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1
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.
Texte intégralRésumé :
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.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.
2
Wright, G. P. « Generation of antigen-specific regulatory T cells by T cell receptor gene transfer ». Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18952/.
Texte intégralRésumé :
Regulatory T cells (Tregs) have shown considerable potential in the treatment of murine models of immuno-pathology. Whilst poly-clonal Tregs are able to suppress immuno-pathology in a number of models, the superiority of Ag-specific Treg treatment has been demonstrated using Tregs from T cell receptor (TCR)- transgenic animals. Translation of these promising results to the clinic has been hampered by difficulties in isolating or enriching the rare Ag-specific Tregs from the polyclonal population. Here I describe two distinct approaches to generate Ag-specific T cells with regulatory ability: firstly, TCR gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs. Secondly, co-transfer of FoxP3 and TCR genes served to convert conventional CD4+ T cells into Ag-specific ‘Treg-like’ cells. Both approaches generated T cells that suppressed in vitro and engrafted efficiently, retaining TCR and FoxP3 expression, when adoptively transferred into recipient mice. Using an established arthritis model, I demonstrate Ag-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a reduction of joint swelling. In animals treated with TCR-transferred natural Tregs this was accompanied by a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, I have described a strategy to rapidly generate Ag-specific Tregs capable of antigen-dependent amelioration of autoimmune damage in the absence of general immune suppression. These approaches could practicably be translated into the clinic in order to treat numerous different immuno-pathologies.
3
Bracq, Lucie. « Analysis of HIV-1 cell-to-cell transfer to macrophages ». Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB063/document.
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Les macrophages sont une cible particulièrement importante de l’infection par le VIH-1 et jouent un rôle crucial dans la physiopathologie de l’infection. Lorsqu’ils sont infectés, leur capacité de survie dans les tissus leur permet de jouer un rôle essentiel dans la dissémination virale et l’établissement de réservoirs viraux au niveau des différents territoires tissulaires. In vitro, les étapes précoces et tardives du cycle de réplication virale dans les macrophages ont été analysées dans le cadre de l’infection par des virus libres. Cependant, les modalités d’infection des macrophages lors d’une transmission intercellulaire reste largement inexplorées. Les travaux présentés ici ont permis d’établir un modèle de transmission intercellulaire du VIH-1 de lymphocytes T infectés vers les macrophages. Nous avons montré que les lymphocytes T infectés sont capables d’interagir étroitement avec les macrophages, conduisant ainsi à la fusion cellulaire de ces deux cellules et permettant le transfert de matériel viral dans les macrophages cibles. Ce transfert viral par fusion cellulaire, rapide et efficace, est restreint aux virus utilisant le corécepteur CCR5 et dépend de l’interaction entre l’enveloppe virale et le récepteur CD4. Les virus transférés sont alors stockés au sein de compartiment cytoplasmique des cellules fusionnées mais nous observons également des évènements précoces d’assemblage et de bourgeonnement du VIH-1 à la membrane plasmique des cellules fusionnées résultant de la fusion des membranes des lymphocytes T infectés et des macrophages cibles. Ces cellules fusionnées acquièrent alors la capacité de fusionner avec les macrophages non infectés environnants permettant la dissémination du VIH-1. L’ensemble de ces résultats met en évidence un nouveau mécanisme de transmission intercellulaire entre lymphocytes T et macrophages via un mécanisme de double fusion cellulaire dépendant de l’enveloppe virale et des récepteurs CD4 et CCR5. Ces évènements successifs de fusion entre lymphocytes T et macrophages puis entre macrophages permettent la formation de cellules géantes multinucléés capables de produire de grande quantité de virus infectieux. Ces cellules multinculées pourraient correspondre aux macrophages multinuclées observés in vivo dans les organes lymphoïdes et le système nerveux central de patients infectés par le VIH-1 ou de singes infectés par le SIV. Ce mécanisme représente donc un modèle de transmission intercellulaire original permettant la dissémination virale et la formation de macrophages réservoirs durant l’infection par le VIH-1Macrophages are important targets of HIV-1 and play crucial roles in physiopathology of infection. Because of their long time survival capacity, infected macrophages participate in virus dissemination and establishment of persistent virus reservoirs in numerous tissues. In vitro, macrophages infection and analysis of the different steps of the virus cycle have been largely documented using cell-free virus infection. However, there is a paucity in knowledge of the mechanisms that control infection and dissemination to macrophages by cell-to-cell transfer. In the work presented here, we establish a model of HIV-1 cell-to-cell transfer from infected T cells to macrophages. We observed that infected T cells are able to interact with macrophages leading to cell fusion for transfer of viral material to macrophages targets. This cell-to-cell fusion transfer, very fast and efficient, is restricted to CCR5-tropic viruses, and mediated by viral envelope-receptor interactions. Transferred viruses can then accumulate in cytoplasmic compartments of newly lymphocyte/macrophages fused cells but we also observed early viral assembly and budding events at the plasma membrane of these fused cells, resulting from the merge of viral material between infected T cells and macrophages. These cells then acquire the ability to fuse with neighboring non-infected macrophages for virus dissemination. Together, these two-sequential envelope-dependent cell fusion process lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in vivo in lymphoid organs and the central nervous system of HIV-1 infected patients and simian immunodeficiency virus-infected macaques. These mechanisms may represent an original mode of virus transmission for viral spreading and formation of macrophage virus reservoirs during HIV-1 infection
4
Böhm, Stefanie [Verfasser], et Lars [Akademischer Betreuer] Nitschke. « Adoptive T-cell-receptor transfer to examine human T-cell immunology in vitro / Stefanie Böhm. Betreuer : Lars Nitschke ». Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2013. http://d-nb.info/1033688193/34.
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Chakupurakal, Geothy. « Preclinical studies of adenovirus-specific T-cells for adoptive transfer to haemopoietic stem cell transplant recipients ». Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/2883/.
Texte intégralRésumé :
Allogeneic stem cell transplantation (SCT) is the only curative treatment option for many haematological malignancies. Adenovirus (Ad) infections are a significant cause of morbidity and mortality post SCT. Lack of effective anti-viral treatment for Ad disease has led to the development of adoptive immunotherapy of Ad-specific T-cells as a promising therapeutic option for patients in this setting. The aim of this project was to establish preclinical criteria for the development of a clinical trial comparing two T-cell enrichment methods- multimer selection and cytokine secretion selection to enrich Ad-specific T-cells for the purposes of adoptive transfer directly without the need for in vitro culture. Eight pHLA tetramers containing HLA class I restricted Ad epitopes were generated and their ability to identify and enrich Ad-specific T-cells investigated. HLA A*01 TDL tetramer consistently detected T-cells in all (13/13) healthy adult donors screened. Frequency and enrichement of Ad-specific T-cells by cytokine secretion and selection was also investigated. Despite the low frequency of Ad-specific T-cells, clinical grade enrichment was feasible by both methods. T-cells selected by both methods were then characterised for homing and proliferative potential. Ad-specific T-cells identified by either method had a high proliferative potential, possessed a novel minimally differentiated memory phenotype, were cytotoxic towards Ad species responsible for infections in SCT recipients and capable of limiting virus replication. In conclusion, Ad-specific T-cells enriched by multimer selection or cytokine secretion selection are suitable for adoptive transfer to patients with Ad infection following HSCT. Both methods also allow the monitoring of Ad-specific immune reconstitution after adoptive transfer.
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Carluccio, S. « GENERATION OF TUMOR-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FROM PEROPHERAL BLOOD OF COLORECTAL CANCER PATIENTS FOR ADOPTIVE T-CELL TRANSFER ». Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/231155.
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Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death in the developed Western countries.
Adoptive T-cell transfer (ACT) refers to an immunotherapeutic approach in which anti-tumor T lymphocytes, usually the tumor infiltrating lymphocytes (TIL), are identified, grown ex vivo and then re-infused into the cancer patient.
ACT of EBV-specific T-cell lines and T Cytotoxic Lymphocytes (CTLs) for the therapy of EBV-induced lymphomas is the best demonstration of clinically efficacious ACT, but there are many evidences also for leukemia and multiple myeloma.
As regards to the solid tumors, ACT using autologous TIL, grown ex-vivo and then re-infused into the cancer patient, has emerged as an effective treatment for metastatic melanoma and renal cell carcinoma (RCC), that are the most immunogenic tumors in humans. Randomized clinical trials are ongoing for gastric cancer, hepatocellular carcinoma and lung cancer.
These approaches mainly use the TIL and the definition of tumor associated antigen (TAA), tumor specific antigen (TSA) or cancer testis antigen (CTA), that are generally correlated with tumor progression and immunogenicity in various types of cancer.
However these antigens are often found to be poorly expressed in CRC, and few is known about their relationship with this type of neoplasia. In addition, although a clear association between TIL and clinical outcome of CRC has been documented, active and adoptive immunotherapy do not play yet an important role in the treatment of advanced CRC.
In order to develop an ACT protocol for CRC treatment, we designed an experimental approach that does not require neither the definition of molecular defined tumor antigens, nor the availability of TIL. Our strategy was based on the in vitro stimulation of patient’s CD8+-enriched T-cells from peripheral blood mononuclear cells (PBMCs) with dendritic cells (DCs), pulsed with apoptotic tumor cells as a source of tumor antigens, in order to generate autologous CTLs with strong anti-tumor activity.
In this study, 78 CRC patients were enrolled. Tumor biopsies were obtained at surgery, together with 100 ml of heparinized peripheral blood (PB). Tumors were mechanically dissociated to a single-cell suspension and cultured to obtain tumor cell line from each patient. DCs were generated from previously separated PBMCs, using a magnetic positive selection of CD14+ monocytes, cultured in presence of recombinant human Interleukin-4 (rh IL-4) and recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rh GM-CSF). Anti-tumor CTLs were elicited in co/micro-culture using DCs as antigen-presenting cells, autologous apoptotic tumor cells as source of antigens and T CD8+ lymphocytes enriched effectors, with weekly stimulation. CTLs Interferon-γ (IFN-γ) secretion was assessed by ELISpot assay to evaluate their activation in response to autologous tumor.
Tumor cell lines were obtained from 20 out of 78 patients (25,6%), because gut intestinal flora had adversly affected the establishment of primary tumor cell line and a loss of expansion of tumor cells was observed. DCs were generated from 26 patients, but only 6 patients had the corresponding tumor cell line, indispensable for the co-culture setting up. This was the reason why co/micro-cultures were set up only for 6 patients. ELISpot assay was performed at the end of co/micro-culture stimulations to evaluate effectors IFN-γ secretion. ELISpot results showed that strong and significant IFN-γ secretion was detected at the third, fourth and fifth stimulations for one patient and at the second for another patient, whereas for three patients a weak secretion was detected during the second and third stimulations.
Although our immunological study must be performed on an increased number of CRC patients, and the CTLs expansion, together with CTLs lytic ability against autologous tumor cells, must be still performed, our results suggested that the generation of tumor-specific CTLs could be useful for supporting an ACT approach in CRC.
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Gräf, Patricia [Verfasser]. « Serial transfer of single cell-derived immunocompetence reveals stemness of CD8+ central memory T cells / Patricia Gräf ». München : Verlag Dr. Hut, 2015. http://d-nb.info/1070124389/34.
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Akhter, Waseem. « Le rôle du transfert de mitochondries des cellules stromales mésenchymateuses (CSM) dans la suppression des réponses des cellules T CD4+ et CD8+ ». Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONT011.
