie zur Verbesserung der Expression humaner TCR, die auf dem Austausch der humanen C-Regionen durch die von Maus TCR basiert (Murinisierung). Ein Problem ist dabei die mögliche Immunogenität dieser hybriden Konstrukte. Deshalb haben wir jene Bereiche der Maus C-Regionen identifiziert, die für die erhöhte Expression verantwortlich sind. In der TCRalpha Kette wurden vier und in der TCRbeta Kette fünf Aminosäuren gefunden, die ausreichend für diesen Effekt waren. Primäre humane T-Zellen mit TCR, die diese neun „Maus“ Aminosäuren enthielten, zeigten eine bessere Funktionalität als T-Zellen mit Wildtyp TCR.<br>The in vitro generation of T cells with a defined antigen specificity by T cell receptor (TCR) gene transfer is an efficient method to create cells for immunotherapy. One major challenge of this strategy is to achieve sufficiently high expression levels of the therapeutic TCR. As T cells expressing an endogenous TCR are equipped with an additional TCR, there is a competition between therapeutic and endogenous TCR. Before this work was started, it was not known which TCR is present on the cell surface after TCR gene transfer. Therefore, we transferred TCR genes into murine and human T cells and analyzed TCR expression of endogenous and transferred TCR by staining with antibodies and MHC-multimers. We found that some TCR have the capability to replace other TCR on the cell surface, which led to a complete conversion of antigen specificity in one model. Based on these findings we proposed the concept of ‘‘strong’’ (well expressed) and “weak” (poorly expressed) TCR. In addition, we found that a mouse TCR is able to replace both “weak” and “strong” human TCR on human cells. In parallel to this result, it was reported that the constant (C)-regions of mouse TCR were responsible for the improved expression of murine TCR on human cells. This led to a strategy to improve human TCR by exchanging the C-regions by their murine counterparts (murinization). However, a problem of these hybrid constructs is the probable immunogenicity. Therefore, we identified the specific parts of the mouse C-regions which are essential to improve human TCR. In the TCRalpha C-region four and in the TCRbeta C-region five amino acids were identified. Primary human T cells modified with TCR containing these nine “murine” amino acids showed an increased function compared to cells modified with wild type TCR. For TCR gene therapy the utilization of these new C-regions will reduce the amount of foreign sequences and thus the risk of immunogenicity of the therapeutic TCR.
11
Smelty, Philippe. "Caractérisation de la chaîne pré-TCR alpha, constituant du pré-TCR, chez les vertébrés non-mammaliens : étude comparative." Paris 6, 2010. http://www.theses.fr/2010PA066740.
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Chez les mammifères, le développement intrathymique des lymphocytes T est conditionné par l’expression d’une molécule clé appelée chaîne pré-TCR alpha (pTa). Celle-ci forme un hétérodimère avec la chaîne TCR pour constituer à la surface des thymocytes le pré-TCR, indispensable à la poursuite de la maturation des lymphocytes T. La fonction du pré-TCR serait ligand indépendant et se déroulerait par homo-oligomérisation du pré-TCR médiée par la pTa. La pTa n’avait été caractérisée que chez 3 espèces mammifères. Mon travail a permis d’identifier la pTa chez toutes les classes de mammifères, mais également pour la première fois, chez les vertébrés non mammaliens, les oiseaux et les reptiles (sauropsidés). L’étude des profils d’expression de la pT aviaire, établie par hybridation in situ et RT-PCR sur cellules triées suggèrent que la fonction de la pT dans le développement des lymphocytes est identique chez les espèces non mammaliennes et mammaliennes. Cependant, les comparaisons des séquences des pTa ont révélé que certains résidus, indispensables pour la fonction de la pTa chez la souris et l’homme, tels que ceux responsables de l’homo-oligomérisation du pré-TCR ou tels que les prolines intracellulaires liées à la transduction du signal, sont localisés différemment ou sont absents au niveau des pTa de sauropsidés et de certaines classes de mammifères. L’hypothèse développée à la suite de ce travail défend ainsi l’idée d’une fonction ancestrale de la pTa bornée à l’adressage du complexe pré-TCR à la membrane, fonction qui s’est complexifiée au cours de l’évolution afin de permettre une plus grande efficacité et une meilleure régulation des signaux de transduction
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Borger, J. G. "Visualising early signaling events during TCR activation." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/758822/.
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T cell activation, differentiation and expansion are induced by signals generated upon engagement of TCR by agonist peptide:MHC. At the site of T cell interaction with an antigen presenting cell, TCR, accessory proteins such as CD28 and LFA-1, and the Src family kinases (SFK) Lck and Fyn, accumulate and form the immunological synapse. Lck is the key signaling molecule, initiating TCR signals by phosphorylating the ITAM in the CD3/ subunits resulting in the recruitment and activation of Zap-70 and further propagation of the signaling cascade. Fyn is a positive mediator of T cell signaling, sharing specific substrates in common with Lck, and also acts as a negative regulator by phosphorylating the adaptor protein, PAG. Phosphorylated PAG binds Csk and its recently identified binding partner, the protein phosphatase PEP, recruiting them to the plasma membrane where they can inhibit the SFK. The differential localisation of the kinases, with Fyn compartmentalised to lipid rafts in mature T cells and the majority of Lck being non-lipid raft associated could explain the unique functions attributed to each kinase. In order to examine the interactions between SFK and their negative regulators in T cell activation, CD8+ T cells from mice expressing the class I MHC-restricted TCR, F5, were investigated. In naïve T cells Lck, Fyn, Csk and PEP were shown to co-localise to the site of activation-induced tyrosine phosphorylation following early TCR crosslinking. In contrast, in memory T cells Csk, PEP and Fyn not only co-localised with the site of TCR activation but also distributed at the distal end of the cell, suggesting differential redistribution of key negative regulators away from the site of TCR engagement in these cells. In the absence of Fyn there was no change in Csk localisation despite the loss of PAG phosphorylation, suggesting another adaptor protein can recruit Csk to the plasma membrane. Caveolin-1, a cholesterol-rich membrane protein was investigated as a possible candidate for recruiting Csk and was shown to be present in CD8+ T cells. Moreover, caveolin-1 was shown to be phosphorylated by Lck, Fyn and Abl kinases upon TCR engagement and to redistribute and polarise to the cSMAC, co-localising with Csk. In summary we have shown that there are alterations in the distribution of the negative regulators Csk, PEP and Fyn, once T cells have encountered antigen, and that this could be associated to the more efficient responses of memory cells upon re-exposure to antigen. Furthermore, its appears there is redundancy of both signaling molecules and adaptor proteins, which may contribute to the fine tuning of the TCR signaling pathway, such that signals derived from the TCR can still modulate an appropriate immune response even in the absence of key regulators.
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Perro, M. "Lentiviral TCR gene transfer for tumour immunotherapy." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/134272/.
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The ability to manipulate the immune system to induce protection against tumour, is one of the most fascinating challenges in immunology. In this regard, TCR gene transfer is an attractive and powerful strategy to generate high numbers of tumour antigen-specific T cells for adoptive transfer treatment. This thesis describes the optimization of lentiviral vectors for TCR gene transfer in the absence of polyclonal activation of the transduced T cells, which may improve subsequent adoptive T cell therapy. The murinised and codon optimised chains of an HLA-A*0201-restricted TCR specific for Wilms` tumour antigen 1 were cloned in lentiviral vector constructs improved with a Leader sequence and the WPRE elements for redirecting T cells specificity. The effects of common gamma chain receptor cytokines IL-2, IL-7, IL-15 and IL-21 were investigated using WT1 TCR-transduced T cells for transduction efficiency, proliferative potential, phenotype and functional activity in response to cognate antigen. Although all cytokines tested allowed transduction, stimulations with IL-15 and IL-15 with IL-7 or with IL-21 promoted a higher efficiency. Expression analysis of CD28 and CD62L showed an important role of IL-21 in maintenance of a naïve phenotype. In addition, all cytokines promoted maintenance of “quality” of T cells as shown by co-expression of IL- 2, IFN-γ and TFN-α after specific stimulation. To further sustain the in vitro results, several in vivo models were tested. Consistently, using F5 transgenic mice recognizing the NP peptide presented on EL4-NP cell line, IL-15 with IL- 21 exposed CD8+ T cells were able to efficiently protect against tumour and to persist longer in tumour bearing mice compared to IL-2 treated T cells. Because previous reports demonstrated that efficient LN homing of T cell correlates with efficient tumour protection in vivo, an imaging approach to study the molecular signalling in vivo during T cell activation in the LN has been developed. In conclusion, in this thesis, it is demonstrated that lentiviral transduction of cytokine exposed T cell can improve gene therapy approach of adoptive therapy.
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Nicholson, E. K. "Enhancing the efficacy of TCR gene therapy." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1418205/.
