Thesis (M.A.)--Trinity Evangelical Divinity School, 2003. / Abstract. Includes bibliographical references (leaves 164-168).

Thesis (M.A.)--Trinity Evangelical Divinity School, 2003. / Abstract. Includes bibliographical references (leaves 164-168).

Anaplastic Large Cell Lymphoma (ALCL) is a peripheral T cell lymphoma divided into three distinct entities: ALCL, Anaplastic Lymphoma Kinase (ALK)+, ALCL ALK- and cutaneous ALCL. In the majority of ALCL, ALK+, ALK is expressed as the result of a chromosomal translocation generating Nucleophosmin 1(NPM)-ALK, which is considered the main driver. ALCL have an unusual immunophenotype; they rarely express a T cell receptor (TCR), but are often positive for CD4 and produce cytotoxic proteins such as perforin and Granzyme B, but in the absence of CD8, questioning the origin and pathogenesis of this malignancy. Expression of NPM-ALK in mice from the T-cell specific CD4 promoter gives rise to thymic lymphomas not modelling human ALCL suggesting that other events and/or expression of NPM-ALK at a defined stage of T cell ontogeny is required for peripheral T cell lymphoma development. Indeed, back-crossing the CD4/NPM-ALK line onto a RAG competent, MHC class I restricted ovalbumin-specific TCR, OTI transgenic line (CD4/NPM-ALK/OTI) permits peripheral lymphoma development mimicking human ALCL (but CD4/NPM-ALK/OTII mice still develop thymic lymphoma); tumours contain cells histopathologically identical to ALCL hallmark cells. Interestingly, peripheral tumours developing in this model also lack cell surface expression of the OTI TCR in fitting with observations of a lack of TCR expression on human ALCL. It follows that stimulation of T cells in vivo by infection with MHV-ova prevents lymphomagenesis suggesting that the TCR is detrimental to tumour growth. Indeed, strong stimulation via the TCR of NPM-ALK-expressing primary T cells in vitro, impedes cell proliferation but cell growth is favoured when a weaker stimulus is employed. Overall, data presented in this thesis identifies a potential mechanism of lymphomagenesis accounting for the unusual immunophenotype of ALCL and an explanation as to why cells lack a TCR and associated proximal signaling.

Themis est une protéine de signalisation des récepteurs des lymphocytes T (TCR) essentielle pour la sélection positive des cellules T. La fonction moléculaire de Themis a été controversée mais de récentes études suggèrent qu'il est un régulateur positif des voies de signalisation des TCR. Nous avons montré dans une étude préliminaire que Themis interagit avec des déubiquitinases et qu'il est ubiquitiné dans les thymocytes. L'objectif de ma thèse était de caractériser les mécanismes moléculaires qui régulent l'ubiquitination de Themis et de déterminer si ces processus affectent la fonction de Themis durant le développement des lymphocytes T. Nous avons montré que si l'expression des ARNm codant pour Themis diminue dans les stades précoces de la sélection positive, son expression protéique est parallèlement augmentée, suggérant une stabilisation de Themis par des modifications post-traductionnelles durant cette étape. Nous avons montré que la déubiquitinase USP9X déubiquitine Themis pour stabiliser son expression durant la stimulation des TCR. L'ensemble de nos résultats proposent qu'USP9X soit activé durant la stimulation des TCR grâce à son recrutement dans les complexes proximaux des TCR par l'intermédiaire de l'adaptateur Grb2 et Themis, entrainant la stabilisation de l'expression de Themis. Nous pensons que ce mécanisme est important pour maintenir l'expression de Themis durant la sélection positive afin de favoriser l'induction d'un signal des TCR soutenu, requis pour l'efficacité de ce processus. / The protein Themis is a new actor of the T cell receptor (TCR) signaling essential for the positive selection of T cells. The molecular function of Themis has been controversial but recent findings suggest that it acts as positive regulator of TCR signaling. We demonstrated in an initial research that Themis interacts with deubiquitylases and is covalently associated to ubiquitin chains in thymocytes. The aim of my PhD project was to characterize the molecular process that regulates the ubiquitination of Themis and to investigate how these post-translational modifications affect Themis function during T cell development. We demonstrated that Themis mRNA expression is progressively decreased after positive selection whereas Themis protein expression is enhanced at the early stages of positive selection, suggesting that Themis is stabilized by post-translational modifications during positive selection. We demonstrated that USP9X allows the deubiquitination of Themis and its stabilization following TCR engagement. Ours results suggest that USP9X is activated during TCR engagement following its recruitment to proximal signaling complexes through Grb2 and Themis, leading to the deubiquitination and stabilization of Themis expression. We believe that this mechanism is important to sustain Themis expression during positive selection and to promote durable TCR signals required for the efficiency of this process.