Texte intégralRésumé :
Les lymphocytes T cytotoxiques CD8+ (LTC) et les cellules T helper CD4+ sont des effecteurs clés dans les maladies auto-immunes, les maladies du greffon contre l'hôte et le rejet de greffe. Les cellules stromales mésenchymateuses (CSM) sont des cellules multipotentes auto-renouvelables qui possèdent des propriétés de réparation tissulaire et d'immunomodulation. En raison de leur capacité à réprimer les réponses immunitaires pathogènes, elles présentent un potentiel thérapeutique pour le traitement des maladies à médiation immunitaire. Les CSMs ont la capacité unique d'exporter leurs propres mitochondries vers les cellules voisines en réponse à une blessure ou une inflammation. Cependant, on ne sait pas si le transfert de mitochondries a lieu et s'il joue un rôle dans la répression des réponses des lymphocytes T CD4+ Th1 et CD8+. Nous avons abordé cette question en utilisant un modèle de souris transgénique de la maladie et des CSMs allogéniques dérivées de la moelle osseuse in vitro et in vivo. Nos résultats ont montré :1) Les CSMs ont inhibé l'expansion, le gain du phénotype effecteur et la production de la cytokine effectrice IFNγ in vitro et le potentiel diabétogène in vivo des cellules CD4+ Th1 après activation. De façon remarquable, les cellules T CD4+ ont absorbé les mitochondries des CSMs pendant la suppression. Le transfert de mitochondries de CSMs aux cellules Th1 CD4+ a entraîné une diminution de la prolifération et de la production d'IFNγ. Enfin, nous avons démontré que les CSMs et les mitochondries de CSMs empêchent la régulation à la hausse du facteur de transcription maître Th1 sur les cellules T CD4+ activées.2) Les CSMs ont diminué l'expansion, le gain du phénotype effecteur et la production de la cytokine effectrice IFNγ dans les cellules T CD8+ après activation in vitro. Notamment, nous avons constaté que pendant leur interaction conduisant à la suppression, les CSMs ont également transféré des mitochondries aux LTC. Le transfert des mitochondries des CSM aux cellules T CD8+ a entraîné une diminution de la prolifération et de la production d'IFNγ lors de l'activation, contribuant à l'effet suppressif global des CSMs. Enfin, nous avons démontré que les mitochondries de CSMs et de CSMs régulent de manière différentielle l'équilibre de deux facteurs de transcription clés pour la différenciation des LTC, T-bet et Eomes, sur les cellules T CD8+ activéesCD8+ Cytotoxic T lymphocytes (CTL) and CD4+ T helper cells are key effectors in autoimmune, graft versus host diseases and graft rejection. Mesenchymal Stromal Cells (MSCs) are self-renewing multipotent cells with tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses they show therapeutic potential for the treatment of immune mediated diseases. MSCs have the unique ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 and CD8+ T cell responses is unknown. We have addressed this issue utilizing a transgenic mouse model of disease and allogeneic bone marrow derived MSCs in vitro and in vivo. Our results showed:1) MSCs inhibited expansion, gain of effector phenotype and the production of the effector cytokine IFNγ in vitro and the diabetogenic potential in vivo of CD4+ Th1 cells after activation. Remarkably, CD4+ T cells took up mitochondria from MSCs during suppression. The transfer of MSC mitochondria to CD4+ Th1 cells resulted in decreased proliferation and production of IFNγ. Finally, we demonstrated that both MSCs and MSC mitochondria prevent the upregulation of the master Th1 transcription factor on activated CD4+ T cells.2) MSCs decreased the expansion, gain of effector phenotype and the production of the effector cytokine IFNγ in CD8+ T cells after activation in vitro. Notably, we found that during their interaction conducting to suppression, MSCs also transferred mitochondria to CTL. MSC mitochondrial transfer to CD8+ T cells resulted in decreased proliferation and production of IFNγ upon activation contributing to the global suppressive effect of MSCs. Finally, we demonstrated that both MSCs and MSC mitochondria differentially regulate the balance of two transcription factors key for CTL differentiation, T-bet and Eomes, on activated CD8+ T cells
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Uhlig, Holm H. « Intestinal bacterial flora and the inflammatory immune response in the T cell transfer model of colitis ». Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403771.
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Petschenka, Jutta [Verfasser]. « Safety and therapeutic efficacy of adoptive p53-specific T cell antigen receptor (TCR) gene transfer / Jutta Petschenka ». Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1052080685/34.
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Weigand, Luise [Verfasser], Heinrich H. D. [Akademischer Betreuer] Meyer et Angela [Akademischer Betreuer] Krackhardt. « Characterization of human MHC II-restricted T cell receptors with reactivity against B cells and tumor cells for therapeutic application in the context of adoptive T cell transfer of transgenic CD4 T cells / Luise Weigand. Gutachter : Angela Krackhardt ; Heinrich H. D. Meyer. Betreuer : Heinrich H. D. Meyer ». München : Universitätsbibliothek der TU München, 2011. http://d-nb.info/1016727798/34.
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Hiraragi, Hajime. « Study of lentiviral vector for in utero gene transfer and functional analysis of human T-lymphotropic virus type p13(II) ». Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1116532636.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xvii, 230 p.; also includes graphics. Includes bibliographical references (p. 200-230). Available online via OhioLINK's ETD Center
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Nihei, Jorge Sadao. « Estudo da migração de células T NK1.1+ no músculo estriado, durante a infecção experímental pelo Trypanosoma Cruzi em animais desprovidos de linfócitos B funcionais ». reponame:Repositório Institucional da FIOCRUZ, 2005. https://www.arca.fiocruz.br/handle/icict/5907.
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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil
Foi anteriormente demonstrado que as células NK (Natural Killer) estão relacionadas às bases para resistência à infecção por Trypanosoma cruzi, pois a depleção de células positivas para NK1.1+ resulta em alta parasitemia de camundongos C57BI/6 infectados pelo T. cruzi. Estudos de nossa equipe indicaram ainda que as células T NKH-t- poderiam induzir a formação de células T efetoras/nnemória, e que a resistência à infecção foi correlacionada com a quantidade de células T CD4-<- CD45RB"®^ presentes antes da infecção. No presente estudo avaliamos a função regulatória de células T NK1.1+ durante a infecção experimental pelo T. cmzi, na ausência de linfócitos B. Utilizamos os seguintes animais: C57BI/6 controles, ^MT C57BI/6, nMT reconstituídos (com células B de C57BI/6 ou B de C67BI/6 IL-10KO) ou tratados com imunoglobulinas. Neste modelo experimental, observamos que os animais p.MT apresentaram menores números de células T efetoras/memória no baço comparados aos controles (C57BI/6), na fase aguda de infecção. A reconstituição com células B ou o tratamento com Ig em animais ^iMT infectados resultou em aumento de células T efetoras/memória, comparado ao controle (jiMT infectado). Da mesma maneira e até fase crônica de infecção, a transferência adotiva de células B em animais ^MT causa persistência de células T efetoras/memória no baço. Como a molécula de CD1 (encontrada sobre células B e dendríticas) é reconhecida por células NK1.1, a expressão desta molécula foi também avaliada durante a infecção. Após a infecção, houve diminuição de células CD1+ no baço de animais C57BI/6, e ausência destas células nos jxMT. A recuperação desta população celular no baço de {aMT infectados após reconstituição com linfócitos B foi concomitante á reposição de células T CD4+ NK1.1 no músculo esquelético destes mesmos animais. Houve ainda aumento de CD4+NK1.1 também no músculo esquelético dos animais ^MT reconstituídos com linfócitos B provenientes de C57BI/6 IL-10KO. De modo interessante, a depleção de NK1.1 durante a fase crônica, causou aumento de células T efetoras/memória encontradas no músculo esquelético de animais |uMT. Esses resultados estão relacionados aos dados de histopatologia, onde foi evidenciado maior infiltrado inflamatória no tecido HfKiscular de animais fxMT tratados com anti-NK1.1, durante a fase crônica da infecção. Nossos resultados indicam desse modo que a presença da célula B estaria ligada à formação de células T CD45RB"^ na fase aguda e manutenção/aumento de memória imune na fase crônica de infecção, conferindo ao grupo de animais reconstituídos com células 6, maior sobrevida. Sugere-se, portanto que as células T CD4+ NK1.1+ poderiam ser regulatórias no sentido de apresentar atividade antiinflamatória e que as células aP+NK1.1+ exerceriam função auxiliar na geração de células T efetoras/memória em nosso sistema experimental.
We have previously demonstrated that NK (Natural Killer) cells have been related to resistance to T. cruzi infection and the depletion of NK1.1+ cells resulted in high mortality and increased parasitemia in C57BI/6 Infected mice. Recently, we suggested that the NK1.1 T cells were involved on memory T cell generation, and resistance to infection was correlated with increased numbers of 004^“'*''^ CD45RB"®^®“'^ T cells, present before infection. In this study we evaluated the regulatory function of NK1.1+ T cells during 7. cruzi infection in ^iMT C57BI/6 infected mice. The following mice were used; C57BI/6, ^MT C57BI/6 and nMT C57BI/6 Imunoglobulin (lg)-treated or adoptively transferred with B cells (obtained from C57BI/6 or from C57BI/6 IL-10KO). In this experimental model. yMT infected mice have show decreased numbers of effector memory T cells, compared to C57BI/6 infected controls, during acute infection. The adoptive transfer of B cells or the treatment with immunoglobulins (Igs), induced increased numbers of effector memory splenic T cells, compared to C57BI/6 controls. Furthermore, Ig administration to p,MT uninfected mice is able to increase ap+NK1.1+ splenic cell population. As NK1.1 cells recc^nize C01 molecule which is expressed on B and dendritic cells, CD1 expression was evaluated in spleens of nMT and C57BI/6 mice to estimate whether the expression of CD1 was modified after infection. When compared to uninfected controls, CD1-presenting cells decreased from both nMT and C57BI/6 mice and were increased following B cell-transfer to laMT recipient mice. Interestingly, the depletion of NK1.1 cells also increased effector memory T cells found on skeletal muscles infiltrates from jiMT, and this was correlated to the increased inflammatory response found in these ^iMT NK1.1-depleted mice, during the chronic phase of infection. In this inflammatory compartment, ^iMT infected mice presented low numbers of CD4+NK1.1 T cells, when compared to C67BI/6. Previous observations from our laboratory suggest that CD4+NK1.1+ T cells (which are decreased in skeletal muscle from infected laMT mice), may be related to the enhanced inflammatory response during the early chronic infection. Finally, these studies suggest that CD4+NK1.1+ T cells may be regulatory with an antiinflammatory activity and that ap+NK1.1+ T cells may be involved on effector memory T cell-generation in our experimental system.
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Norris, Paula Suzanne. « Retrovirus-mediated transfer of wild-type p53 and p16INK4a suppresses cell growth in a mouse model for T-cell acute lymphoblastic leukemia via independent mechanisms / ». Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9904819.
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BLYTH, Emily Margaret. « The reconstitution of cellular immunity via adoptive transfer of multi-pathogen specific donor-derived T cells in recipients of allogeneic haemopoietic stem cell transplantation ». Thesis, The University of Sydney, 2013. http://hdl.handle.net/2123/9478.
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Allogeneic haemopoietic stem cell transplantation is the only curative therapy for a number of haematological diseases. Infection remains a leading cause of non-relapse mortality due to impaired recipient immunity. The reconstitution of cellular immunity via adoptive T cell transfer is a promising way to address this problem. Establishment of feasibility, safety and efficacy via clinical translation and expansion of the repertoire of pathogens targeted by adoptive T cell transfer strategies are the central goals of this thesis. Chapter 2 describes a clinical trial of 50 patients treated with cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) for prevention of CMV in recipients of haemopoietic stem cell transplants. Chapter 3 describes a method for generating CTLs from peripheral blood stem cell harvest product in order to improve the logistic feasibility of adoptive immunotherapy. Chapters 4 and 5 describe methods for generating BK virus and varicella zoster virus-specific CTLs. It is hoped that the work described in this thesis will facilitate clinical trials examining the role of adoptive T cell transfer in immune reconstitution in immunocompromised patients thus bringing adoptive T cell transfer as a therapeutic strategy closer to incorporation into routine clinical practice.
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Shippy, Daniel. « Effects of Adoptive Transfer of Beta-Amyloid Sensitive Immune Cells in a Mouse Model for Alzheimer’s Disease ». Scholar Commons, 2005. https://scholarcommons.usf.edu/etd/861.
Texte intégralRésumé :
One major therapeutic target for preventing and treating Alzheimer's Disease (AD) is removal of excess β-amyloid (Aβ) from the brain. Both active and passive immunotherapies targeting Aβ have proven effective in reducing brain Aβ levels and improving cognitive function in mouse transgenic models of AD. However, these approaches can induce adverse neuropathologic effects and immunologic over-activation. Indeed, clinical trials of active Aβ immunotherapy in AD patients were halted due to development of meningoencephalitis, apparently resulting from wide-spread neuroinflammation. Here we show that a more restricted and specific immune re-activation through a single adoptive transfer of Aβ-specific T cells can provide long-term benefits to APPsw+PS1 transgenic mice that last at least 1 1/2 months. Aβ-sensitive splenocytes and lymphocytes were generated in normal mice, re-stimulated with Aβ in vitro, and then adoptively transferred into cognitively-impaired APPsw+PS1 mice. Compared to control transgenic mice through 1 1/2 month post-infusion, those mice that received Aβ-sensitive T cells exhibited a reversal of pre-infusion working memory impairment and demonstrated superior basic mnemonic processing. Step-wise forward Discriminant Function Analysis of behavioral results clearly demonstrated that T cell infused mice performed comparably to wild-type non-transgenics, further emphasizing the extent of cognitive benefit this therapeutic technique afforded. Importantly, a global inflammatory response did not accompany these benefits. Though no overall reductions in Aβ deposition were noted for T cell recipient mice, a subset of T cell infused mice that benefited most in cognitive function had reduced hippocampal burdens, suggesting that hippocampal Aβ burdes did play a role in determining performance capabilities of these mice. Since chronically high levels of beta-amy
loid such as those found in APPsw+PS1 mice cause immune hypo-responsive/tolerance to Aβ, our results indicate that adoptive transfer of Aβ-sensitive T-cells can supercede such immune tolerance to Aβ, and may provide a safe, long-lasting therapy for AD.