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TCR gene therapy allows redirection of the antigen specificity of T cells by the introduction of novel TCR α and β chains by retroviral transduction. These TCR gene modified T cells can be adoptively transferred to target defined tumour antigens. The majority of TCR gene therapy studies has focused on the adoptive transfer of CD8+ T cells but there is increasing recognition of a central role for CD4+ T cells in effective immunotherapy protocols. The use of CD4+ T cells has been limited by the lack of well defined class II restricted TCR and also because the majority of tumours don’t express class II MHC. As a result research has focused on introducing class I restricted TCR into CD4+ T cells. Initial work has demonstrated that class I restricted CD4+ T cells often have reduced functional avidity compared to the parental CD8+ T cell. In particular, CD4+ T cells transduced with CD8 dependent TCRs are often of much lower functional avidity when introduced in the absence of a CD8 co-receptor. In order to improve the functional avidity of class I restricted CD4+ T cells, murine CD4+ T cells were co-transduced with F5 TCR (specific for influenza peptide, NP, in the context of H2-Kb) and additional CD3 molecules. The amount of CD3 within in a cell is rate limiting for the expression of introduced TCR and thus when cells are transduced with additional CD3 it removes this rate limiting step and thus enhances the surface expression of the TCR. TCR surface expression is one of the key determinants of T cell functional avidity. CD4+ T cells co-transduced with F5- TCR and CD3 had increased surface expression of F5-TCR and increased pentamer binding. This translated in vitro into increased functional avidity compared to CD4+ T cells transduced with F5-TCR only. When adoptively transferred in vivo into irradiated tumour bearing syngeneic recipients, F5- TCR + CD3 CD4+ T cells had greater expansion and persistence and trafficked to the tumour site at higher and faster rates than F5-TCR only CD4+ T cells. In addition, F5-CD3 CD4+ T cells demonstrated superior control of tumour growth. Unexpectedly mice that received adoptive transfer of F5-TCR + CD3 CD4+ T cells developed marked lethal toxicity. Further experiments to try to determine the nature of this toxicity suggest a multifactorial cause including mispairing of the introduced TCR α and β chains with the endogenous TCR and development of autoreactive T cells in the presence of additional CD3 mediated either by upregulation of the introduced TCR or the endogenous TCR.
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Bruger, Annika Målin. "TCR signalling in response to affinity stimulation." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5f9c1001-6c43-472f-a495-c9573b54a84a.
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T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1<sub>156-165</sub> peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1<sub>156-165</sub> peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.
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Trinquand, Amélie. "Leucémies Aigues Lymphoblastiques T et signalisation TCR." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05S008.
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Les Leucémies Aigues Lymphoblastiques T (LAL-T) sont des hémopathies malignes causées par la prolifération de cellules immatures T bloquées à un stade donné de leur différenciation. Leur oncogenèse est multigénique. Une anomalie de type A (à l’origine du blocage de différenciation) ainsi que des anomalies de type B (gains de fonctions de type prolifération, métabolisme, résistance à l’apoptose…) sont souvent retrouvées chez le même patient. Ces leucémies reproduisent individuellement les différentes étapes de la maturation thymique humaine. En fonction de l’immunogénétique (phénotype et réarrangement des loci des TCR), 3 groupes de LAL-T sont ainsi identifiables : les LAL-T immatures (correspondant aux formes non T-restreintes), les LAL-T corticales (bloquées autour de la b-sélection) et les LAL-T matures TCR/CD3+. Mon travail de thèse a consisté à déterminer à quel point l’activation et la signalisation du TCR pouvaient être impliquées dans la biologie de cette hémopathie. Nous avons utilisé le modèle transgénique Marilyn TCR-HY et montré in vitro (lignée TLX+) et in vivo (modèle de leucémogénèse TEL-JAK2) que l’activation du TCR par la reconnaissance de son peptide spécifique (DBY, peptide présent uniquement dans les souris mâles) empêche le développement et le maintien de la leucémie. L'induction de la signalisation du TCR par des anticorps monoclonaux dirigés contre la chaîne de signalisation CD3e (anti-CD3e humain, OKT3 ; anti-CD3e murin, 145-2C11) entraîne également une mort massive des cellules leucémiques en induisant un programme d'expression génique et de phosphoproteomique ressemblant à la sélection négative thymique. In vitro dans des LAL-T primaires, la stimulation du complexe CD3/TCR par un anticorps anti-CD3 entraîne la mort cellulaire des LAL-T CD3/TCR+ et non des TCR/CD3- quelque soit leur mécanisme oncogénétique sous-jacent. Finalement, le traitement anti-CD3 in vivo empêche la leucémogenèse chez les souris transplantées avec des LAL-T murines et humaines. Ces données fournissent un fort rationnel en faveur d’une thérapie ciblée, basée sur le traitement anti-CD3 des LAL-T matures CD3/TCR+. Ce travail démontre aussi que des étapes-clés du développement (comme la sélection négative) peuvent être des cibles thérapeutiques et sont actionnables malgré l’accumulation d’altérations géniques et épigénétiques dans les cellules cancéreuses. Par ailleurs, j’ai étudié la fréquence et l’impact pronostique des anomalies de la signalisation du TCR/pré-TCR dans une grande série protocolaire de LAL-T de l’adulte (GRAALL-2003 et -2005). Les voies de prolifération RAS/MAPK et PI3K/PTEN/AKT participent à la signalisation du TCR/pré-TCR et ont été rapportées comme dérégulées dans des séries de LAL-T pédiatriques. Dans notre série, j’ai identifié des délétions/mutations perte de fonction de PTEN (12 %) ou des mutations activatrices de KRAS/N-RAS (11%) montrant que les anomalies du pré-TCR/TCR sont fréquentes dans les LAL-T puisqu’elles sont retrouvées dans 23% des cas. Les anomalies de RAS/PTEN sont associées à un pronostic défavorable. Leur impact pronostique en fonction du statut mutationnel NOTCH1/FBXW7 (N/F) a également été étudié et montre que les anomalies de RAS/PTEN abrogent le bon pronostic des mutations N/F. Ce travail permet de proposer une classification oncogénétique basée sur les anomalies de N/F et RAS/PTEN. Cette classification définit les patients de bas risque comme ceux ayant N/F muté mais sans anomalie de RAS et PTEN (51 %) et les hauts risques qui regroupent tous les autres patients (49 %). Cette classification oncogénétique est dorénavant utilisée dans le nouveau protocole GRAALL-2014 des LAL-T de l’adulte<br>T-cell acute lymphoblastic leukemias (T-ALL) are rare lymphoid neoplasms characterized by the proliferation of T lymphoblasts arrested at specific stages of maturation. Leukemic transformation of maturating thymocytes is caused by a multistep pathogenesis involving numerous genetic abnormalities that drive normal T cells into uncontrolled cell growth and clonal expansion. Depending on immunogenetic, T-ALLs are classified in 3 groups: immature, cortical (blocked around b-selection) and mature (CD3/TCR+) T-ALL. My work was to determine if activation and TCR signalling are involved in the biology of this disease. We demonstrate in T-ALL that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent TCR signalling has anti-leukemic properties and enforces a molecular and phosphoproteomic program resembling thymic negative selection, a major developmental event in normal T cell development. Using mouse models of T-ALL, we show that induction of TCR signalling by high affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3e chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of TCR-expressing T-ALL patients and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. Besides, I studied frequency and prognostic impact of anomalies concerning pre-TCR/TCR signalling in a large cohort of adult T-ALL included in GRAALL trials. RAS/MAPK and PI3K/PTEN/AKT pathways are involved in pre-TCR/TCR signalling and are reported as deregulated in pediatric T-ALL. I identified deletion/mutation loss-of-function of PTEN (12%) and activating mutations of KRAS/N-RAS (11%) in 23% of patients. These anomalies predict poor outcome and abrogate the good prognosis of NOTCH1/FBXW7 mutations. We proposed a purely genetic stratification of patients based on N/F/RAS/PTEN status, identifying low-risk patients (51%) with N/F mutations without RAS/PTEN anomalies and high-risk patients (49%) composed by the remaining cohort. This stratification will be used for the next protocol of adult-T-ALL
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Mancini, Stéphane. "Différenciation des lymphocytes T et recombinaison des gènes du TCR : quel rôle pour le pré-TCR dans la régulation du réarrangement des gènes codant pour la chaîne [alpha] du TCR ?" Université Joseph Fourier (Grenoble), 2000. http://www.theses.fr/2000GRE10116.