Lors d’une réponse immune, les lymphocytes T et les cellules présentatrices d’antigènes (CPA) interagissent entre elles. La synapse immunologique (SI), interface de contact entre les deux cellules, est le site où une cascade de signalisation se met en place. Les lymphocytes T subissent alors un profond réarrangement au niveau de la membrane plasmique et du cytoplasme : les protéines impliquées dans cette signalisation sont alors recrutées à la synapse immunologique. Nous nous intéressons à une nouvelle structure appelée “l’antisynapse” qui se localise au pôle opposé à celui de la synapse immunologique. L’objectif de notre étude est de déterminer la composition de cette nouvelle structure et sa cinétique d’apparition et de disparition. Afin d’étudier cette structure, nous faisons des conjugués in vitro entre des CPA et des lymphocytes T et nous observons la formation de ces contacts sur cellules vivantes ou cellules fixés. L’antisynapse est composée de molécules de signalisations que l’on retrouve classiquement à la synapse immunologique, tels que LAT, CD3, lck ou la PI3K. Grâce à la sonde fluorescente ROZA récemment développée au laboratoire1, nous avons montré que la kinase ZAP-70 est activée à l’antisynapse. Ces observations sont cohérentes avec le fait que nous avons déjà observé la présence de protéines avec des tyrosines phosphorylées à ce pôle. Cette structure précoce et transitoire s’observe fréquemment et apparaît très souvent avant que la synapse ne puisse être détectée. Son apparition est indépendante de la signalisation en aval du TCR et peut être déclenchée par un signal d’adhésion. D’autre part, le cytosquelette de microtubules semble jouer un rôle majeur dans sa disparition. Le rôle de l’antisynapse est toujours en cours d’étude mais nous avons déjà pu montrer qu’elle constituait un point de stockage pour les protéines destinées à former la synapse immunologique au moment de sa formation. Grâce à cette structure nous essayons de mieux comprendre comment le signalosome s’assemble dans la cellule T. Nous voulons également comprendre comment une telle structure peut apparaître aussi rapidement et quelles sont les voies de signalisation mises en jeu dans sa formation. / During the immune response, T lymphocytes and antigen presenting cells (APC) are known to develop strong interactions. The immunological synapse (IS), structure established at the interface between the two cells, is the site where a cascade of signaling events is initiated and may lead to a physiological response. T lymphocyte undergoes a profound rearrangement in the plasma membrane and in the cytoplasm: proteins which are involved in the signaling are recruited to the immunological synapse. We have recently described a new structure that we have called antisynapse (ASI), located at the cell pole opposite to the synapse1. The purpose of this work is to characterize the components of this new structure and their kinetic of appearance and disappearance. To study this structure, we made in vitro contact between APC and T lymphocytes and we observed these conjugates either in live or fixed conditions. Surprisingly, the antisynapse contains most of the signaling molecules classically reported as components of the immunological synapse such as LAT, CD3, Lck or PI3K. By using the fluorescent probe ROZA that we recently developed1, we have shown that ZAP-70 is activated at the antisynapse. This observation is consistent with the fact that we have also observed the presence of tyrosine-phosphorylated proteins at the ASI. Interestingly, we have observed that LFA-1, a protein involved in the adherence, is also found at the ASI. Our results indicate that this transient structure develops frequently and appears rapidly after the contact between the T cell (around one minute) and the APC. Surprisingly, antisynapse formation is independent on TCR signaling but is triggered by adhesion. Furthermore, it disappears using the microtubule network. The role of the antisynapse is currently under investigation but we have shown that it constitutes a stock of proteins ready to go to the forming immune synapse. We currently try to take advantage of this structure to better understand how the T cell signalosome may assemble and to find out if, functionally, the T cell takes advantage of this structure. We also try to understand how this paradoxical structure can appear so rapidly and what are the signaling pathways involved in its establishment.

No description available.

No description available.

A T helper cell clone, referred to as clone 9, was derived from an allogeneic mixed lymphocyte culture. Clone 9, as well as supernatant factor(s) derived from it, could help the cytotoxic T lymphocyte (CTL) responses of H-2 Db (Db) responder cells to alloantigens, or they could help the CTL responses of non- Db responder cells to Db alloantigens. Clone 9 cells or their factor(s) were active only when added during the first 24 hours of a five-day culture period. Clone 9 or its factor(s) could also synergize with interleukin-2 (IL-2)-containing medium in mounting cytotoxic responses to alloantigens. The helper activity in clone 9 supernatant was not due to IL-2 and it was specifically absorbed out by Db -spleen cells. The characterization of the Db -specific helper factor(ASHF) was facilitated by the isolation of a T hybridoma clone (clone 25), obtained from fusion of clone 9 cells with the T cell lymphoma, BW5147, and a B cell hybridoma that produced an IgM monoclonal antibody (clone 30 IgM) which bound ASHF. An additional monoclonal antibody (F23.1), which recognizes a determinant of the Vβ8 family of the T cell receptor, was also particularly useful for the characterization of ASHF. Analysis with these reagents showed that both clone 30 IgM and F23.1 immunoadsorbents could retain ASHF activity. Preabsorption of the ASHF with Db spleen cells prior to affinity purification over a clone 30 IgM column resulted in the absorption of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band on SDS-PAGE under reducing conditions. Furthermore, affinity purification of ASHF over the F23.1 immunoadsorbent, but not an irrelevant monoclonal antibody (mAb) column, also yielded a 50,000 MW molecule. Taken together, these findings suggest that the 50,000 MW molecule is a component of the ASHF and it is intimately related to the B chain of the T-cell receptor. The mode of action of clone 9 and its products in the induction bfCTL responses was also investigated. It was found that clone 9 and ASHF could help CTL responses by inducing IL-2 production in B6-stimulated cultures. In addition to ASHF, clone 9 cells also produced an additional factor(s) which participated in the induction of CTL responses. This additional factor(s) was referred to as IL-X. IL-X synergized with excess human recombinant IL-2 in the activation of CTL precursors (CTL-P) in the absence of antigenic stimulation. A model which involves the participation of ASHF, T helper cells, IL-2 and IL-X in the induction of CTL responses is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19267号 / 医博第4031号 / 新制||医||1011(附属図書館) / 32269 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 生田 宏一, 教授 松岡 雅雄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

No description available.