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Proust, Alizé. « Etude du transfert du VIH-1 des cellules présentatrices d'antigènes aux lymphocytes T CD4 primaires et inhibition par les anticorps neutralisants ». Thesis, Strasbourg, 2013. http://www.theses.fr/2013STRAJ107/document.
Texte intégralRésumé :
Les cellules présentatrices d'antigènes (APCs) présentes dans les muqueuses comptent parmi les première cibles du VIH-1 et participent à sa dissémination dans l'organisme. Durant ma thèse, j'ai étudié le transfert du VIH des macrophages (Mφ) et des cellules dendritiques (DCs) aux lymphocytes T. J'ai montré que ces APCs transfèrent efficacement le virus aux lymphocytes par le biais de différents mécanismes: transfert direct en trans dans les coculture Mφ/T, et transfert en cis (suite à la production de nouveaux virions) dans les DCs/T. Ces deux modes de transfert sont inhibés par les anticorps neutralisants (AcN). De manière intéressante, certains AcN anti-gp120 inhibaient plus efficacement le transfert du VIH dans les cocultures Mφ/T que dans les cocultures DCs/T et l'infection des cellules T par le virus libre. Ces résultats suggèrent que les APCs participent activement au transfert et à la dissémination du VIH et que les AcN sont capable d'inhiber ces différents modes de transfertAntigen-presenting cells (APCs) present at mucosal sites are among the first HIV-1 target cells and contribute to the spread of infection. During my thesis, I studied HIV transfer from macrophages (Mφ) and dendritic cells (DCs) to CD4-T lymphocytes. I showed that APCs were able to efficiently transfer HIV particles to lymphocytes, but through different mechanisms: Mφ rapidlytransferred HIV by direct trans-transfer, whereas DCs were mainly implicated in cistransfer (after production of de novo HIV). Moreover, I have demonstrated that these two modes of transfer were inhibited by neutralizing antibodies (NAb) in both type ofcocultures. Very interestingly, I showed that anti-gp120 NAb inhibit more efficiently HIV transfer in Mφ/T than in DCs/T cocultures and T cells infection by free viral particles. These findings highlight the major contributions of various mucosal target cells in HIV transfer and demonstrate the potent role of NAb on inhibition of cell-to-cell transfer
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Singh, Ogesh. « Regulatory T cell diversity analysis and a gene transfer approach to cellular immunotherapy in a murine model of type one diabetes ». Thesis, Royal Veterinary College (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522749.
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Edes, Inan. « Targeted transduction of T cell subsets for immunotherapy of cancer and infectious disease ». Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17669.
Texte intégralRésumé :
Das Ziel der vorliegenden Arbeit bestand darin, ein Vektorsystem zu entwickeln, dass den simultanen Transfer verschiedener Transgene in CD8+ und CD4+ T-Zellen und dadurch die Herstellung eines immunotherapeutischen T-Zell-Produkts ermöglicht, welches aus zwei unterschiedlich modifizierten T-Zell-Subtypen besteht. Im ersten Teil der Arbeit wurde die Targeting-Technologie von lentiviralen auf γ-retrovirale Vektoren übertragen. Anschließend wird die Herstellung von Vektoren beschrieben, die spezifisch für murines CD4 oder CD8 sind. Deren Spezifität wurde zum einen durch die exklusive Expression von GFP in CD4+ oder CD8+ Zellen und zum anderen durch den Dosis-abhängigen Verlust des GFP-Signals nach Inkubation dieser Zellen mit CD4- und CD8-blockierenden Antikörpern nachgewiesen. Im dritten Teil der Arbeit wird gezeigt, dass MVm8 und MVm4 primäre T-Zellen spezifisch transduzieren. MVm8-vermittelter Transfer des Ovalbumin (OVA)-reaktiven TZRs OT-I führte zu T-Zellen, die OVA+ Tumor-Zelllinien erkannten und Interferon-γ sezernierten. Der vierte Teil dieser Arbeit beschäftigt sich mit der in vivo Transduktion primärer T-Zellen mithilfe von MVm8, welches den OT-I-TZR und eine Luciferase transferiert (MVm8/OT-I-luc). Durch systemische Applikation von MVm8/OT-I-luc wurden T-Zellen in vivo transduziert. Durch Immunisierungen konnten antigen-spezifisches Homing, Expansion und eine anschließende Kontraktion in vivo transduzierter T-Zellen gezeigt werden. Mäuse mit starker OT-I-luc-Expression waren gegenüber einer Infektion durch OVA-transgene listeria monocytogenes geschützt. Zusammenfassend lässt sich sagen, dass das in dieser Arbeit entwickelte Vektorsystem in der Lage ist zwischen Subtypen von T-Zellen zu unterscheiden und sie simultan mit unterschiedlichen Transgenen auszustatten. Für MVm8 konnte gezeigt werden, dass es T-Zellen direkt in vivo transduzieren kann.The aim of this thesis was to generate a vector system that allows the simultaneous transfer of different transgenes into CD8+ and CD4+ T cells, allowing the generation of a immunotherapeutic T cell product comprised of two differently engineered T cell subsets. The first part of the thesis describes the transfer of the measles virus (MV) envelope-based targeting technology from lentiviral (LV) to γ-retroviral (gRV) vectors. The second part reports the generation of two targeting vectors specific for murine CD4 or CD8. The exclusive specificity of MVm4 and MVm8 was proven by expression of GFP in CD4+ and CD8+ reporter cells, respectively, but not in CD4-CD8- cells after transduction, and by a dose-dependent loss of GFP signal after incubation of reporter cells with CD4 or CD8 blocking antibodies before transduction. The third part shows that MVm8 but not MVm4 transduced primary T cells. MVm8-mediated transfer of the ovalbumin (OVA)-reactive TCR OT-I resulted in T cells secreting interferon-γ (IFNγ) upon recognition of OVA+ tumor cell lines. The final part of this thesis describes the in vivo transduction of primary T cells using MVm8 transferring OT-I and a luciferase (MVm8/OT-I-luc). To this end, B6 mice deficient for Rag2 have been repopulated with either polyclonal (B6) or monoclonal T cells derived from P14-TCR transgenic mice (P14). One day later the transferred T cells were transduced in vivo by systemic application of MVm8/OT-I-luc. Upon immunization in vivo-transduced T cells homed, expanded and contracted repeatedly in an antigen-dependent manner. Finally, mice exhibiting strong luc-signals showed improved protection against infections by OVA-transgenic listeria monocytogenes (LM-OVA). In conclusion, the viral vector system developed within this thesis is able to discriminate between the two main T cell subsets and to equip them with distinct transgenes simultaneously.
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Bôle-Richard, Elodie. « Développement d'outils innovants et sécurisés de thérapie cellulaire et génique basés sur la reprogrammation de lymphocytes T dans un contexte d'immunothérapie anti-tumorale ». Thesis, Besançon, 2016. http://www.theses.fr/2016BESA3001/document.
Texte intégralRésumé :
La thérapie cellulaire est basée sur l'administration de cellules immunocompétentes dans le but d'induire une réponse thérapeutique. Le transfert de gène est un moyen d'optimiser et de sécuriser la thérapie cellulaire. Récemment, plusieurs essais cliniques d'immunothérapie ont montré l'efficacité de lymphocytes T reprogrammés pour le traitement des cancers. De plus, le transfert de gène « suicide » permet de sécuriser les effecteurs immunitaires utilisés en thérapie cellulaire. Cependant, les capacités des cellules pourraient encore être améliorées par l'expression de cytokines et de récepteurs aux chémiokines. Dans ce contexte, l'objectif de ce projet de thèse était le développement et la caractérisation d'outil innovants de thérapie génique. Ce travail a demandé le développement de vecteurs sécurisés pour 1 reprogrammation des lymphocytes T avec un CAR et leur persistance in vivo. Ces outils pourront par la suite être mis à la disposition de la recherche clinique afin de promouvoir de nouvelles stratégies de thérapi cellulaire anti-tumoraleCell therapy is based on administration of immunocompetent cells in order to induce a therapeutic response. Gene transfer can optimize and secure the cell therapy. Recently, several clinical trials of immunotherapy have shown the efficacy of reprogrammed T cells for the treatment of cancers. Moreover, the transfer of suicide gene enables th use of secure immune effectors in cell therapy. However, capacities of cells could be further improved by the expression of cytokines and receptors chemiokines. ln this context, the aim of this thesis project was the development and the characterization of innovative tools for gene therapy. This work required the development of safe retroviral vectors for reprogramming T cells with Chi me rie Antigen Receptor (CAR). These tools will be made available for clinical research in order to promote new anti-tumor cell therapy strategies
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Mak'Anyengo, Rachel [Verfasser], et Christian [Akademischer Betreuer] Bauer. « Role of the NIrp3 inflammasome in regulation of the tolerogenic function of CD103+ dendritic cells in CD4+CD45RbHigh T cell transfer colitis and in steady state / Rachel Mak'Anyengo ; Betreuer : Christian Bauer ». München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1165503905/34.
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Schirrmann, Thomas. « Tumorspezifische Targeting der humanen natürlichen Killerzellinie YT durch Gentransfer chimärer Immunglobulin-T-Zellrezeptoren ». Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2005. http://dx.doi.org/10.18452/15246.
Texte intégralRésumé :
Die spezifische adoptive Immuntherapie ist ein hoffnungsvoller Ansatz zur Behandlung von Tumoren. Die aufwendige individuelle Bereitstellung primärer Effektorlymphozyten könnte durch den Einsatz etablierter tumorantigenspezifischer Effektorzellinien vermieden werden. In dieser Arbeit wurde untersucht, ob sich ein Tumortargeting der humanen Natürlichen Killer-(NK)-Zellinie YT durch den Gentransfer chimärer Immunglobulin-T-Zellrezeptoren (cIgTCRs) erreichen läßt. Die cIgTCR-Konstrukte wurden aus single-chain-Fv-Fragmenten (scFv), dem IgG1-Fc-Teil und der CD3-Zeta-Signalkette erzeugt. Die scFv-Fragmente wurden aus den humanisierten Antikörpern BW431/26 und HuM195, die spezifisch für das karzinoembryonale Antigen (CEA) bzw. CD33 sind, konstruiert und zeigten als scFv-hFc-Fusionsproteine eine spezifische Bindung an Tumorzellen. Die YT-Zellen wurden mit den cIgTCR-Genkonstrukten über Elektroporation transfiziert und über immunologische Verfahren angereichert. In-vitro-Studien ergaben eine spezifische Lyse von CEA+ Kolonkarzinomzellinien durch die scBW431/26-hFcZeta+ YT-Zellen. Die Zytotoxizität korrelierte mit der Expression des cIgTCR-Antigens auf den Tumorzellen und wurde durch zirkulierendes CEA nicht gehemmt. Die scHuM195-hFcZeta+ YT-Zellen zeigten eine spezifische Lyse der CD33+ myeloischen Leukämiezellinie KG1. Die Bestrahlung wurde zur Wachstumsbegrenzung der YT-Zellen eingesetzt. Die spezifische Zytotoxizität der scBW431/26-hFcZeta+ YT-Zellen gegenüber CEA+ Tumorzellen war einen Tag nach Bestrahlung unverändert. Die Koinjektion von CEA+ Tumorzellen mit bestrahlten scBW431/26-hFcZeta+ YT-Zellen führte zu einer signifikanten Hemmung des Tumorwachstums in NOD/SCID-Mäusen. Die cIgTCR+ YT-Zellen zeigten in vitro eine geringe Sensibilität gegenüber allogenen Blutlymphozyten. Die Ergebnisse zeigen, daß die Zytotoxizität der NK-Zellinie YT tumorantigenspezifisch durch cIgTCR-Gentransfer erweitert wird und ein Potential zur Behandlung minimaler Tumorerkrankungen besteht.The specific adoptive immunotherapy is a promising strategy for cancer treatment. The utilization of established tumor antigen specific effector cell lines could bypass the expendable individual preparation and often limited specificity of primary effector lymphocytes. This study investigated the tumor targeting of the human Natural Killer (NK) cell line YT by gene transfer of chimeric immunoglobulin T cell receptors (cIgTCRs). The cIgTCR constructs were generated of single chain antibody fragments (scFv), the IgG1 Fc part and the CD3 Zeta chain. The scFv fragments were constructed of the humanized antibodies BW431/26 and HuM195 with specificity for the carcinoembryonic antigen (CEA) and CD33, respectively, and showed as scFv-Fc fusion proteins a specific binding to tumor cells. YT cells were transfected with the cIgTCR gene constructs by electroporation and enriched by immunological cell separation. In vitro studies revealed a specific lysis of CEA+ colon carcinoma cell lines by scBW431/26-hFcZeta+ YT cells. The cytotoxicity correlated with the expression of the cIgTCR antigen on the tumor cells and was not inhibited in the presence of soluble CEA. The scHuM195-hFcZeta+ YT cells mediated a specific lysis of the CD33+ myeloic leukemia cell line KG1. The irradiation was used to limit the growth of the YT cell line. The specific cytotoxicity of the scBW431/26-hFcZeta+ YT cells against CEA+ tumor cells was unaltered one day after irradiation. The coinjection of CEA+ tumor cells and irradiated scBW431/26-hFcZeta+ YT cells led to a significant growth inhibition in NOD/SCID mice. The cIgTCR+ YT cells showed a low susceptibility to the cytotoxicity of allogeneic blood lymphocytes in vitro. The results demonstrated that the cytotoxicity of the human NK cell line YT can be specifically extended to tumor antigens by cIgTCR gene transfer. The employment of receptor gene modified YT cells could be a useful tool for the adoptive immunotherapy of minimal tumor diseases.