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Les genes codant pour les chaines alpha et beta du recepteur a l'antigene des cellules t (tcr) sont rearranges sequentiellement durant la differenciation intrathymique des lymphocytes t, caracterisee par l'expression de plusieurs molecules membranaires, dont cd4 et cd8. Le rearrangement des genes tcrb, codant pour la chaine beta, intervient durant le stade cd4 8 (dn), et permet l'expression d'un pre-tcr comprenant la chaine beta, ptalpha et le complexe cd3, responsable de la transduction des signaux d'activation. L'assemblage du pre-tcr induit la maturation des thymocytes jusqu'au stade cd4 +8 + (dp). Il est generalement admis que les genes tcra, codant pour la chaine alpha, sont rearranges a la transition dn/dp, apres signalisation par le pre-tcr. Ma these a pour objet l'etude de la regulation des rearrangements des genes tcra, et du role du pre-tcr dans ce processus. L'inactivation du gene codant pour ptalpha entraine chez la souris un blocage partiel de la differenciation des lymphocytes t au stade dn. Nous avons montre que le repertoire des chaines tcralpha exprimees chez ces animaux est essentiellement normal. Il semble donc que le pre-tcr ne soit pas requis pour le rearrangement et l'expression des genes tcra, et que ces evenements peuvent potentiellement se produire au stade dn. Nous avons poursuivi ces travaux en analysant le statut des genes tcra dans d'autres modeles de souris genetiquement modifiees ou la differenciation des thymocytes est bloquee au stade dn. Nous avons pu formellement demontrer, pour la premiere fois, la presence de rearrangements tcra des le stade dn, en absence de signaux issus de cd3, du pre-tcr ou du tcr. Enfin, nous avons etudie la reponse a l'irradiation des thymocytes de souris n'exprimant pas cd3. L'irradiation conduit a l'induction des rearrangements tcra et a la reprise de la differenciation a la fois par des voies p53 dependantes et independantes.
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Trenh, Peter. "An Examination of MHC, Peptide, and TCR Interactions." eScholarship@UMMS, 2018. https://escholarship.umassmed.edu/gsbs_diss/989.
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T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV.
First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1.
Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops.
Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity.
Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation.
The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.
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Reuß, Simone. "Safety analysis of TCR gene-modified T cells." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16503.
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T-Zellrezeptor (TZR)-Gentherapie zeigte erste Erfolge in klinischen Studien, jedoch wurden gleichzeitig Risikofaktoren deutlich. Ein Risikofaktor ist das falsche Paaren der transferierten TZR-Ketten mit den endogenen, was zu TZR-Molekülen von unbekannter Spezifität führt und die Oberflächenexpression und somit auch die Funktionalität des transgenen TZR reduziert. Dieser Aspekt wurde in generierten T-Zellklonen mit einer konstitutiven/endogenen TZR-Expression sowie einer zweiten induzierbaren/transgenen TZR-Expression untersucht. Es konnte gezeigt werden, dass nach Induktion der transgenen TZR-Expression der endogene TZR seine Funktionalität verlor, obwohl er noch auf der Oberfläche detektierbar war. Als Ursachen wurden neben einer reduzierten Oberflächenexpression des endogenen TZR auch falsch gepaarte TZR-Moleküle, die mit Hilfe der Fluoreszenz-Resonanz-Energie-Transfer-Methode detektiert wurden, gefunden. Die Modifikation des TZR durch den Einbau einer zweiten Cystein-Brücke, was das Paaren der korrespondierenden TZR-Ketten stabilisieren soll, führte in den T-Zellklonen zu keiner Reduktion der falsch-paarenden TZR-Moleküle. In primären Wildtyp-T-Zellen verbesserte sich das richtige Paaren des transgenen TZR leicht und konnte durch Codon-Optimierung der TZR-Gene weiter verbessert werden. Der zweite untersuchte Risikofaktor ist die Insertionsmutagenese durch den retroviralen Vektor. Die sichere Verwendbarkeit von differenzierten T-Zellen für die TZR-Gentherapie wurde in einem Tiermodel mit wiederholter T-Zellstimulierung, um weitere Mutationen während der Zellteilung zu provozieren, analysiert. Im Laufe der Zeit reicherten sich die transferierten T-Zellen in den Tieren dramatisch an, aber entwickelten sich nicht zu T-Zelllymphomen. Die Proliferationskapazität und die Funktionalität der transferierten T-Zellen wurden bestätigt. Die Polyklonalität der TZR-gen-modifizierten T-Zellen wurde mit Hilfe der linear-amplifizierten Polymerasekettenreaktion nachgewiesen.<br>T cell receptor (TCR) gene therapy is a new therapy for cancer which showed first clinical success but at the same time risk factors evolved. One risk factor is the mispairing of the TCR chains with the endogenous TCR chains which leads to TCRs with unknown specificities and to a reduced expression and functionality of the transferred TCR. This aspect was analyzed in dual TCR T cell clones which had one constitutive/endogenous TCR expression as well as a second inducible/transgenic TCR expression. It could be shown that the endogenous TCR lost its functionality after induction of the transgenic TCR expression although it was still detectable on the cell surface. The reason was found in the lower surface expression level of the endogenous TCR as well as in mispaired TCR dimers detected by fluorescence resonance energy transfer (FRET) technique. Modification of the TCR by insertion of a second cysteine bridge which should stabilize the pairing of the corresponding TCR chains did not reduce the TCR mispairing in the T cell clones. In primary wild-type cells, the pairing of the transgenic TCR improved slightly and could be further improved by codon-optimization of the TCR genes. The second analyzed possible side effect of TCR gene therapy is the insertional mutagenesis by the retroviral vector. The safety of differentiated T cells for TCR gene therapy was analyzed in an animal model with a repetitive T cell stimulation to provide the opportunity for mutations to occur during cell division. Over time, transferred T cells increased dramatically in the recipient mice, but did not lead to T cell lymphomas. The proliferative capacity and the functionality of transferred T cells were confirmed. The polyclonality of the TCR gene-modified T cells could be confirmed by linear amplification-mediated polymerase-chain reaction.
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Stewart-Jones, Guillaume B. E. "Structural and functional analyses of human pMHC/TCR complexes." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413213.
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Moysi, Eirini. "T-cell receptor (TCR) usage in HIV-2 infection." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:ea3a066f-0043-4c71-88ec-2369de642460.
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Long-term non-progressors (LTPNs) in HIV infection target the structural protein Gag more frequently than individuals who progress to disease. However, the targeting of Gag per se does not always distinguish these two groups. Various factors have been put forth as likely explanations for this discrepancy including differences in the breadth and magnitude of observed responses, the HLA type of the host, the nature of the individual epitopes targeted and the ability of the virus to mutate these antigenic regions. The purpose of this thesis was to examine, using PBMCs isolated from HIV-2 infected LTNPs and CTL clones established in vitro, the clonotypic architecture and quality of an immunodominant HIV-2 Gag-specific response directed towards the HLA-B*3501-restricted epitope NPVPVGNIY (NY9: Gag245-253). The data presented in this thesis show that in spite of the expression of multiple inhibitory receptors on the surface of NY9-specific CD8+ T-cells, the NY9-response, which is a clonotypically 'private' response, bears a signature characterised by an increased cytotoxic sensitivity and the production of an array of cytokines, most notably IFN-γ and MIP-1β. Moreover, the results of this thesis indicate that the NY9-specific CD8+ T-cells are able to cross-recognise and lyse target B-cells pulsed with the corresponding HIV epitope PY9 and its variants at functional avidities (EC50) that are close to those exhibited by PY9-specific T-cells. However, not all mobilised TCR clonotypes are equally sensitive or equally cross-reactive. When individual CTL clones were studied it emerged that dominant clonotypes within the NY9-specific CD8+ T-cell memory pool possessed a higher avidity for tetramer and sensitivity for antigen than subdominant ones and demonstrated a better cross-reactive potential towards variants of the HIV-2 epitope. Hence, future HIV vaccine strategies may benefit from the inclusion of epitopes like NY9, the presentation of which appears to mobilise CD8+ T-cells with superior functional profiles.
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Gallagher, Maighréad. "Régulation de l'expression des gènes codant les chaînes TCR[alpha] et TCR[delta] au cours de la différenciation des lymphocytes T chez la souris." Université Joseph Fourier (Grenoble), 2000. http://www.theses.fr/2000GRE10150.
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Le recepteur a l'antigene des lymphocytes t (tcr) se compose de deux modules : le module de transduction du signal, ou complexe cd3 ; et le module de reconnaissance du peptide antigenique, compose de deux chaines clonotypiques, tcralpha et tcrbeta ou tcrgamma et tcrdelta. Celles-ci sont codees par des genes ayant subi une recombinaison somatique de segments variables (v), de diversite (d) pour tcrbeta et tcrdelta uniquement et de jonction (j). Chez la souris, les genes codant les chaines tcralpha et tcrdelta partagent un meme locus, le locus tcrad. L'organisation de ce locus permet l'utilisation des segments v (ou adv) par les deux chaines, tcralpha et tcrdelta, mais aucune etude definitive de la question existe a cette date. Cette these a pour objet l'etude du rearrangement et de la transcription des genes tcra et tcrd chez la souris dans differentes conditions. L'inactivation du gene cd3epsilon chez la souris permet l'etude des transcrits tcrdelta en l'absence de transcrits tcralpha. Les resultats de cette etude demontrent : un controle independant du rearrangement et de la transcription des chaines tcralpha et tcrdelta ; et l'absence d'un effet lie a la position des segments adv sur le locus sur les rearrangements tcrd permis. En analysant les sequences d'un grand nombre de transcrits adv2-ac chez la souris de type sauvage adulte nous avons pu detecter des rearrangements preferentiels entre segments adv2 et aj selon leurs positions relatives sur le locus tcrad. L'analyse des transcrits de l'ensemble des 23 familles adv presentes sur le locus tcrad par des techniques de pcr classique et quantitative a permis : la definition d'un repertoire tcrdelta plus etendu que precedemment suppose ; la confirmation de l'absence d'un effet due a la position des segments adv sur le locus ; la mise en evidence d'une evolution du repertoire tcralpha au cours de l'ontogenie ; et une grande difference entre les repertoires adultes tcralpha et tcrdelta chez la souris.