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MERCURI, ELISABETTA. « PRECLINICAL MODELING HIGHLIGHTS THE THERAPEUTIC POTENTIAL OF THE ADOPTIVE TRANSPLANT OF GENE CORRECTED T CELLS IN X-LINKED HYPER-IGM IMMUNODEFICIENCY ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2020. http://hdl.handle.net/10281/263922.
Texte intégralRésumé :
La terapia genica di cellule staminali ematopoietiche (HSC) ha prodotto benefici clinici in diversi pazienti affetti da una varietà di malattie genetiche. Tuttavia, l’uso di vettori che si integrano nel genoma in modo semi-casuale pone il rischio di mutagenesi inserzionale e di una espressione del transgene ectopica/non regolata. Quest’ultimo problema è particolarmente rilevante quando si trattano geni strettamente regolati attivi sulla proliferazione cellulare, come il gene CD40LG, la cui espressione sulle cellule T attivate porta all’attivazione contatto-dipendente delle cellule B, alla loro proliferazione ed allo scambio di classe delle immunoglobuline. Poichè le sue mutazioni causano l’immunodeficienza legata all’X con iper-IgM (HIGM1), il trasferimento genico in HSC è stato proposto come potenziale trattamento per questa sindrome. Anche se piccole quantità di cellule trasdotte hanno ripristinato la funzione immunitaria umorale e cellulare in un modello murino di HIGM1, l’espressione costitutiva di CD40LG in timociti o in cellule T periferiche ha portato a linfoproliferazioni, molte delle quali sono progredite a linfom. Le strategie di riparazione genica che preservano il controllo fisiologico dell’espressione del gene corretto potrebbero quindi rappresentare un approccio più promettente per il trattamento di HIGM1. In questo studio sfruttiamo il meccanismo Homology Directed Repair (HDR) per la correzione in situ della maggior parte delle mutazioni responsabili della sindrome HIGM1 presenti nel gene CD40L, con l’obiettivo di ristabilirne la funzione e il controllo dell’espressione. In particolare sfruttiamo il sistema CRISPR/Cas9 per promuovere l’integrazione sito-specifica di una copia funzionale di parte del gene CD40L a valle del suo promotore endogeno, correggendo così la maggior parte delle mutazioni responsabili della malattia. Dato che il difetto genetico non è deleterio per le cellule T, questo tipo di malattia ci offre l’opportunità unica di sviluppare una terapia genica basata sulla correzione di cellule T autologhe. Al fine di stabilire quali sono le dosi terapeutiche e le condizioni di trapianto necessarie per ottenere la ricostituzione immunitaria e il ripristino delle funzioni immunologiche con cellule corrette, abbiamo infuso diverse dosi di cellule T WT in topi HIGM1 pre-condizionati o meno con diversi regimi linfodepletanti ed eseguito trapianti competitivi di cellule staminali ematopoietiche WT e Cd40lg - / - nel modello animale. Mentre l’ analisi del sangue periferico ha dimostrato la persistenza a lungo termine di cellule T in tutte le condizioni, sono stati ottenuti livelli di attecchimento più elevati nei topi trapiantati dopo il trattamento chemioterapico con ciclofosfamide (CPA). Tutti i topi trapiantati hanno mostrato un parziale ripristino della risposta IgG specifica dopo immunizzazione con TNP-KLH, ma è stato osservato un ripristino più elevato nei topi pre-condizionati con CPA. Questi topi hanno anche mostrato la presenza di centri germinativi nella milza. Topi HIGM1 ricostituiti con dosi crescenti di HSPC WT hanno mostrato un ripristino dose-dipendente della risposta immunitaria T dipendente. In particolare, abbiamo dimostrato che il 10% di HSPC funzionali è sufficiente a ripristinare parzialmente la capacità di produrre anticorpi specifici contro diversi antigeni oltre che ad attenuare l'infezione in topi HIGM1 inoculati con il patogeno Pneumocystis murina. Il nostro obiettivo futuro è quello di dimostrare il ripristino della risposta immunitaria contro l'infezione da pneumocystis murina in topi HIGM1 trapiantati con cellule T CD4 + WT. In caso di successo, i nostri risultati saranno strumentali per stabilire il potenziale terapeutico di un approccio di correzione genica basato sulle cellule T per il trattamento della sindrome HIGM1 che potrebbe fungere da terapia ponte per una terapia definitiva basata sul trapianto di HSPC corrette.Background The X-linked hyper-IgM syndrome type I (HIGM1) is caused by inactivating mutations in the CD40 ligand gene (CD40LG) that disrupt the T cell helper function on B cells and macrophages. This disease represents an ideal candidate for a gene correction strategy because preclinical studies of Hematopoietic Stem Cell (HSC) gene therapy have already shown i) evidence of potential efficacy even with few amounts of transduced cells; ii) critical safety issues due to unregulated transgene expression. Since in HIGM1 the genetic defect is not lethal to T cells, we aim to apply our gene editing strategy on autologous T cells that could be used to provide immediate therapeutic benefit to the patients by resolving pre-existing infections prior to a definitive HSPC transplant. Methods To establish which are the therapeutic threshold levels and transplant conditions required to achieve immune reconstitution and functional immunologic restoration with corrected cells, we infused different doses of WT T cells into HIGM1 mice pre-conditioned or not with different lymphodepleting regimens and performed competitive transplants of WT and Cd40lg-/- HSPC in the mouse model. Results While longitudinal blood analyses showed a long-term, stable T cell engraftment in all the conditions, highest engraftment rates were obtained in mice transplanted after chemotherapy treatment with cyclophosphamide (CPA). All the transplanted mice showed a partial rescue of the antigen-specific IgG response after immunization with Keyhole Limpet Hemocyanin (TNP-KLH) but a higher rescue was observed in mice pre-conditioned with CPA. These mice also showed the presence of TNP-KLH specific IgG producing B cells and germinal centers within splenic lymphoid follicles. HIGM1 mice reconstituted with increasing proportions of WT HSPC displayed a dose-dependent rescue of the T cell mediated immune response. In particular we found that 10% of WT HSPC is sufficient to partially restore serologic immunity against different antigens as well as to attenuate infection in HIGM1 mice challenged with Pneumocystis murina. Conclusions Our current efforts are aimed to demonstrate functional restoration of the immune response against Pneumocystis murina infection in HIGM1 mice that received adoptive transfer of WT CD4+ T cells. If successful, our findings will be instrumental to establish the therapeutic potential of a T cell gene correction approach for the treatment of the HIGM1 disease that could act as a bridge therapy to the HSPC-based strategy.
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ANNONI, ANDREA. « Strategies for tolerance induction to gene therapy derived products ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/763.
Texte intégralRésumé :
I vettori lentivirali (LV) rappresentano una concreta realtà per lo sviluppo di protocolli di terapia genica. I LV, infatti, hanno la capacità di trasdurre ed integrare stabilmente il proprio genoma anche in cellule che non sono in attivo stato di replicazione. Tuttavia, uno dei maggiori ostacoli al successo degli approcci di terapia genica che prevedono l’utilizzo di vettori virali è rappresentato dalla risposta immune innata ed adattativa da essi indotta. Le risposte immuni cellulo-mediate ed anticorpali dirette verso antigeni associati alle particelle virali e alla proteina derivante dal transgene eliminano le cellule geneticamente modificate, rendendo inefficacie la terapia genica.
Nello studio qui descritto, abbiamo sviluppato un modello murino di terapia genica, che prevede somministrazione sistemica in topi immunocompetenti di LV codificante per una proteina fluorescente. Tale modello sperimentale è funzionale alla caratterizzazione della cinetica d’espressione del transgene e della risposta immunitaria contro di esso e allo stesso tempo è utile per lo sviluppo di nuovi approcci per la modulazione di tale risposta.
Diverse strategie sono state esplorate per la modulazione della risposta al transgene, come l’immunosoppressione e l’utilizzo di vie di somministrazione meno immunogeniche. In questo studio abbiamo indagato la possibilità di ristabilire la tolleranza immunologica nei confronti del prodotto di terapia genica attraverso due differenti modalità: una terapia cellulare da associare al trasferimento genico o l’utilizzo di LV con una regolazione d’espressione dipendente da micro-RNA endogeni.
E’ stato ampiamente dimostrato che le cellule CD4+CD25+ T regolatorie (Tregs) rappresentano una sottopopolazione linfocitaria con la capacità di controllare le risposte immunitarie in vivo garantendo la tolleranza agli antigeni “self”. Con l’ausilio del nostro modello animale abbiamo testato se dette cellule fossero in grado di sopprimere la risposta al transgene. Abbiamo così definito che il trasferimento di Tregs, derivanti da topi wild type (wt) o da topi GFP-transgenici (GFP-tg), tolleranti per GFP, non è in grado di controllare la risposta al transgene. Contrariamente, il trasferimento di un’ alta dose di cellule presentanti l’antigene (APC), derivate da topi GFP-tg, sono risultate efficaci nel controllo della risposta immune estendendo significativamente i tempi di espressione del transgene.
La risposta immune verso il transgene si genera poiché, dopo la somministrazione del vettore lentivirale, una frazione delle APC esprime il transgene e ne presenta epitopi con modalità immunogenica attivando le cellule T. Promotori tessuto-specifici, come quello per l’albumina, sono stati testati per ridurre l’espressione del transgene nelle APC, restringendola ai soli epatociti. Recentemente, Brown et al. (Nat. Med. 2006; 12:585-591) hanno dimostrato che inserendo ripetute sequenze riconosciute da un micro-RNA, specifico della linea ematopoietica, mir142-3p, nel vettore codificante per GFP (LV.PGK.GFP.mir142-3pT) l’espressione del transgene era abrogata in tutte le cellule di derivazione ematopoietica ma era stabile e persistente nel fegato sia negli epatociti che nelle cellule endoteliali (LSEC). Così, abbiamo studiato come il sistema immunitario reagisce alla somministrazione di tale LV micro-RNA regolato. I dati ottenuti indicano che si genera uno stato di tolleranza verso il transgene, mediata da cellule Tregs.
Questi risultati nel loro insieme potranno avere importanti sviluppi per le future applicazioni di protocolli di terapia genica.Due to their ability to transduce non-dividing cells and stably integrate, lentiviral vectors (LV) are a candidate system for therapeutic gene transfer in a number of genetic diseases. However, the use of LV may be hampered by their ability to trigger innate and adaptive immunity. Immune responses against genetically modified cells represent a major obstacle to the success of gene therapy. Cellular and humoral immune responses to vector associated and transgene-derived proteins lead to prompt elimination of transgene expressing cells abrogating, the benefits of gene transfer. A non-therapeutic murine model of gene therapy has been established. LV, encoding for green fluorescent protein (GFP) under the control of an ubiquitous promoter, was intravenously administrated into immunocompetent mice in order to define the kinetics of transgene expression, characterize the anti-transgene immune response and develop different approaches to overcome the anti-transgene immunity and achieve long term transgene expression. Several strategies have been used to limit immune response following gene transfer, including immunosuppression, and the use of less immunogenic routes of administration. In the present work we focused our attention in inducing immunological tolerance to transgene expressing cells as first approach, by a cellular therapy, and second, through the use micro-RNA (mir) regulated lentiviral vectors. Regulatory T cells (Tregs), which have been demonstrated to control immune responses in vivo, were tested for their ability to suppress anti-transgene response leading to stable long-term gene expression. Adoptive transfer of naturally occurring CD4+CD25+ Tregs (nTregs) isolated from wt mice or from transgene tolerant transgenic (tg) mice did not suppress the anti-transgene immune response after LV delivery. These data demonstrate that neither increasing the endogenous pool of nTregs nor transferring nTregs selected in a transgene expressing thymus can modulate the immune response and mediate sustained transgene expression. Conversely, adoptive transfer of antigen presenting cells (APC) isolated from transgene tolerant tg mice efficiently reduced the immune response leading to stable LV-encoded protein expression in vivo. Most of the immunogenicity of the LV-based gene transfer protocols depends on the transduction and transgene expression by professional APC that prime T cells inducing an anti-transgene immune response. The use of tissue specific promoters, such as the albumin promoter, has been explored in order to target transgene expression in hepatocytes and drastically reduce transgene expression in APC. Recently, in the same direction, Brown et al. (Nat. Med. 2006; 12:585-591) demonstrated that addition of target sequences for an hematopoietic-specific micro-RNA, mir-142-3p, into LV encoding for GFP under transcriptional control of the ubiquitous PGK promoter (LV.PGK.GFP.142-3pT) prevented transgene expression in all hematopoietic lineages and led to stable transgene expression in the liver. Here, we set out to elucidate the immunological events that enabled stable gene transfer with this microRNA-regulated LV. Results demonstrate that systemic gene transfer by the mir-142-regulated LV can provide robust tolerance to a specific antigen which is mediated by CD4+ Tregs. These findings will have important implications for developing future gene therapy strategies.
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Cobbold, Mark. « Direct selection and adoptive transfer of cytomegalovirus-specific cytotoxic T cells ». Thesis, University of Birmingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433688.
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Pospori, C. « WT1 TCR gene transfer into haematopoietic stem cells : in vivo functional analysis of WT1-specific T cells ». Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1369532/.
Texte intégralRésumé :
The Wilms tumour antigen is a promising target for T cell-based tumour immunotherapies. Vaccines against WT1 peptides tested in cancer patients showed immunological and molecular responses. However, the clinical responses observed were partial and it is currently not known whether physiological levels of WT1 expression in some healthy tissues results in the deletion or tolerance induction of WT1-specific T cells. In this PhD project, TCR gene transfer into purified haematopoietic stem cells (HSCs) was used to study the thymic development of WT1-specific T cells and their fate in the periphery. Lentiviral constructs containing the genes for an HLAA2 allorestricted, murinised WT1 TCR or the genes for a control, viral peptide-specific, LMP2 TCR, were generated. The conditions for lentivirally-transduced HSC transplants were optimised. The results obtained from WT1 TCR tranduced HSC transplants in HLA-A2Kb transgenic mice demonstrated that thymocytes expressing this high-avidity WT1 TCR were positively selected into CD8 T cells and emerged in the recipient’s periphery. WT1-specific T cells exhibited a memory, CD44hi phenotype correlating with rapid antigen specific killing, proliferation and cytokine secretion of WT1-specific T cells in the absence of vaccination. LMP2-specific T cells exhibited a naive-like, CD44low phenotype without any antigen specific function. WT1-specific T cells persistent long-term in the periphery of transplanted mice, and no autoimmunity was noted. The results presented in this thesis show for the first time that T cell specificity for a tumour-associated, self-antigen did not result in tolerance induction, but instead mediated the spontaneous generation of functionally competent, memory phenotype T cells.
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Klysz, Dorota. « Impact of lymphopenia-inducing regimens and energetic resources on the fate of adoptively transferred T cells ». Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20184/document.
Texte intégralRésumé :
Les thérapies anti-tumorales se sont considérablement améliorées au cours de la dernière décennie. Toutefois, les traitements utilisés actuellement rencontrent d'importantes limitations, notamment dans le cas de cancers métastatiques, révélant l'urgence de développer de nouvelles approches. Ainsi, l'immunothérapie par transfert adoptif de cellules T représente une approche innovante particulièrement prometteuse. Son principe s'appuie sur l'injection de cellules T autologues spécifiques d'antigènes tumoraux, préalablement manipulées et amplifiées ex vivo, chez des patients rendus lymphopéniques par chimiothérapie et/ou radiothérapie. Toutefois, même si l'état lymphopénique est induit par ces 2 protocoles de conditionnements, leurs effets sur l'environnement de l'hôte ainsi que sur le devenir des cellules T greffées étaient, jusqu'à nos travaux, mal connus. Par le biais de modèles murins, nous avons pu démontrer que le devenir des cellules T diffère après transfert dans des souris irradiées ou traitées par chimiothérapie (Bu/Cy). Ainsi, après transfert dans des animaux irradiés, on observe une prolifération préférentielle des cellules T CD8, dépendante de l'IL-7, est observée alors qu'un transfert chez des souris traitées Bu/Cy se traduit par une prolifération rapide, indépendante de l'IL-7, des cellules T CD4. De plus, ces comportements sont associés à d'importantes modifications de l'environnement généré chez l'hôte. Plus spécifiquement, nous avons démontré, dans les organes lymphoïdes secondaires, que la localisation et la représentation des différentes sous-populations de cellules dendritiques présentes étaient différentiellement modulées par le type de conditionnement utilisé. Par ailleurs, l'élimination spécifique des cellules CD11c+ chez des souris traitées Bu/Cy était accompagnée d'une inhibition importante de la prolifération rapide des cellules T CD4 greffées. L'ensemble de nos travaux montrent que les traitements lymphopéniques génèrent des environnements distincts capables de moduler le devenir des cellules T greffées.Durant ma thèse, nous avons également abordé de façon originale un aspect novateur de l'environnement en étudiant le rôle potentiel des nutriments comme régulateurs métaboliques des fonctions effectrices des cellules T. La glutamine est l'acide aminé le plus abondant du plasma, pouvant contribuer aux besoins bionénergétiques et biosynthétiques des cellules T en prolifération. Nous avons démontré dans nos travaux qu'une carence en glutamine lors de l'activation de cellules T CD4 par leur TCR entrainait un délai dans l'activation de la voie mTOR, une réduction de la production intracellulaire d'ATP aux temps précoces et se traduisait par une diminution de la prolifération. De plus, ces conditions étaient associées à une augmentation de la conversion de cellules CD4 T naïves, via TGFβ, en cellules régulatrices Foxp3+ , y compris en condition de polarization Th1. Par contre, la carence en glutamine n'a pas inhibé la différenciation Th2. Les cellules T Foxp3+ ainsi générées en condition limitante de glutamine présentaient in vivo des fonctions suppressives aussi efficaces que celles des cellules régulatrices nTregs. En effet, elles ont la capacité de bloquer l'induction de la colite provoquée par la greffe de cellules T effectrices dans des souris Rag2-/- . Nos travaux démontrent ainsi que l'environnement métabolique peut être un régulateur clé de la différenciation des cellules T CD4. L'ensemble de mes travaux de thèse ont mis en évidence de nouveaux paramètres capables de potentiellement modifier la survie et la réactivité des cellules T grefféesAnti-tumor therapies have improved significantly over the decade. However, the currently used treatments have important limitations, notably for metastatic cancers, and the development of new approaches is therefore a high priority. Adoptive T cell therapy (ACT) represents an innovative strategy that has shown much promise. This therapy is based on the infusion of tumor-specific T cells, which have been manipulated and expanded ex vivo, into patients who have been rendered lymphopenic by chemotherapy and/or irradiation. It is interesting to note that while lymphodepletion is attained by the vast majority of conditioning regimens, the effects of these protocols on the host environment and potentially, on the destiny of adoptively-transferred T cells had not been elucidated prior to the studies which we initiated. Using a murine model, we found that the fate of adoptively-transferred T cells differs markedly in mice rendered lymphopenic by sub-lethal irradiation as compared to a busulfan/cyclophosphamide (Bu/Cy) chemotherapy regimen. Irradiation-mediated lymphopenia resulted in a skewed IL-7-dependent proliferation of donor CD8+ T cells, whereas Bu/Cy treatment led to an increased IL-7-independent, rapid CD4+ T cell proliferation. These alterations in T cell proliferation were associated with striking changes in the host microenvironment. More specifically, we demonstrated that the proportion and localization of different dendritic cell (DC) subsets in lymphoid organs were differentially affected by the type of conditioning. Furthermore, we found that these DC controlled the rapid donor CD4+ T cell division detected in Bu/Cy-treated mice as depletion of CD11c+ DC inhibited this proliferation. Altogether, our studies demonstrate that lymphopenic regimens generate distinct host environments which modulate the fate of adoptively-transferred T cells. Durind my PhD, we also investigated an original and novel aspect of the microenvironement by studying the potential role of nutrients as metabolic regulators of T cell effector function. Glutamine is the most abundant amino acid in the plasma and contributes to the bioenergetic and biosynthetic requirements of proliferating T cells. Here, we demonstrated that activation of CD4+ T cells under glutamine-deprived conditions results in a delayed mTOR activation with reduced early ATP production and decreased proliferation. Moreover, these conditions resulted in the conversion of naïve CD4+ T cells into Foxp3+ regulatory T cells (Tregs). This de novo Treg differentiation occurred even under Th1-polarizing conditions and was TGFβ-dependent. Interestingly, glutamine deprivation did not inhibit Th2 differentiation. Importantly, these converted Foxp3+ T cells showed enhanced in vivo persistence and were highly suppressive, completely protecting Rag-deficient mice from the development of autoimmune inflammatory bowel disease as efficiently as natural-occuring Tregs. Thus, our data reveal the external metabolic environment to be a key regulator of a CD4 T lymphocyte's differentiation. Altogether, the data generated during my PhD provide new insights into the identification of parameters that can potentially alter the survival and reactivity of adoptively-transferred T cells
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Ghenassia, Alexandre. « Induction de réponses mémoires lymphocytaires T CD8 et protection vaccinale après transfert de gènes par le vecteur AAV recombinant ». Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T032/document.
Texte intégralRésumé :
La mémoire immunologique est le mécanisme biologique fondamental à la base du développement de la vaccination. La compréhension de ce mécanisme ainsi que de ses interactions avec les différents acteurs du système immunitaire a permis l’élaboration de vaccins qui sont aujourd’hui les garants d’une protection accrue face à l’émergence de maladies infectieuses potentiellement mortelles. La voie d’injection et le mode de transfert de ces vaccins sont des paramètres majeurs à prendre en considération car ils définissent une modulation des réponses immunitaires et de leurs spécificités d’action. De nos jours, seule la voie intramusculaire demeure la voie majoritaire d’administration de vaccins lors de la prophylaxie primaire en santé humaine. Au cours de notre étude, nous nous sommes intéressés à comparer l’injection d’un antigène (l’ovalbumine) selon deux voies d’administration : la voie intramusculaire et la voie intradermique. Nous nous sommes également appuyés sur une technologie du laboratoire qui consiste à transférer des gènes par des vecteurs AAV2/1 recombinants. Nous disposions de deux constructions de ces vecteurs ayant une spécificité pour cibler les cellules musculaires et permettant l’apport d’un effet auxiliaire par les lymphocytes T CD4+ lors d’injections dans des souris femelles. De plus, une de ces constructions nous permettait d’éviter la voie de présentation directe de l’antigène par les cellules dendritiques (DCs) aux lymphocytes T CD8+. Les capacités modulatrices de ces vecteurs nous permirent de montrer pour la première fois que le vecteur AAV2/1 recombinant était capable de faire exprimer un transgène au sein de la peau et d’y générer une réponse cellulaire forte. Nous avons également montré qu’il existait une synergie d’action entre l’effet auxiliaire et la voie intradermique qui améliorait considérablement les réponses cellulaires issues de la présentation croisée d’antigène. Enfin, nous avons pu démontrer que les lymphocytes T CD8+ générés suite à cette synergie d’action présentaient un profil phénotypique de cellules mémoires polyfonctionnelles et capables de protéger l’hôte face à un challenge pathogéniqueImmunological memory is the fundamental biological mechanism at the beginning of the development of vaccination. Understanding this mechanism and its interactions with the various players of the immune system has allowed the development of vaccines that are today the most effective barrier against the emergence of life-threatening infectious diseases. Route of injection and the nature of carriers of these vaccines are key parameters to be taken into consideration because they define a modulation of immune responses and their specific features. Nowadays, only the intramuscular injection route remains the major route of vaccines injection in the context of primary prophylaxis in human health. During our study, we were interested in comparing the injection of antigen (ovalbumin) following two routes of administration: intramuscular and intradermal routes. We also relied on a technology in the laboratory that involves the transfer of genes by rAAV2/1 vectors. We had two constructs of these vectors having specificity to target skeletal muscle cells and allowing us to provide a helper effect from CD4+ T cells during injections into female mice recipients. Moreover, one of these constructs enabled us to avoid the direct presentation of antigens by dendritic cells (DCs) to CD8+ T cells. The capacity of modulation of these vectors allowed us to show for the first time that the rAAV2/1 vector was able to trigger the expression of a transgene in the skin, and there to generate a strong cellular response. We have also shown that CD4+ T cell help and the intradermal route of immunization synergize to improve greatly cellular responses from the cross-presentation of antigens. Finally, we have demonstrated that CD8+ T cells generated following this synergism exhibited a phenotypic profile of polyfunctional memory cells and able to protect the host against a pathogenic challenge
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Balducci, Estelle. « Microvésicules et microARNs : rôle dans le transfert d'informations biologiques entre les lymphocytes T CD4 et l'endothélium au cours de l'infection par le VIH-1 ». Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0423.