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McMahon, Roisin M. "Structures of autoimmune peptide-MHC and TCR peptide-MHC complexes." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526503.
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Hotblack, A. C. "Characterising the role of dendritic cells in TCR gene therapy." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1537265/.
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Adoptive transfer of T cell receptor (TCR) gene-modified T cells is a promising field of tumour immunology. However whilst these cells can potently control tumour growth, this occurs in only a subset of patients. In order to devise new strategies to improve the anti-tumour response we need a clear understanding of the mechanisms by which transferred T cells are activated to kill tumours. Whilst dendritic cells (DC) are required to activate and control T cell function in adaptive immune responses it is not known whether control of tumours by adoptively transferred T cells depends on similar interactions with endogenous DC. To address this the CD11c.DTR model was used to transiently deplete DC in mice with established B16.F10 sub-cutaneous tumours after having been treated with TCR-transduced T cells. Unexpectedly we found that depletion of CD11c+ cells facilitates enhanced expansion and effector-phenotype differentiation of the transferred T cells. This appears to be mediated by depletion of a DC population with regulatory capabilities. However depletion of DC in the closely related CD11c.DOG model fails to promote accumulation of TCR-transduced T cells. Indeed, in this model DC depletion leads to less T cell infiltration into tumours. These contrasting results following depletion likely reflect the heterogeneous CD11c+/DC compartment in the tumour, where different populations contribute pro- or anti-inflammatory roles. Nonetheless these data suggest that TCR-transduced T cells interact with the endogenous immune system, although the exact nature of this interaction requires further investigation.
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Guillet, Marina. "Analyse de la régulation du TCR dans différentes situations immunologiques." Nantes, 2002. http://www.theses.fr/2002NANT12VS.
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Grâce à l'utilisation d'une nouvelle méthode d'analyse de la régulation de la chaine β du TCR, nommée TcLandscape, nous avons observé que les cellules T nai͏̈ves stimulées in vitro dans des conditions de reconnaissance " directe " stricte présentaient un profil TCR particulier. En effet, la reconnaissance directe du CMH allogénique par des cellules T nai͏̈ves est associée à une forte accumulation des transcrits Vβ, sans altération de la distribution de taille du CDR3. . . . . Ces profils mettant en évidence des cellules T présentant une distribution altérée de la taille du CDR3 permettent d'identifier et de hiérarchiser des populations impliquées dans la réaction immunitaire. Une telle capacité s'avère particulièrement utile pour suivre dans le temps la réponse immune dans divers contextes. Un exemple ayant trait à des patients infectés par le VIH et vaccinés avec un mélange de lipopeptides est développé<br>Using a new global approach referred to as TcLandscape, we previously observed that direct -type pathway of allorecognition was associated with a strong accumulation of V~ transcript without skewing of CDR3 length distribution. Such a pattern was also observed in vivo in acute rejection of cardiac allografts and in acute delayed rejection of " accommodated " cardiac xenografis in rats. In contrast, T cell infiltrating cardiac tolerated allografis showed an altered pattern of TCR V~ chain that might represent the molecular signature of regulatory T cells. This pattern allowed to attribute quantitative difference in the response of different T cell families. This quantitative/qualitative approach allowed to follow over time the effect of an immune-based therapy in HIV-1 infected patients vaccinated with a mixture of lipopeptides
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Frazer, Gordon Lee. "How TCR signal strength controls CTL polarisation for target killing." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277687.
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Cytotoxic T lymphocytes (CTL) are major effector cells in the adaptive immune response against intracellular pathogens and cancers, killing targets with high precision. Precision is achieved through the specificity of the clonally expressed T cell receptor (TCR). TCRs recognise a specific peptide chain loaded into a major-histocompatability complex, triggering signalling, inducing the CTL to attach and kill target cells. Key stages in this attack are the initial conjugation followed by polarisation and docking of the centrosome to the junction of the two cells, the immune synapse (IS). This focuses secretion of the cytolytic components, perforin and granzyme, from modified lysosomes to kill the target cell. My PhD has utilised amino acid substitutions in the target peptide to alter its signal strength and shown this alters the subsequent killing efficiency of a target population. I developed new imaging and analysis techniques to investigate the effect of TCR signal strength at each step of the killing process. I show the first step, conjugation, is reduced for a percentage of cells with dwell times decreasing as TCR signal strength decreased. The next key step of centrosome polarisation and docking at the IS was also impaired for an increasing proportion of cells as TCR signalling reduced. Impaired centrosome docking reduced efficient granule recruitment to the IS, necessary for target killing. Centrosome docking was linked with the TCR-induced intracellular calcium flux, the duration of which increases with the strength of TCR signalling. This demonstrates how the process of CTL killing can be fine-tuned by the quality of antigen.
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Bartelt, Rebekah Ruth. "Characterization of GRB2 and SOS1 binding downstream of TCR activation." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3257.
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Despite their essential role in protection, T cells can be dangerous if unregulated. Dysfunctional T cell activity has been implicated in numerous diseases, including the failure of organ transplants, allergic reactions, multiple sclerosis, and coronary artery disease. Signal transduction pathways activated by the T cell receptor (TCR) are good targets for the development of therapies. However, we must first better understand the mechanisms of intracellular signaling that occur when a T cell is activated.
This thesis focuses on the scaffold protein LAT and its role in T cell activation. The localization of signaling proteins into LAT-nucleated complexes and subsequent aggregation of these complexes into microclusters is vital for the activation of intracellular signaling pathways, and the effector functions of T cells. Following TCR stimulation, LAT is phosphorylated and binds SH2 domain containing molecules, such as the adaptor protein Grb2. One LAT molecule is capable of binding up to three Grb2 molecules at one time. Grb2 also binds to the proline rich regions of several proteins, including SOS1. Recent studies indicate that at physiological ratios of Grb2 and SOS1, two Grb2 molecules bind to one SOS1 proline rich region, and this 2:1 stoichiometry is essential for LAT oligomerization and cluster formation.
The interaction of Grb2 and SOS1 is considered to be a model SH3 domain interaction, and has biased our understanding of these relationships for decades. Many studies have focused on the association between the Grb2 SH3 domains and the proline rich region of SOS1. This previous work identified four consensus-binding sites for Grb2 in the proline rich region of SOS1 using short 10-15 amino acid peptides, and indicated that this interaction has a low affinity. Interestingly, the interaction of full SOS1 with Grb2 appears to be at least 100-fold stronger than these peptide-based studies imply. While informative, the use of short peptides leaves the physiological relevance of the peptide-SH3 domain interaction ambiguous.
In this thesis, we specifically emphasize the LAT multi-protein complex and its role in the activation of T cells. First, we compare the differences in the phosphorylation states of various LAT-proximal molecules in two T cell lines and peripheral blood T cells. We then focus on the formation of this complex by investigating the essential interaction between Grb2 and SOS1. Using biochemical and biophysical techniques, we clearly demonstrate that although the previously identified consensus binding sites are important in the context of short peptides, they do not facilitate the interaction of full length Grb2 with the full proline rich region of SOS1. We also attempt to ascertain the role of LAT microclusters in T cell signaling.
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Potthast, Kerstin. "Interaktion des humanen CD8 mit MHC Klasse I und CD3-TCR." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=967744253.
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Mutluer, Bilge Halas. "Design, Implementation And Engineering Aspects Of Tcr For Industrial Svc Systems." Phd thesis, METU, 2008. http://etd.lib.metu.edu.tr/upload/12609424/index.pdf.
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Design and implementation of TCR (Thyristor Controlled Reactor) for industrial SVC (Static VAr Compensator) systems require special design. Both power stage and control system design and implementation are thoroughly investigated in this thesis. Engineering aspects of TCR design are emphasized and supported with case studies. As the first case study<br>a novel, unified and relocatable SVC for open cast lignite mining in Turkey is designed, implemented and commissioned. The second case study is the first 12 pulse TCR design and implementation for ladle furnace compensation in the world. The SVC simulation results are verified by data acquired in the field. Real time data are also simulated in EMTDC/PSCAD program to verify the control system responses of the commissioned systems.
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Oble, Darryl. "A TCR transgenic model of infection-induced autoimmune psoriasiform skin disease." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/31199.