Texte intégralRésumé :
Le virus de l’immunodéficience humaine de type 1 (VIH-1) induit une activation généralisée des réponses de l'hôte impliquant les lymphocytes T mais aussi les cellules du microenvironnement comme les cellules endothéliales. Les microvésicules (MV) sont des vésicules extracellulaires impliquées dans la communication intercellulaire décrites comme des vecteurs de microARNs (miARNs). Dans ce travail, nous avons émis l'hypothèse que l'infection par le VIH-1 induit l'expression de miARNs dans les lymphocytes T CD4 qui peuvent être vectorisés par les MV et transférés de manière paracrine aux CE. Ces MV joueraient un rôle important dans la pathogenèse de l’infection en contrôlant à distance l'homéostasie endothéliale. Nos résultats montrent que le miR-146-5p est uprégulé à la fois dans les lymphocytes T CD4 de patients infectés par le VIH-1, naïfs de traitement et dans les MV issues de ces lymphocytes. En utilisant un modèle de MV d’une lignée lymphocytaire T enrichie en miR-146-5p (miR-146b-MV), nous montrons que ces MV sont capables de : 1) de protéger leur contenu en miARNs de la dégradation par les RNases, 2) de transférer le miR-146b-5p mimic à des HUVEC et 3) réduire la réponse inflammatoire endothéliale in vitro et in vivo, dans les poumons de souris qui ont reçu une injection systémique de miR-146b-MV. Ce transfert est responsable d’une diminution de l’expression d’ICAM-1 et VCAM-1, à travers une down-régulation d’IRAK1 et de TRAF6. L’ensemble de ces résultats montre que le miR-146-5p transféré par des MV peut diminuer les réponses inflammatoires endothéliales et constituer ainsi un mécanisme de défense de l’hôte contre les altérations vasculaires induites par le VIH-1Human immunodeficiency virus type 1 (HIV-1) promotes a generalized activation of host responses that involves CD4 T cells, but also cells of the micro-environnement that are not directly infected such as endothelial cells. Microvesicles (MV), implicated in cell-to-cell communication, have been recently described as vectors of microRNAs (miRNAs). We hypothesized that HIV-1 infection induce cellular miRNAs expression in CD4 T cells which may be vectorized by MV and transferred in a paracrine manner to endothelial cells to regulate vascular homeostasis. Using a miRNome quantitative RT-PCR analysis, we showed that HIV-1 infection leads to a dysregulation of several miRNAs and identified miR-146b-5p as upregulated in both CD4 T cells and CD4 T cells derived-MV from antiretroviral therapy (ART)-naive HIV-1 infected patients, compared to age- and sex-matched healthy subjects. Using a CEM T cell line transfected with miR-146b-5p mimic, we demonstrated that MV from CEM overexpressing miR-146b-5p mimic (miR-146b-MV): 1/ protect their miRNA cargo from RNase degradation, 2/ transfer miR-146b-5p mimic into HUVEC, and 3/ reduce endothelial inflammatory response in vitro and in vivo, in lungs from mice injected with miR-146b-MV. This paracrine control of endothelial inflammatory response mediated by MV involved a decreased expression of NF-κB responsive molecules ICAM-1 and VCAM-1, through down-regulation of IRAK1 and TRAF6. Collectively, these findings demonstrate that miR-146b-5p transferred by MV counteract IRAK1- and TRAF6-mediated endothelial inflammatory responses in HIV-1 infection and could be considered as a host defence mechanism against HIV-1-associated vascular alterations
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Gagnepain, Anaïs. « Évaluation de nouveaux pseudotypes de vecteurs lentiviraux pour le transfert de gènes dans les cellules hématopoiétiques ». Thesis, Lyon, École normale supérieure, 2014. http://www.theses.fr/2014ENSL0939/document.
Texte intégralRésumé :
Le transfert de gènes dans les cellules souches hématopoïétiques par des vecteurs lentiviraux s’inscrit dans les protocoles actuels de traitement par thérapie génique de plusieurs maladies monogéniques (B-thalassémie, Adrénoleucodystrophie, SCID…). De même, le transfert de gènes dans les lymphocytes T et B ouvre des perspectives tant au niveau de la thérapie génique que pour l’immunothérapie. Nous avons mis au point des vecteurs lentiviraux pseudotypés par des glycoprotéines chimérique (BaEV/TR) et mutante (BaEVRLess) du rétrovirus endogène de babouin. Nous avons montré que ces nouveaux vecteurs peuvent transduire de manière plus efficace les cellules souches hématopoïétiques stimulées et quiescentes que les vecteurs pseudotypés par la glycoprotéine du virus de la stomatite vésiculaire (VSV-G). Il en est de même pour les vecteurs développés récemment et pseudotypés par les Glycoprotéines H et F du virus de la rougeole. Nous avons aussi comparé la capacité de ces derniers vecteurs à ceux pseudotypés par les glycoprotéines BaEV/TR et BaEVRLess dans le transfert de gènes dans les lymphocytes B et T ainsi que dans l’ensemble des cellules de la lignée T. Nous sommes désormais en mesure de proposer des vecteurs adaptés au transfert de gènes à chaque étape de la différenciation des cellules CD34+ en thymocytes ainsi qu’en lymphocytes T matures. Ceci pourrait permettre de proposer de nouveaux protocoles cliniques en thérapie génique avec une co-transplantation de cellules souches génétiquement modifiées et de cellules T différenciées à partir de ces cellules. Ceci permettrait notamment de réduire les phases d’aplasie actuellement nécessaires pour la greffe de cellules souchesLentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs). We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols
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Kozlov, Andriy. « T-type Ca channels as pathways of Ca2+ entry into the cell. : Role of ion-channel interactions in channel gating ». Université Louis Pasteur (Strasbourg) (1971-2008), 2001. http://www.theses.fr/2001STR13186.
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Zeng, Yi [Verfasser], et Stefan [Akademischer Betreuer] Endres. « Gene expression profiles of T cells after adoptive transfer in a mouse model of pancreatic carcinoma / Yi Zeng ; Betreuer : Stefan Endres ». München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1126407313/34.
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Cabrera, Aulestia Francisco Javier. « Treating Cellular Stress and Damage : Use of Healthy Mitochondria Isolated from Donor Cells in the Artificial Mitochondria Transfer / Transplant (Amt/T) to Repair Mitochondrial Disfunction in Differentiated (Peripheral Blood Mononuclear Cells) and Germinal Cells (Oocytes) ». Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT073.
Texte intégralRésumé :
Selon la théorie endosymbiotique, la mitochondrie est un organite dérivé d'une ancienne alpha-protéobactérie qui développa une symbiose avec un ancêtre eucaryote. L'ADN mitochondrial (ADNmt) existe dans des centaines à des milliers d'exemplaires dans chaque cellule et code pour 13 protéines structurelles qui sont des sous-unités de la chaîne respiratoire. Les mitochondries génèrent de l'énergie pour les processus cellulaires en produisant de l'ATP par phosphorylation oxydative. Ils contrôlent également d’autres processus tels que la synthèse de nucléotide et d’hème, l’équilibre rédox, le métabolisme du calcium, la gestion des déchets (urée et ROS) et l’apoptose. Les délétions d'ADNmt, les mutations ponctuelles, les dimères de thymine et les déplétions d'ADNmt sont fortement liés à la maladie chez l'homme et d'autres mammifères. Certaines altérations de l'ADNmt peuvent survenir spontanément pendant le spam de la vie, d'autres peuvent être héritées de la lignée maternelle sous forme de mutations spécifiques. Ainsi, les mutations de l’ADN nucléaire peuvent produire des troubles mitochondriaux car, alors que l’ADNmt code pour 13 protéines, les mitochondries ont besoin de près de 2000 protéines dotées de rôles structurels et fonctionnels. Dans ces cas, un modèle d'héritage mendélien peut être observé. Des altérations de l'ADNmt peuvent être produites par l'exposition à des substances toxiques ou à des rayonnements UV et à haute énergie. Les mutations de l'ADNmt sont cumulatives car les mitochondries sont dépourvues de mécanismes de réparation. Les ADNmt normaux et mutants peuvent coexister dans la même cellule, une condition appelée hétéroplasmie. L'hétéroplasmie permet la persistance d'une mutation par ailleurs mortelle à travers les générations. Les troubles mitochondriaux peuvent apparaître sous forme de myopathies, cardiomyopathies, diabète sucré, l’acidose lactique, hypofertilité féminine, lipodystrophie, neuropathies comme l’autisme ou la maladie d’Alzheimer ou des troubles hématologiques et rénaux. En raison de l'hétéroplasmie, ces troubles peuvent apparaître avec une vaste gamme d'intensités, car l'ADNmt mutant nécessaire pour provoquer un trouble varie selon les organismes, les systèmes organiques et au sein d'un tissu donné, et dépend d'un équilibre délicat entre l'offre et la demande en ATP. Un autre type de problème survient au niveau des tissus soumis à une lésion liée à l'hypoxémie, où les mitochondries jouent un rôle important dans la survie et le rétablissement des cellules. Enfin, le rôle joué par les mitochondries dans la survie et le traitement du cancer est concentré dans de nombreuses recherches.Les troubles mitochondriaux semblent sous la forme d’une large gamme de signes cliniques. Dans la plupart des cas de maladies mitochondriales, le traitement symptomatique améliore légèrement la qualité de vie. La plupart des abords expérimentales cherchent à prévenir ces maladies en réduisant le pourcentage d'ADNmt mutant dans l'embryon par transfert nucléaire. Le transfert/transplantation artificiel mitochondrial (AMT/T) parait comme traitement alternatif. Nous montrons une méthode AMT/T -MitoCeption- dans un modèle cellulaire pour le traitement du trouble acquis de l'ADNmt défié par la radiation UV en usant des leucocytes plus la viabilité de Mitoception pour AMT/T sur des embryons murins. Ces résultats représentent une mise à niveau des applications AMT/T. Nous montrâmes que les leucocytes pouvaient être utilisées comme source de mitochondries et le transfert mitochondrial de multiples donneurs vers des cellules altérées. La MitoCeption usée sur des embryons semble être une méthode sûre pour la AMT/T en utilisant des mitochondries humaines. Des chiots murins saines furent obtenus, indiquant que les mitochondries humaines avaient été retirées des embryons pendant leur développementAccording to the endosymbiotic theory, mitochondria is an organelle derived from an ancient alpha-proteobacteria that developed a symbiosis with a eukaryotic ancestor. Mitochondrial DNA (mtDNA) exists in hundreds to thousands of copies in each cell and encodes for 13 structural proteins which are subunits of respiratory chain. Mitochondria generate energy for cellular processes by producing ATP through oxidative phosphorylation. Also, they control other processes as nucleotide and heme syntheses, redox balance, calcium metabolism, waste management (urea and ROS) and apoptosis. mtDNA deletions, point mutations, thymine dimers and mtDNA depletions are strongly related with disease in humans and other mammals. Some mtDNA alterations can arise spontaneously during life spam, other can be inherited by maternal lineage as specific mutations. So, nuclear DNA mutations can produce mitochondrial disorders because while mtDNA encodes 13 proteins, mitochondria need almost 2000 proteins with structural and functional roles. In these cases, a mendelian inheritance pattern can be observed. mtDNA alterations can be produced by exposure to toxic substances or UV and high-energy radiations. mtDNA mutations are cumulative because mitochondria lack reparative mechanisms. Normal and mutant mtDNA can coexist in the same cell, a condition known as heteroplasmy. Heteroplasmy allows the persistence of an otherwise lethal mutation through generations. Mitochondrial disorders can appear as myopathies, cardiomyopathies, lactic acidosis diabetes mellitus, female’s subfertility, lipodystrophy, neuropathies as autism or Alzheimer’s diseases or haematological and renal disorders. Due to heteroplasmy, these disorders can appear with a wide range of intensities, because the mutant mtDNA needed to cause a disorder varies among organisms, among organ systems and within a given tissue, and depends on a delicate balance between ATP supply and demand. Another kind of problem surges at tissues under hypoxemic-related damage, where mitochondria play an important role in cell survival and recovery. Finally, the role played by mitochondria in cancer survival and treatment is focused in many researches.Mitochondrial disorders have not a single treatment. In the most serious cases of inherited mitochondrial diseases, the supportive treatment only improves the life quality slightly. Nowadays, the most of experimental approaches search prevents the clinical manifestations of these diseases by reducing the mutant mtDNA percentage into the oocyte or the early embryo via nuclear transfer. Artificial Mitochondrial Transfer/Transplant (AMT/T) rises as an alternative to many acquired or inherited mitochondrial disorders, both ex vivo, in vitro and in vivo conditions. The present work shows the variation of an AMT/T method -MitoCeption- in a cellular model for in vitro treatment of acquired mtDNA disorder caused by UV Radiation by using Peripheral Blood Mononuclear Cells (PBMCs) and the feasibility of the same method for ex vivo AMT/T to murine oocytes and early embryos. In the in vitro model of cell damage by UV radiation, the main results represent an upgrading in the applications of AMT/T. We showed that PBMCs could be used as a primary allogeneic mixed source of mitochondria. We also showed that these mitochondria can be transferred in a mix from different donors (PAMM) to UVR-damaged, non-adherent primary cells. Additionally, the duration of the MitoCeption protocol was reduced. On the other hand, Mitoception used on murine oocytes and early embryos probed to be a safe method for AMT/T by using human mitochondrial mix (PAMM). Murine 0ocytes’ and embryos’ exogenous mitochondrial content was observed by fluorescence microscopy and exogenous mtDNA was quantified by qPCR and 2ΔCT method. Finally, healthy murine new-borns were obtained by embryo transfer, probing that human mitochondria were removed from murine cells during embryo’s development after implantation
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Rothe, Katherina. « Einfluss CD4+CD25+ regulatorischer T-Zellen auf die hämatopoetische Rekonstitution nach syngener und allogener Stammzelltransplantation in einem dreifach transgenen Mausmodell ». Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-68783.