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Psoriasiform skin diseases are a poorly understood group of disorders. Recent data has implicated the immune system with a central role in disease pathogenesis. In this thesis, various T cell populations were studied in a TCR transgenic model of psoriasiform disease, whose abnormal interactions culminate in distinctive psoriasiform pathology. The model is based upon the expression of the transgenic 2C TCR on the H-2[sup d] -expressing DBA/2 inbred strain (referred to as D2C mice). The 2C TCR recognizes a peptide (p2Ca) derived from the ubiquitous mitochondrial protein 2-oxoglutarate dehydrogenase, presented by the MHC class I molecule, L[sup d]. In D2C mice, expression of the 2C TCR led to a comprehensive depletion of immature T cell progenitors, which express the 2C TCR, and a resultant lymphopenia of mature peripheral T cells. The lymphopenia of CD8⁺ cytotoxic and CD4⁺ helper T cells predisposes to the overgrowth of opportunistic pathogens, resulting in inflammatory skin disease restricted to the sebum rich areas of the skin, resembling the human psoriasiform disease seborrheic dermatitis. In D2C mice, there is also a deficiency in T regulatory (T[sub reg]) cells as a result of slowed thymic output of mature T cells. The reduced T[sub reg] function results in a lymphoproliferation of polyclonal CD4⁺CD25⁻ cells, many of which home to the aforementioned cutaneous inflammatory sites. This expansion of "helper" cells was likely due to antigenic stimulation, presumably against immunogenic determinants of opportunistic pathogens. Reconstitution of the T[sub reg] compartment by adoptive transfer abrogates the development of psoriasiform pathology and precludes the lymphoproliferation of CD⁺CD25⁻ cells. These data suggest that T[sub reg] may downregulate the response of mature cells to ubiquitous commensal organisms as a means to maintain immunological homeostasis. In TCR transgenic mice which express the cognate Ag of the transgenic TCR, a large population of transgenic cells exist in the peripheral lymphoid tissues. These cells are anergized and fail to respond to stimulation with cognate ligand; however, they express a memory immunophenotype, including the intermediate affinity IL-2 receptor. The expression of this receptor enables these cells to use bystander IL-2 or IL-15 to overcome this inactivation, revealing enhanced functional properties induced by the high-affinity interaction with cognate ligand. These observations suggest that such clonally anergized cells may represent an in vivo autoimmune hazard; and, interestingly, a further consequence of the CD4⁺CD25⁻ lymphoproliferation in D2C mice is the bystander activation of these transgenic T cells. After undergoing acute activation, 2C T cells exacerbate the cutaneous pathology in these animals, a consequence that can be abrogated by the administration of a blocking mAb against the 2C TCR. Interestingly, the combination of immunodeficiency, T[sub reg] lymphopenia, the presence of CD4⁺CD25⁻ cells capable of undergoing vigorous expansion, and a large population of memory phenotype 2C transgenic cells was insufficient to induce disease when D2C bone marrow was adoptively transferred to lethally irradiated syngeneic DBA/2 mice. Examination of these animals revealed that bone marrow transfer did not deplete the skin of DBA/2-derived cutaneous γδ cells. Sentinel intraepithelial γδ lymphocytes have been shown to have an important role in surveying the epithelium for signs of infection and malignancy as well as in maintaining epithelial integrity. The development of these cells is curtailed in D2C mice due to the forced expression of the 2C TCR; however, the persistence of these cells in the aforementioned bone marrow chimeras may have protected these animals from the development of the disease phenotype. While generated DBA/2 TCRδ⁻/⁻ mice did not develop spontaneous disease, the transfer of D2C bone marrow to lethally irradiated DBA/2 TCRδ⁻/⁻ recipients successfully transferred the disease phenotype, confirming the importance of these cells in protecting against the development of psoriasiform pathology. This result also demonstrated that a compromised cutaneous barrier is necessary for disease pathogenesis, as disease does not develop when the skin is populated by sentinel intraepithelial lymphocytes. While considerable research efforts have been focused on human psoriasiform disease, a solid understanding of disease pathophysiology is severely lacking. This limited knowledge of psoriasiform disease is highlighted by the ongoing uncertainty of whether these diseases represent primary diseases of the epithelium, or whether these diseases represent tissue specific autoimmunity occurring in normal skin as a result of dysfunctional immunity. One explanation for this failure to understand basic principles of psoriasiform disease pathophysiology can be attributed to the limited numbers of appropriate model systems to carefully study disease. The D2C model of psoriasiform disease has been shown to be an accurate model system which has demonstrated that a complex interplay between various immunocytes and epithelium culminates in psoriasiform disease. The insight that the D2C model has generated will lead to a better understanding of these poorly characterized psoriasiform conditions.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate
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Gorman, Claire. "Genetic, phenotypic and functional analysis of the TCR zeta (s) chain." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503779.
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何沛德 and Pui-tak Ho. "Control and operation of high-performance thyristor-controlled-reactor(TCR) compensators." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1988. http://hub.hku.hk/bib/B31231160.
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Wikstrom, Matthew E. "The regulation of peripheral T cell responses in TCR transgenic mice." Thesis, The University of Sydney, 1997. https://hdl.handle.net/2123/27641.
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Transgenic mice expressing a T cell receptor specific for cytochrome C in association
with I-Ek were employed to study the mechanisms regulating the decision between
tolerance and immunity. Tolerance and immunity to the same peptide antigen were
. induced by using different routes of administration. Thus tolerance was induced by the
intravenous route and immunity by the subcutaneous route. The results presented in
this thesis provide a detailed map of the cellular events that occur during these two
distinct responses.
Intravenous immunisation induced activation of CD4+ cells expressing the transgenic
TCR (CD4+Tg0L+ cells), resulting in CD69 expression within two hours, priming of T
cell proliferation and Th1 cytokine production by 24 hours. Clonal expansion peaked 3-
4 days after immunisation. The number of CD4+Tg0t+ cells decreased rapidly after day
four, such that only 50% of initial cell numbers remained 7—10 days after immunisation.
Intravenous peptide also upregulated CD44 expression, increasing the proportion and
number of CD4+Tg0t+CD44hi cells at the peak of the response, but having no net effect
at the resolution of the response. When labelled CD4+Tg0t+ cells were adoptively
transferred to syngeneic non-transgenic hosts, deletional tolerance to intravenous
peptide was still seen, demonstrating that it was not an artefact of the high precursor
frequency in TCR transgenic mice. Antigen re-challenge experiments demonstrated that
CD4+Tg0t+ cells remaining at the resolution of the primary response to intravenous
peptide could not respond as vigorously as naive cells either in vitro and in vivo.
Interestingly, intravenous administration of intact cytochrome C also stimulated T cell
activation, but failed to induce peripheral deletion, and resulted instead in a minor
increase in the final proportion of CD4+Tg0t+CD44hi cells.
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Bunse, Mario [Verfasser], Wolfgang [Akademischer Betreuer] Uckert, Thomas [Akademischer Betreuer] Blankenstein, and Christian [Akademischer Betreuer] Schmitz-Linneweber. "RNAi-mediated knockdown of the endogenous TCR improves safety of immunotherapy with TCR gene-modified T cells / Mario Bunse. Gutachter: Wolfgang Uckert ; Thomas Blankenstein ; Christian Schmitz-Linneweber." Berlin : Lebenswissenschaftliche Fakultät, 2015. http://d-nb.info/106856895X/34.
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Hazgui, Hana. "Modélisation de la dynamique des réseaux biologiques : applications en génétique et immunologie." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAS040/document.
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Dans cette thèse, nous nous intéressons à la modélisation statistique de données biologiques, et plus particulièrement à l'étude de l'information génétique et protéique.Dans un premier volet, nous avons amélioré un modèle statistique des données immunologiques existant chez la souris, que nous avons transposé à l'homme, afin d'étudier les différentes recombinaisons qui apparaissent au sein du thymus, à la fin de la vie embryonnaire, entre les segments des gènes de la portion V(D)J du chromosome 14 humain, appelées recombinaisons V(D)J.Dans un deuxième volet, nous avons étudié l'information génétique par le biais des réseaux de régulations génétique, celui de la maladie familiale « atrésie biliaire », ainsi que dans les réseaux de contrôle du système immunitaire, que nous avons appelés « Immunetworks ».Dans un troisième volet, nous proposons une nouvelle approche de la compression des données biologiques, qui intègre une étape de modélisation des processus dynamiques qui leur ont donné naissance : nous avons appelé cette approche la transformée Dynalet et nous l'appliquons, entre autres, à des signaux de spectrométrie RMN (Résonance Magnétique Nucléaire) des protéines et acides nucléiques. Cette méthode consiste à convertir les signaux de spectrométrie en sons, afin de construire une lutherie anharmonique permettant de reproduire les pics de relaxation périodisés, issus des spectres RMN des 20 acides aminés, ainsi que de ceux des 4 bases nucléiques azotées<br>In this thesis, we focus on statistical modeling of biological data, and more particularly on the study of genetic and protein information.First, we have improved a statistical model of existing immunological data in mice, we have transposed it to human, in order to study the various recombinations that occur within the thymus, at the end of the embryonic life, between segments of genes from the portion V (D) J of the human chromosome 14, called recombinations V (D) J.Secondly, we studied the genetic information through genetic regulatory networks, for example in the case of a family illness called the "biliary atresia," as well as in the immune system control networks, which we have called "Immunetworks".In a last part, we propose a new approach for biological data compression, which includes a step of modeling the dynamic processes that gave rise to them: we call this approach the "Dynalet" transform and we apply it, among others, to NMR spectrometry signals, i.e., Nuclear Magnetic Resonance spectra of proteins and nucleic acids. This method consists in converting the peaks of the spectrometric signals into sounds, in order to construct an anharmonic instrument capable to reproduce periodized relaxation peaks from the NMR spectra of the 20 amino acids, as well as those of the 4 nucleic bases
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Gangadharan, Denise Michelle. "Thymic development of CS8[alpha][alpha]⁺ TCR[alpha][beta]⁺ agonist selected IEL." Diss., Connected to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3189998.