Texte intégralRésumé :
Regulatorische CD4+CD25+ T-Zellen (Tregs) stellen eine kleine Zellpopulation dar (1-5% der peripheren Blutzellen), die hauptsächlich für die Regulierung von Immunreaktionen verantwortlich ist. In der vorliegenden Arbeit wurden diese Zellen gemeinsam mit Stammzellen syngen und allogen kotransplantiert, um ihren Effekt auf das Anwachsen der Spenderzellen und die Rekonstitution der Hämatopoese nach Ganzkörperbestrahlung zu untersuchen. Es wurden humanisierte dreifach transgene Empfängermäuse (C57Bl/6-TTG) verwendet (human CD4+, murin CD4-, human HLA-DR+), wodurch sowohl bei syngener als auch bei allogener Transplantation eine Unterscheidung zwischen Spender- und Empfängerzellen möglich ist. Zunächst wurden CD4+CD25+ T-Zellen durch Separation aus Milzzellen bzw. Buffy Coats gewonnen und in vitro mittels Durchflusszytometrie und ELISpot charakterisiert. Anschließend fanden syngene und allogene Transplantationen mit einer Laufzeit von 61 Tagen statt. Überleben und Gewicht wurden täglich ermittelt und außerdem wurden wöchentlich Blutbilder erstellt und durchflusszytometrische Chimärismusanalysen (murines und humanes CD4, CD8, MHC (H2Db, H2Kd)) durchgeführt. Durch die magnetische Separation konnte die FoxP3-Expression der murinen Zellen (Transplantat) von 1,6% in der Ausgangspopulation auf 68,5% in der CD4+CD25+ Population gesteigert werden. In den ELISpot-Assays zeigten diese separierten Zellen, wie für Tregs typisch, keine Produktion von Interleukin-2. Nach syngener Transplantation (Spender: wildtyp C57Bl/6) von 2x106 Knochenmarkzellen und 1x106 CD4+CD25+ T-Zellen überlebten 100% der Tiere, wie zu erwarten war. Dabei setzte bei Tregs-kotransplantierten Tieren die Blutbildung nach bestrahlungsbedingter Leukozytopenie aufgrund bisher nicht bekannter Mechanismen früher wieder ein und der Donor-Zell-Chimärismus war an Tag 19 nach Transplantation signifikant höher als in der Kontrollgruppe. Dies zeigt, dass regulatorische T-Zellen im syngenen Transplantationsmodell einen positiven Effekt auf die Akzeptanz bzw. das Anwachsen des Transplantats haben. Dieses Modell entspricht klinisch einer autologen Transplantation. Nach einer knochenmarkzerstörenden Therapie werden dem Patienten eigene Stammzellen reinfundiert, um die Blutbildung und das Immunsystem wieder in Gang zu bringen. Der Zusatz von regulatorischen T-Zellen zum autologen Stammzelltransplantat könnte das Anwachsen der Zellen beschleunigen und die gefährliche Phase der Immunsuppression, in der es häufig zu Sekundärinfektionen kommt, verkürzen. Die Transplantation der gleichen Zahl von allogenen Spenderzellen (wildtyp Balb/c) führte überraschend zum Tod aller dreifach transgenen Empfängertiere. Der Vergleich zu Experimenten mit wildtyp C57Bl/6-Empfängertieren zeigte, dass dreifach transgene Mäuse sehr viel höhere Zellzahlen im Transplantat zum Überleben benötigen (Daten nicht gezeigt). Das Ausbleiben der Blutbildung nach der Bestrahlung führte zu vermindertem Allgemeinbefinden, gestörter Futter- und Wassseraufnahme und Exsikkose bis zum Tod bzw. aus Tierschutzgründen zur Euthanasie. Durch Erhöhung der Zellzahl im Transplantat auf 1x107 Knochenmark + 5x106 Milzzellen überlebten 25% der Mäuse, bei 3x107 Knochenmark + 5x106 Milzzellen waren es 50%. Anders als im syngenen Modell führte die Kotransplantation 1,5x106 allogener CD4+CD25+ T-Zellen zu 3x107 Knochenmark + 5x106 Milzzellen zum Versterben der Tiere. Dies verdeutlicht, dass regulatorische T-Zellen in diesem allogenen Transplantationsmodell das Anwachsen des Transplantats behindern (Transplantatversagen). Hier gilt es zu klären, ob dieser Effekt spezifisch für die gewählten Mausstämme ist und welche Mechanismen für das Transplantatversagen verantwortlich sind. In einem dreifach transgenen Mausmodell konnte ein positiver Effekt von regulatorischen T-Zellen auf die Rekonstitution der Hämatopoese bei syngener Kotransplantation nachgewiesen werden. Im allogenen Transplantationsmodell hingegen führte die Kotransplantation CD4+CD25+ T-Zellen zum Versterben der Empfänger. Der beschriebene und schon publizierte positive Effekt spenderspezifischer Tregs zur Behandlung von Graft versus Host Disease nach allogener Stammzelltransplantation widerspricht diesen Ergebnissen nicht, da es bei diesen Patienten schon zum Engraftment von hämatopoetischen Stammzellen gekommen ist. Dies hat weitreichende Konsequenzen für die therapeutische Anwendung regulatorischer T-Zellen bei hämatologischen Erkrankungen in der Human- und VeterinärmedizinRegulatory CD4+CD25+ T cells (Tregs) represent a small cell population (1-5% of peripheral blood cells) mainly responsible for the regulation of the immune system. In the present work, these cells were cotransplanted with syngeneic and allogeneic stem cells in order to analyze the effect of Tregs on the reconstitution of hematopoiesis after total body irradiation. Humanized triple transgenic hosts (C57Bl/6-TTG) (human CD4+, murine CD4-, human HLA-DR+) were applied allowing differentiation of donor and host cells in syngeneic and allogeneic transplantation settings. Murine and human CD4+CD25+ T cells were magnetically separated out of splenocytes or buffy-coats and characterized in vitro by means of flow cytometry and ELISpot. Afterwards syngeneic and allogeneic transplantation experiments were performed for a period of 61 days. Survival and weight were assessed daily and once a week blood parameters and chimerism analyses (murine and human CD4, CD8, MHC (H2Db/ H2Kd)) were carried out. FoxP3 expression increased from 1,6% in the initial murine cell fraction to 68,5% in the separated CD4+CD25+ T cells. ELISpot assays showed the typical lack of interleukin 2 production of Tregs. After syngeneic transplantation (donor: wildtype C57Bl/6) of 2x106 bone marrow cells and 1x106 CD4+CD25+ T cells, 100% of mice survived what was to be expected. Cotransplanted animals showed earlier reconstitution of hematopoiesis after leukocytopenia and significant higher donor-cell-chimerism on day 19 after transplantation. The mechanisms for this positive effect of Tregs in syngeneic transplantation on the engraftment have to be investigated. This model clinically correspond an autologous transplantation where patients are treated with their own stem cells after a myeloablative treatment (chemotherapy or irradiation). The addition of regulatory T cells to the transplant could accelerate the engraftment and shorten the risky period of immunosuppression. Injection of the same numbers of allogeneic cells (donor: wildtype Balb/c) did not preserve hosts from mortality. Compared to experiments with wildtype recipients, results showed that triple transgenic mice need much higher cell numbers in the transplant for survival (data not shown). The failure of hematopoiesis after irradiation led to reduced general condition, disordered ingestion and exsikkosis leading to death respectively to euthanasia for reasons of protection of animals. By scaling up the cell number in the inoculum to 1x107 bone marrow cells + 5x106 splenocytes 25% of mice survived, with 3x107 bone marrow cells + 5x106 splenocytes survival was 50%. In contrast to syngeneic experiments, cotransplantation of 1,5x106 allogeneic CD4+CD25+ T cells and 3x107 bone marrow cells + 5x106 splenocytes did not prevent animals from mortality. In this allogeneic transplantation model Tregs restrain engraftment (graft failure). It has to be clarified if this effect is specific for the utilized mouse strains and which mechanisms are responsible for the graft failure. In the syngeneic triple transgenic mouse model cotransplantation of CD4+CD25+ T cells showed a positive effect on reconstitution of hematopoiesis after irradiation. In the allogeneic setting however cotransplantation of allogeneic regulatory T cells avoided the engraftment of transplanted cells. The described and published effect of donor-specific Tregs for treatment of graft versus host disease after allogeneic transplantation does not contradict the presented results because treated patients already possessed engrafted hematopoietic stem cells. The results have wide consequences for the therapeutic appliance of regulatory T cells in hematological diseases in human and veterinary medicine
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Monjezi, Razieh [Verfasser], et Michael [Gutachter] Hudecek. « Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing / Razieh Monjezi ; Gutachter : Michael Hudecek ». Würzburg : Universität Würzburg, 2018. http://d-nb.info/1162062231/34.
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Perche, Federico. « Transfert d'ARNm par des lipopolyplexes et vaccination antimélanome : ciblage des cellules dendritiques à l'aide de lipopolyplexes mannosylés ». Phd thesis, Université d'Orléans, 2010. http://tel.archives-ouvertes.fr/tel-00665118.
Texte intégralRésumé :
Précédemment, il a été démontré au laboratoire qu'une vaccination des souris avec des lipopolyplexes (LPR) contenant l'ARNm de l'antigène de mélanome MART1 permet d'induire la formation de lymphocytes T cytotoxiques spécifiques et de retarder le développement de mélanomes B16F10 et de métastases pulmonaires. Les LPR sont des complexes ternaires constitués d'ARNm, d'un polymère cationique histidylé et de liposomes cationiques histidylés. L'objectif de ma thèse était d'améliorer cette vaccination antitumorale en développant de nouveaux liposomes capables de cibler les cellules dendritiques (DC). Le ciblage a été réalisé en incorporant un glycolipide mannosylé aux liposomes afin de favoriser leur reconnaissance par le récepteur mannose. A partir de ces liposomes, des formulations de complexes ternaires à base d'ADN (LPD mannosylés ou Man11-LPD100) ou à base d'ARN (LPR mannosylés ou Man11- LPR100) ont été mis au point. Les résultats montrent que : in vitro les formulations Man11-LPD100 sont mieux internalisés et transfectent plus efficacement les DC que les LPD100 non mannosylés. Les formulations Man11-LPR100 transfectent avec une plus grande efficacité les DC par rapport aux Man11- LPD100. Par ailleurs, une forte réduction de la toxicité des formulations a été obtenue en dialysant les liposomes. Il est également possible de conserver les formulations sous forme déshydratée. Une imagerie par scintigraphie effectuée chez la souris a permis de constater que 9% des LPD sont captés dans la rate après une injection IV. Nous avons mis en évidence après un isolement de DC spléniques que les formulations Man11-LPR100 transfectent 4 fois plus de DC que les LPR non manosylés. Enfin, l'immunisation des souris avec Man11-LPR100 contenant l'ARNm MART1 permet une vaccination plus efficace contre la tumeur B16F10 et une meilleure survie. En conclusion, les LPR Man11-LPD100 sont de bons vecteurs pour cibler et transfecter les DC spléniques avec l'ARNm d'un antigène tumoral et pour induire la réponse immune contre les cellules tumorales.