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Thesis (Ph. D.)--University of California, San Diego, 2005.<br>Title from first page of PDF file (viewed Marc 6, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 100-116).
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Cheng, Gordon W. "Functions of CD45 in TCR signaling in CD4§+CD8§+ double-positive thymocytes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29256.pdf.
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Paletta, Daniel Sylvester [Verfasser], and Thomas [Gutachter] Herrmann. "Die Variabilität des Ratten iNKT TCR / Daniel Sylvester Paletta. Gutachter: Thomas Herrmann." Würzburg : Universität Würzburg, 2015. http://d-nb.info/1111783810/34.
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Bronnimann, Heather. "Functional Analysis of Interactions within the TCR-CD3-pMHC-CD4 Macro-complex." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/612427.
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CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.
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Villarese, Patrick. "Apport de l'analyse des réarrangements du TCR dans l'oncogenèse et l'ontogénie T." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05S007.
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Les cellules T maturent dans le thymus lors d’un processus très régulé par l’intermédiaire de facteurs intrinsèques, comme des facteurs de transcription, et des facteurs extrinsèques (par exemple des cytokines des cellules stromales). L’acquisition du potentiel T au cours de la thymopoïèse, à partir d’un précurseur médullaire, se réalise grâce à des étapes successives définies par l’expression de molécules de surface et par les différents réarrangements des gènes du TCR qui sont ordonnés : le TCRd étant le premier à se produire, suivi par le TCRg et TCRb, pour finir par le TCRa. Les réarrangements du TCR restent également parfaitement ordonnancés dans les leucémies aigues lymphoblastiques T et ce malgré l’accumulation successive d’évènements oncogéniques. Il est ainsi possible de définir trois sous-groupes immunogénétiques de LAL-T ; (i) les formes immatures n’exprimant pas de TCRb cytoplasmique (ii) les LAL-T matures exprimant un TCR de surface et enfin (iii) les LAL-T intermédiaires, dites pré-ab, exprimant le TCRb en intracytoplasmique sans expression membranaire d’un hétérodimère ab ou gd. Dans ce dernier sous groupe de LAL-T de phénotype cortical, deux oncogènes à homéodomaines, TLX1 et TLX3, appartenant à la famille des gènes homéotiques orphelins NKL, sont souvent dérégulés. Nous avons précédemment mis en évidence le rôle direct des oncoprotéines TLX dans le processus de l’arrêt de maturation, grâce à leur interaction avec le facteur de transcription ETS1, bloquant l’expression et les réarrangements du TCRa. Néanmoins, une partie des LAL-T corticales ne surexprime ni TLX1 ni TLX3, posant la question de l’implication potentielle d’autres gènes de la famille NKL dans le blocage de maturation. Nous avons donc réalisé une analyse transcriptionnelle de l’ensemble des 46 gènes de la famille NKL dans une large série de LAL-T et comparé les résultats avec ceux obtenus dans des sous populations thymiques humaines triées. Nous avons ainsi identifié 10 gènes dérégulés de manière ectopique dans notre série de LAL-T, incluant 6 gènes dont la dérégulation était inconnue dans ce contexte. Par ailleurs, nous avons mis au point une approche complexe combinant une analyse en CGH-array haute résolution, le dosage allélique du locus TCRa et un système de RT-PCR multiplex afin d’étudier de manière exhaustive le statut du locus TCRa dans cette même série de LAL-T. Nos résultats ont ainsi montré que ces nouveaux gènes NKL aboutissent aussi à une répression du TCRa par un mécanisme similaire à celui observé avec les oncoprotéines TLX. Les lymphomes anaplasiques (ALCL), qui sont caractérisés par une expression aberrante d’ALK, issue de la t(2;5), expriment des marqueurs d’activation T (CD30), cytotoxique (granzyme, perforin, TIA1) et des réarrangements clonaux du TCR, mais sans signalisation du TCR/CD3. Le stade du développement lymphoïde T où est initié la lymphomagenèse est inconnu, il est possible que cette translocation se produise avant l’expulsion thymique. Pour étudier cette hypothèse, nous avons analysé l’ensemble des TCR (d,g,b,a) par PCR et CGH array dans une série d’ALCL humain et utilisé un modèle murin de lymphomagénèse T dans lequel NPM-ALK est exprimé à l’aide du promoteur de CD4. Nous avons croisé ce premier modèle avec des souris transgéniques RAG déficient et/ou en présence d’un transgène TCR (OT1), afin d’étudier le rôle du TCR dans le développement tumoral. Le modèle de lymphomagenèse identifié est basé sur une expression de NPM-ALK dès les stades précoces de la différenciation thymique, lorsque le transcrit de fusion peut remplacer le TCRb, et lors de l’expansion des thymocytes corticaux au niveau de la « b-sélection ». Un TCR est cependant nécessaire pour la sortie du thymus, bien que perdu lors du développement des ALCL en périphérie. En conclusion, nous avons montré l’implication du TCR dans deux modèles d’oncogenèse. (...)<br>T cells mature in the thymus through a highly regulated process mediated by intrinsic factors (e.g. transcription factors) and extrinsic factors (e.g. cytokines or stromal cells). The acquisition of T lymphoid commitment during thymopoiesis, originating from a bone marrow precursor, is carried out through successive stages defined by the expression of various surface molecules and the precisely ordered TCR gene rearrangements; TCRd being the first to occur, followed by the TCRg and TCRb, and finally TCRa. TCR rearrangements are also highly coordinated in T acute lymphoblastic leukemia (T-ALL) despite the successive accumulation of oncogenic events. It is thus possible to define three immunogenetic subgroups of T-ALL; (I) the immature forms that do not express cytoplasmic TCRb, (ii) mature T-ALL which express a surface TCR and finally (iii) intermediate T-ALL, termed preab, which express intracytoplasmic TCRb without membrane expression of a TCR ab or gd Complex. In the latter subgroup, classically termed cortical T-ALL, two oncogenic transcription factors belonging to the NKL family of homeobox genes, TLX1 and TLX3, are commonly deregulated. We have previously demonstrated the direct role of TLX oncoproteins in the process of maturation arrest through their interaction with the ETS1 transcription factor, which blocks expression and rearrangements of TCRa. Not all cortical T-ALL cortical overexpress TLX1 nor TLX3, however, suggesting that other NKL family genes might be involved in the maturation arrest. We therefore, conducted a transcriptional analysis of all 46 NKL family genes in a large series of T-ALL and compared the results with those obtained in sorted human thymic subpopulations. We identified 10 ‘ectopic’ deregulated genes in T-ALL, including 6 genes whose deregulation was previously unknown in this leukemia. By combining high resolution CGH array, allelic of TCRa locus dosage and a novel TCRa RT-PCR multiplex, we show that these deregulated NKL genes also lead to inhibition of TCRa rearrangement, similar to that observed with TLX. These date demonstrate that homeobox inhibition of TCRa rearrangement is likely to explain the maturation arrest in the majority of cortical T-ALL, the commonest and most emblematic subgroup in this leukemia. Anaplastic lymphoma (ALCL), which are characterized by t(2;5) driven aberrant expression of ALK, express T activation markers (CD30), cytotoxic (granzyme, perforin, TIA1), and clonal TCR rearrangements in the intriguing absence of TCR/CD3 signaling. It is not clear at what stage of development ALCL lymphomagenesis is initiated, but as the expression of NPM is ubiquitous, it is possible that this translocation occurs before thymic egress. To investigate this, we analyzed all TCR(a,b,g,d) by PCR and CGH array in a series of human ALCL and compared these results with a T lymphomagenesis murine model in which NPM-ALK is regulated by the CD4 promoter. We crossed this first model with RAG deficient transgenic mice in the presence or not of a TCR transgene (OT1), to study the role of the TCR in tumor development. NPM-ALK expression from the earliest stages of thymic differentiation allow the fusion transcript to replace TCRb during the cortical thymic cellular expansion process known as "beta-selection". A TCR is, however, necessary for thymus egress, but is subsequently lost during the development of ALCL in the periphery, suggesting that the coexistence of TCR and NPM-ALK signaling is not compatible with lymphomagenesis and that the TCR may act as a tumor suppressor gene. In conclusion, we have delineated the involvement of TCRa in two models of oncogenesis. In T-ALL, NKL oncoproteins NKL prevent TCRa rearrangements and block cells at the highly proliferative TCRb-selection cortical thymic stage. In ALCL, a functional TCR appears to act as a tumor suppressor gene. Both models pave the way to differentiation therapy via TCR modulation
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Okabe, Namiko. "Suppressor of TCR signaling-2 (STS-2) suppresses arthritis development in mice." Kyoto University, 2018. http://hdl.handle.net/2433/232089.
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Majri, Sonia. "Regulation of CD4⁺ memory T cell homeostasis by STAT5 during TCR restimulation." Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC139.
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Les facteurs de transcriptions Signal Transducer and Activator Transcription (STAT) sont essentiels pour la régulation de gènes impliqués dans plusieurs fonctions biologiques, y compris la réponse immunitaire. Nous avons étudié un patient ayant une nouvelle mutation ponctuelle hétérozygote dans le gène STAT5B. Les symptômes cliniques majeurs observés chez ce patient sont thrombocytopénie immune (TPI), lymphadenopathies, concentration élevée en anticorps IgM dans le sérum et hypergammaglobulinémie. Nous avons observé une accumulation anormale de lymphocytes T mémoires effecteurs (LME). Une analyse du transcriptome du patient a permis d'observer une sous-expression des gènes régulés par STAT5. De plus, nous avons révélé que les LME du patient étaient résistants à la mort induite par la restimulation du TCR. Ces résultats démontrent un rôle important et inattendu de STAT5 dans la régulation de l'homéostasie des lymphocytes T mémoires pendant la restimulation du TCR<br>Signal transducer and activator transcription (STAT) proteins are essential transcription factors regulating gene expression involved in many biological functions especially immune responses. Here we report a patient with a de novo heterozygous missense mutation in STAT5B gene resulting in altered STAT5 transcriptional function. The patient presented with immune thrombocytopenia, lymphadenopathy, splenomegaly, an antibody class switching defect and granulocytosis with necrotizing granulomas. We found a specific dysregulation of CD4+ T cell subsets with an abnormal accumulation of effector memory T (TEM) cells. Transcriptome analysis in patient's T cells revealed a selective downregulation of the STAT5-dependent IL-2 signaling pathway. We found that TEM cells from the patient were resistant to in vitro TCR restimulation-induced cell death. These results demonstrate a key role of STAT5 in memory T cell homeostasis by regulating cell death during TCR restimulation
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Bachelez, Hervé. "Analyses moleculaires et fonctionnelles du tcr au cours des lymphoproliferations t cutanees." Paris 7, 1999. http://www.theses.fr/1999PA077017.
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La premiere partie des travaux presentes concerne l'analyse moleculaire de clonalite et l'etude du repertoire des segments variables de la chaine du tcr au cours des lymphomes cutanes epidermotropes (syndrome de sezary et mycosis fongoide). Les etudes de clonalite ont montre l'existence de rearrangements dominants du locus tcrg mis en evidence par pcr, dans une proportion de lesions cutanees analysees qui dependait du stade clinique. Ainsi, alors qu'un clone t unique ou tres predominant etait detecte dans les stades des tumeurs, aucun clone t dominant n'etait identifie dans pres d'un cas sur deux dans les lesions initiales. Ces resultats suggerent l'existence d'une selection progressive du clone qui semble parallele a la progression clinique, ainsi que le role d'un mecanisme de carcinogenese par etapes dans la pathogenese de ces lymphomes. En etudiant le repertoire des segments tcrbv utilises par les cellules de sezary, nous avons pu eliminer l'hypothese d'une stimulation superantigenique particuliere dans l'expansion lymphocytaire cd4 des lce. La deuxieme partie du travail concerne un syndrome caracterise par une infiltration lymphocytaire t cd8 pseudotumorale de la peau et des ganglions lymphatiques chez des malades infectes par le vih. Nous avons pu montrer que ce syndrome etait en rapport avec une infiltration tisulaire par des lymphocytes t cytotoxiques specifiques du vih, et non a une processus de transformation neoplasique. Ces donnees physiopathologiques ont ete renforcees par l'evolution favorable de l'infiltration cutanee sous tritherapie antiretrovirale hautement active.
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Peixoto, Andreia Liliana da Silva. "Análise da diversidade do repertório do TCR Vß na infecção pelo VIH." Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/3997.
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Mestrado em Biologia Molecular e Celular<br>Um repertório completo do TCR indica uma população de células T intacta, sem alterações, com potencial de reconhecer um vasto número de imunogénios.
Durante a infecção primária do VIH, a activação e expansão de células T específicas contra o vírus afecta globalmente o repertório do TCR, causando perturbações ao nível da representação individual de cada família V.
A diversidade do repertório TCR V pode ser analisada pelo método Spectratyping. Este método molecular permite detectar expansões clonais no repertório do TCR, através da visualização do tamanho da região CDR3 dentro de cada família V.
O presente trabalho teve como principal objectivo analisar a diversidade do repertório dos receptores das células T, TCR V, nas várias subpopulações celulares T.
O estudo incidiu em 22 indivíduos infectados pelo VIH, antes da adesão à terapêutica anti-retroviral, e em 22 indivíduos seronegativos para o VIH.
A análise do repertório em indivíduos com infecção pelo VIH evidenciou que a maioria das famílias V do TCR era policlonal e apresentava um perfil de distribuição dos picos anormal ou não-gaussiano. Algumas dessas famílias V apresentavam expansões dominantes de 1 ou 2 picos de CDR3, principalmente na população T CD8+. Esta subpopulação T também foi caracterizada pela ausência de expressão de algumas famílias V e pela presença de outras em expansões oligoclonais.
Os resultados da análise do repertório da cadeia V do TCR sugerem uma mudança na composição do repertório de células T em indivíduos com infecção pelo VIH nas diferentes subpopulações T.<br>A full TCR repertoire indicates a T cell population intact, unchanged, with potential to recognize a wide number of immunogens.
During the primary infection of HIV, activation and expansion of specific T cells against the virus affects the overall TCR repertoire, causing disorders at the level of individual representation of each V family.
The diversity of the TCR V repertoire can be analyzed by the Spectratyping method. This molecular method can detect clonal expansions in the TCR repertoire, through the visualization of CDR3 region size within each V family.
The main objective of this work was to study the diversity of the repertoire of T cells receptors, TCR V, in the several subpopulations of T cells. This study focused on 22 individuals with HIV infection, before the access to antiretroviral therapy, and on 22 healthy individuals.
The analysis of the repertoire in individuals infected with HIV showed that the majority of the V families of the TCR were polyclonal and presented a distribution profile of abnormal peaks or non-Gaussian. Some V families showed dominant expansions of one or two peaks of CDR3, mainly in the T CD8+ population. This T subpopulation was also characterized by the absence of some V families expression and by the presence of others in oligoclonal expansions.
The result of analysis of the repertoire of the TCR V chain suggest a change in the composition of the T cells repertoire in individuals with HIV infection in the different T subpopulations.
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Bethune, Michael T., Marvin H. Gee, Mario Bunse, Mark S. Lee, Eric H. Gschweng, Meghana S. Pagadala, Jing Zhou, et al. "Domain-swapped T cell receptors improve the safety of TCR gene therapy." ELIFE SCIENCES PUBLICATIONS LTD, 2016. http://hdl.handle.net/10150/622825.
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T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.
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Brabb, Thea. "The fate of MBP-specific T cells in MBP TCR transgenic mice /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/10853.
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Saini, Manoj. "The role of TCR and cytokine signals in naïve T cell homeostasis." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1446076/.
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The peripheral T cell compartment is maintained at a constant size, resulting from a balance of cell development, survival, proliferation and death. Transmission of signals through the TCR and IL-7R on the T cell surface is involved in regulating all these processes, however the precise manner in which these signals together maintain T cell homeostasis is unclear. To investigate the contribution of TCR and IL-7 signals to naive T cell homeostatic responses we established two model systems. To specifically address the role of homeostatic TCR signalling in the development and maintenance of the T cell compartment, we generated transgenic mice that conditionally express the Syk family tyrosine kinase Zap70. The transduction of TCR signals by Zap70 is essential for thymic development and T cell activation. Given the importance of Zap70 expression in T cell antigen receptor signalling, we investigated whether Zap70 was also essential for the transmission of TCR signals, required for the steady state survival of the peripheral naive T cell compartments. Zap70 deficient mice exhibit a complete block in thymopoiesis at the DP stage in the thymus and as a consequence lack mature peripheral T cells. For this reason we generated mice that express Zap70 in a conditional manner, using the tetracycline responsive gene regulatory system. Thymic selection proceeded normally in these mice, however ablation of Zap70 expression resulted in the disappearance in the peripheral naive CD4+ and CD8+ T cells, with the naive CD8+ T cell compartment appearing most affected by the loss of Zap70 expression. This data suggests an important role for Zap70 signalling in the transmission of homeostatic TCR survival signals. Unexpectedly we also found that TCR signals transmitted by Zap70 had the capacity to influence IL-7R expression and importantly revealed a novel role for positive selection signals in the regulation of peripheral IL-7R expression and therefore the competitive fitness of peripheral T cell clones. The second model system examined the homeostatic responses of class I MHC restricted F5+/+ TCR transgenic Rag1A CD8+ T cells following transfer into MHC class I or IL-7 deficient hosts, to specifically quantify the contribution of TCR and IL-7 signals in naive T cell homeostasis. Our data reveals that IL-7 signals are more essential than homeostatic TCR signals for the survival of the naive T cell compartment in conditions of lymphopenia. Interestingly we also demonstrate that IL-7 may exert part of its homeostatic effects by modulating TCR engagement with MHC ligands by regulating T cell - APC interactions in vivo. In conclusion the data presented in this thesis confirms that TCR and IL-7 signals are essential for naive T cell homeostasis, but also reveals extensive cross-talk between homeostatic TCR and IL7 signals in the control of peripheral T cell homeostasis.
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Doig, Cassandra Ann. "Development of MHC Class II Tetramer for Detection of Influenza Specific TCR." Thesis, The University of Arizona, 2015. http://hdl.handle.net/10150/579393.
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T cells play a critical role in adaptive immunity by utilizing genetic recombination to produce diverse TCRs that strongly recognize specific antigens. The TCR recognizes peptides presented by APCs via the MHC class II molecules on their surface. The recognition of a specific peptide stimulates the CD4+ T cell to signal downstream processes, which result in the mounting of an antigen specific immune response. With age the available naïve CD8+ T cell pool declines, making the aged population more vulnerable to infections and less responsive to immunotherapies such as vaccines. We hypothesize there is a similar decline in CD4+ T cells, further reducing response to pathogens, such as influenza, with age. To identify CD4+ T cells specific for influenza we engineered an HLA-DR class II tetramer presenting influenza peptide. DR4 was chosen due to the frequency in the Caucasian population. The tetramer reagent will not only allow us to study the changes in the diversity of influenza specific CD4+ T cells, but will also be a valuable tool for the analysis of effector functions for a variety of adaptive immune cell populations in response to influenza virus.
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Chouaki-Benmansour, Nassima. "Analyse du rôle des PIP2 dans l'initiation de la signalisation TCR et l'activation lymphocytaire." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4052.
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L'activation des lymphocytes T est un événement fondamental de la réponse immunitaire adaptative. Elle est déclenchée par la transduction du signal médiée par le complexe TCR/CD3.Le mécanisme de déclenchement du signal via le TCR reste, mal compris. Mon projet de thèse vise à examiner la contribution des PI(4,5)P2 dans le mécanisme du déclenchement du signal TCR. L'expression ectopique d'une phosphatase spécifique de PIP2 a permis de réduire de 50% les PI(4,5)P2 membranaires. L'analyse du profil de phosphorylations spécifiques des tyrosines montre que l'expression ectopique de cette 5-phosphatase augmente les événements de phosphorylation à l'état basal comparés à des conditions analogues pour des cellules contrôles. En revanche, alors que suite à l'engagement du TCR par le complexe MHC-peptide indépendamment du co-récepteur CD4 nous observons une augmentation des phosphorylations, l'activation par le complexe MHC-peptide CD4 dépendant semble affectée. Nous avons analysé la contribution des PI(4,5)P2 dans la dynamique membranaire du TCR grâce à la technique svFCS. PIP2 peuvent jouer un rôle essentiel dans la régulation de la dynamique latérale du TCR à la membrane plasmique. Enfin, nous avons observé que l'inhibition par la néomycine (aminoglycoside qui en tant que polycation peut se lier et neutraliser le groupement anionique de PI(4,5)P2), aboutit à des changements similaires dans la dynamique membranaire du TCR et la régulation proximale dans des cellules T primaires murines CD4+. Ensemble, nos données révèlent le rôle régulateur fondamental de PI(4,5)P2 dans la dynamique membranaire du TCR et de CD4, pour le contrôle de l'initiation des voies de signalisation du TCR<br>PI(4,5)P2 plays important roles in a large spectrum of membrane-based cellular activities . It is therefore surprising that it is currently not known if PI(4,5)P2 is also involved in the T cell receptor (TCR) signal transduction mechanism. We investigate here the role of PI(4,5)P2 in the regulation of the TCR membrane dynamics and signaling initiation using a combination of biophysical, biochemistry and cell biology approaches. Ectopic expression of the Inp54p, a 5-phophatase that hydrolyzes PI(4,5)P2 into PI(4)P, with a membrane targeting signal specifically decreased by 50% of the PI(4,5)P2 in a CD4+ T cell hybridoma. Interestingly, we observed that this decrease caused modified TCR (and CD4 co-receptor) dynamics in the plasma membrane. The lateral diffusion switched from a regime dominated by dynamic partitioning in the cholesterol- and sphingolipid-dependent nanodomains into one dominated by dynamic partitioning in the actin cytoskeleton-assisted nanodomains. This switch was associated with a change in activation of the TCR and proximal signaling pathways both at the basal level and upon stimulation. Upon pMHC engagement, the CD4-independent activation of the TCR signaling pathways was found significantly augmented while that of CD4-dependent was affected. We further provided evidence for the involvement of PI(4,5)P2 in the Finally, we found that inhibition of interactions between PI(4,5)P2 and endogenous proteins with neomycin resulted in the modified TCR membrane dynamics and proximal signaling in primary murine CD4+ T cells. Altogether, our data reveal that PI(4,5)P2 is crucially involved in the control of the activation of TCR early signaling pathways
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Carras, Sylvain. "Rôles de la stimulation chronique du TCR et de la reprogrammation cellulaire dans les lymphomes T périphériques." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1322/document.
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Les lymphomes T périphériques (ou PTCL) sont des lymphomes malins non Hodgkiniens ayant pour cellules d’origine des lymphocytes T (LT) ou Natural Killer matures. Ces lymphomes sont rares, hétérogènes et méconnus. Des arguments issus de la littérature suggérant l’implication de la stimulation chronique du récepteur T à l’antigène (TCR) dans la transformation des LT, nous ont conduits à développer un modèle murin basé sur la stimulation chronique du TCR pour adresser spécifiquement cette question. Dans ce modèle, le transfert de LT p53-/- dans des souris CD3e-/- entraine l’apparition de lymphomes T périphériques (PTCL) clonaux dans 60% des cas avec une médiane de survenue de 230 jours alors que les souris transférées avec des LT wt ne développent pas de lymphomes. Ces PTCL présentent un phénotype T effecteur-mémoire CD62LLo-CD44hi-CD122lo-CD25lo ainsi qu’une profonde downrégulation de l’expression des gènes impliqués dans la voie du TCR illustrant l’impact de la stimulation chronique dans la lymphomagénèse. L’étude de ces lymphomes a révélé qu’ils ne dépendent plus, pour la plupart, de l’engagement du TCR pour leur survie et qu’ils acquièrent des caractéristiques « innate-like » avec notamment l’expression de récepteurs NK inhibiteurs (NKiR) et de récepteurs NK activateurs (NKaR) ainsi que des protéines adaptatrices DAP12 et FceRIg. Cette expression est associée à celle de Syk et PLC?2, impliquées dans la signalisation des NKaR. Nous montrons que les NKaR et leurs voies de signalisation associées sont fonctionnelles et participent à la survie des cellules lymphomateuses, le blocage de certains NKaR retardant notamment le développement lymphomateux in vivo. Nous avons par la suite exploré l’expression des NKR, de Syk et de PLCg2 au sein des PTCL humains et nous montrons ou confirmons que certaines entités expriment des panels variés de NKR ainsi que les effecteurs Syk et PLCg2 suggérant l’existence de mécanismes de lymphomagénèse similaires à ceux identifiés dans notre modèle au sein d’un certain nombres de PTCL humains<br>Peripheral T-cell lymphomas (PTCL) are rare non Hodgkin malignant lymphomas emerging from mature T or NK cells. PTCL are highly heterogeneous and mainly misunderstood. As several evidences pointed the potential role of TCR chronic stimulation in human T-cell lymphomagenesis, we developed a murine model based on chronic TCR stimulation to address this question. In this model, transfer of p53-/- T-cells into T-cell deficient mice (CD3e-/-) triggered PTCL development in 60% of cases with a median survival of 230 days while transfer of wt T-cells in CD3e-/- mice did not lead to PTCL development. These PTCL exhibited an effector-memory phenotype CD62LLo-CD44hi-CD122lo-CD25lo associated with a dramatic downregulation of TCR pathway genes expression consistent with a chronic TCR stimulation highlighting it’s implication in lymphomagenesis. The analysis of these PTCL revealed that a large majority of cases (80%) do not depend anymore on TCR stimulation for their growth and survival and that they acquire innate-like features with expression of inhibitory NKR (NKiR) and activating NK receptors (NKaR) as well as the adaptor proteins DAP12 or FceRIg. Expression of these receptors is associated with the expression of SYK and PLC?2, which are classical key effectors downstream of NKaR. We show that these NKaR are functional and can mediate TCR-independent activation in mPTCL and that this signaling is involved in cell survival/proliferation as in vivo blockade of NKG2D and NKp46 delays PTCL development in PTCL transplantation experiments. In parallel, we studied NKR, Syk and PLCg2 expression in human PTCL and found that some entities express a large range of these receptors as well as Syk and PLCg2, suggesting similar lymphomagenesis mechanisms in some human PTCL