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Kuruc, Jiří. « Podpora kvalitativních požadavků služeb v rádiových přístupových sítích vysokorychlostních variant mobilních sítí ». Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2009. http://www.nusl.cz/ntk/nusl-218103.
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The work deals with the developmental stages (Releases) of the third generation UMTS net-works. It explains a partial improvement from Release 99 to Release 5 and Release 6 to im-prove the quality parameters of a mobile data transmission. In the practical part, there are measured qualitative parameters of the third generation networks that are available in the Czech Republic: UMTS, HSDPA, CDMA 1xEV-DO Rev.0 and Rev.A and UMTS-TDD. The measurements give reasonably achievable data rates in both directions (downlink & uplunk), a response time, quality of VoIP calls, a dispersion, a data-loss and an ability to transmit video with different data flows. The other aspects especially an availability of coverage and end-user terminals have been assessed wihin the each network. The work is supplemeted with a laboratory, which is focused on a practical comparison of the basic parameters of data transmissions of 2G and 3G networks.
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Lederle, Alexandre. « Infection des cellules dendritiques plasmacytoïdes par le VIH : mécanisme d'inhibition par les anticorps et étude des modifications fonctionnelles ». Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00766677.
Texte intégralRésumé :
Les cellules dendritiques plasmacytoïdes (pDC) sont infectées par le VIH-1 et la diminution de leur nombre dans la circulation sanguine est corrélée avec la virémie des patients. Au cours de mes travaux de thèse, nous avons montré que les anticorps neutralisants (AcN) spécifiques du VIH-1 inhibent l'infection des pDC par des isolats primaires de VIH-1. Contrairement aux mDC, le mécanisme d'inhibition de l'infection des pDC est indépendant du RFcγII présent à leur surface. En parallèle, nos résultats indiquent que les pDC produisent de l'interféron-α et d'autres cytokines et chimiokines en réponse au VIH-1, même lorsque l'infection des cellules est inhibée par les AcN. Enfin, nous avons observé l'inhibition du transfert en cis et en trans du VIH-1 des pDC aux lymphocytes T CD4 par les AcN.Dans un contexte d'induction d'AcN par vaccination, l'inhibition de la réplication du VIH-1 dans les pDC associé au maintien de la sécrétion de cytokines pro-inflammatoire par ces cellules pourrait favoriser l'élimination du virus et ralentir sa dissémination dans l'organisme.
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Aganj, Ehsan. « Multi-view Reconstruction and Texturing=Reconstruction multi-vues et texturation ». Phd thesis, Ecole des Ponts ParisTech, 2009. http://pastel.archives-ouvertes.fr/pastel-00517742.
Texte intégralRésumé :
Dans cette thèse, nous étudions les problèmes de reconstruction statique et dynamique à partir de vues multiples et texturation, en s'appuyant sur des applications réelles et pratiques. Nous proposons trois méthodes de reconstruction destinées à l'estimation d'une représentation d'une scène statique/dynamique à partir d'un ensemble d'images/vidéos. Nous considérons ensuite le problème de texturation multi-vues en se concentrant sur la qualité visuelle de rendu..
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Terschlüsen, Joachim A. « Constructing and Commissioning HELIOS – A High Harmonic Generation Source for Pump-Probe Measurements with sub 50 fs Temporal Resolution : The Development of Experimental Equipment for Extreme Ultraviolet Spectroscopy ». Doctoral thesis, Uppsala universitet, Molekyl- och kondenserade materiens fysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-281298.
Texte intégralRésumé :
This thesis presents HELIOS, an in-house laboratory for time-resolved pump-probe spectroscopy with extreme-ultraviolet (XUV) probe radiation. A wide span of pump wavelengths can be generated using commercial laser equipment while XUV probe radiation is generated via a high harmonic generation process in a noble gas delivering probe photons with energies between 20 eV and 72 eV. The XUV beam path features a time-preserving monochromator and was constructed and built in-house. HELIOS features an overall time resolution of about 50 fs when using 800 nm pump and 41 eV probe photons. An energy resolution of 110 meV at 41 eV photon energy can be achieved. HELIOS features two beamlines. One µ-focus beamline with an XUV focal size of about 20 µm can be used with experiments that require such a small XUV focal size as well as with different end stations. The other beamline features a semi-permanently mounted end station for angle-resolved photoelectron spectroscopy under ultra-high vacuum conditions. Experiments demonstrating the usability of HELIOS and the two beamlines are presented. A pump-probe measurement on graphene demonstrates the capability of determining a large part of the k-space in only one measurement due to the use of an ARTOF angle-resolved time-of-flight electron spectrometer. A non-angle-resolved pump-probe measurement on the conducting polymer PCPDTBT demonstrates the high signal-to-noise ratio achievable at this beamline in non-angle-resolved photoelectron-spectroscopy pump-probe measurements. The usability of the µ-focus beamline is demonstrated with time-resolved measurements on magnetic samples employing an in-house-designed spectrometer. These experiments allow the retrieval of element-specific information on the magnetization within a sample employing the transversal magneto-optical Kerr effect (T-MOKE). Additionally, a Fourier transform spectrometer for the XUV is presented, the concept was tested at a synchrotron and it was used to determine the longitudinal coherence of the XUV radiation at HELIOS.
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Sommermeyer, Daniel [Verfasser]. « Generation of dual T cell receptor (TCR) T cells by TCR gene transfer for adoptive T cell therapy / Daniel Sommermeyer ». 2010. http://d-nb.info/1002067073/34.
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Wang, Fu-Hwei, et 王復輝. « Gene transfer into antigen-specific T cells induced by dendritic cell stimulation ». Thesis, 2001. http://ndltd.ncl.edu.tw/handle/23981444690757150066.
Texte intégralRésumé :
碩士東吳大學
微生物學系
89
The infusion of antigen-specific T lymphocytes is a potential therapy against certain cancers and viral diseases. To increase their effectiveness, we examined whether the combined use of retroviral vector, which only infects dividing cells, and in vitro sensitization of T cells with antigen-loaded dendritic cells (DCs) could selectively modify antigen- specific T cells, and whether the transfer of bcl-2 gene could enhance the survival of antigen-specific T cells. The surface and core antigens of hepatitis B virus (HBV) were used as model antigens. DCs transfected with HBV antigens stimulated autologous T cell proliferation. Importantly, these proliferating autologous T cells could be selsctively transduced with bcl-2-retroviruses. After being subjected to apoptotic death by growth factor withdrawal, bcl-2-transduced T cells displayed enhance survival. These survival T cells were demonstrated to contain integrated bcl-2 provirus and exhibited antigen-specific interferon-g secretion. Therefore, the combined use of retroviral vector and T cell activation by antigen-loaded dendritic cells may selectively modify antigen-specific T cells and bcl-2 gene transfer into antigen-specific T cells may enhance their survival.
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Seehar, Mehwish. « Adoptive cell transfer : examining the potential of a T cell-mediated therapy for metastatic melanoma ». Thesis, 2016. https://hdl.handle.net/2144/19481.
Texte intégralRésumé :
Adoptive cell transfer techniques identify and isolate patient anti-tumor lymphocytes in vitro followed by ex vivo expansion of these tumor specific T cells. Identification and isolation of lymphocytes from patient tumors allows for the selection of anti-tumor lymphocytes that are highly specific for individual tumor antigens. Furthermore, recombinant technology allows for engineering of chimeric antigen receptors (CARs) which allow these T cells to target multiple tumor antigens. Techniques involving ex vivo growth lead to a 1,000- to 5,000-fold increase in numbers of lymphocytes. Cultured lymphocytes can then be infused via IV and growth maintained with administration of exogenous IL-2. Cancer patients are then monitored for both immunological activity as well as any adverse cytokine reactions. We looked at several trial studies for the application of adoptive cell transfer in metastatic melanoma compare the efficacy of the regimen to other established melanoma treatments. Adoptive transfer has proven to be effective for patients with late stage melanoma, however, the aim of this study was to examine some of the challenges in creating an effective standard protocol for adaptation in clinical settings, including difficulty in obtaining significant cell populations from tumors, challenges in the proliferation of these tumor-infiltrating lymphocytes (TIL) and determination of antigen-specificity, i.e. facilitation of a simplified and quicker approach to the therapy.
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Jaleco, Sara Monteiro Primo. « Gene transfer and homeostasis of neonatal and adult T cell subsets ». Doctoral thesis, 2004. http://hdl.handle.net/10316/10151.
Texte intégralRésumé :
Tese de doutoramento em Biologia (Biologia Celular) apresentada à Fac. de Ciências e Tecnologia de CoimbraA utilização de células T geneticamente modificadas representa uma poderosa esperança para o tratamento de diversas doenças do foro hematológico. Os vectores virais derivados de ‘Murine Leukemia Virus’ (MuLV) podem ser utilizados para introduzir, de forma estável, material genético, uma vez que integram o genoma do hospedeiro. Contudo, o uso destes vectores é limitado às células em divisão; como a maioria dos linfócitos T se encontra em estado quiescente, estas células têm de ser activadas antes de serem sujeitas à introdução do gene mediada por um vector MuLV. Conseguido o estabelecimento prévio de condições optimizantes de transferência genética utilizando um vector deste tipo, aplicou-se esta estratégia à subpopulação T de células naïve, já que a sua transdução é essential para a maioria dos protocolos de terapia genética. Numa primeira fase avaliaram-se diferentes técnicas de isolamento de células T naïve, uma vez que a obtenção de populações puras e que não foram afectadas (ou apenas minimamente) pela técnica de purificação, é extremamente importante. Após conveniente purificação e estimulação de células T naïve de recém-nascido (e indivíduo adulto) através do seu receptor ‘T-Cell Receptor’ (TCR), alcançaram-se, com vectores derivados de MuLV, eficácias de transdução superiores a 50%. No entanto, aquele tipo de estimulação parece alterar a função das células T naïve, activando-as. Por esta razão averiguámos em seguida o efeito da citoquina IL-7 na transdução de células T de recém-nascido, uma vez que esta citoquina é um factor de sobrevivência das células T que parece não alterar significativamente o seu estado de activação. Observámos níveis de transdução de cerca de 20%. Embora encorajadores, estes resultados revelaram-se aquém dos obtidos após activação das células pelo TCR, o que nos conduziu, posteriormente, à utilização dum vector derivado de HIV-1, no qual tem sido notada a capacidade de infectar algumas células não proliferantes. Nestas condições observámos então, em subpopulações T (CD4+) estimuladas pela IL-7, que a população adulta de células-memória é significativamente mais susceptível à transdução com um vector derivado de HIV-1 do que as populações naïve, quer de recém-nascido, quer de indivíduo adulto. Neste contexto, verificámos que os linfócitos T de recém-nascido e adulto não respondem da mesma forma à IL-7: as células T naïve de recém-nascido efectuam várias divisões celulares enquanto que as subpopulações naïve e memória de origem adulta progridem no ciclo celular mas proliferam apenas minimamente. Estes resultados sugerem que o contexto celular global e não apenas a progressão no ciclo celular/proliferação per se, regula a transdução daquelas células por um vector derivado de HIV-1. Além disso, estes estudos indicam ainda um controle diferencial da homeostasia das diferentes subpopulações T pela IL-7. Consequentemente, o papel de determinadas citoquinas na regulação homeostática das células T de recém-nascido e adulto tornou-se particularmente relevante. Sendo asssim, procedemos ao estudo da função de IL-7 e IL-2, outra citoquina pleiotrópica para a célula T, na regulação do equilíbrio entre a proliferação e a morte celulares. Como resultado, observámos que a combinação de IL-2 e IL-7 induz nas células T adultas, quer naïve quer memória, um número mínimo de divisões celulares, enquanto que as células T naïve de recém-nascido efectuam muito mais divisões. No entanto, ambas as populações T naïve, quer de recém-nascido quer de adulto, são extremamente susceptíveis à apoptose mediada por Fas, após estimulação pelas IL-2/IL-7, fenómeno este que se apresentou muito menos pronunciado nas células T memória de adulto. Assim, podemos concluir que IL-2 e IL-7 regulam de forma distinta o equilíbrio homeostático entre a proliferação e a apoptose induzida por Fas, nas células T de recém-nascido e nas células T adultas, naïve e memória. O conjunto destes resultados constitui não só um utensílio indispensável para uma melhor compreensão do papel de IL-2 e IL-7 na biologia celular humana, mas também uma esperança para o aperfeiçoamento de técnicas de terapia celular que utilizam a célula T, nomeadamente em protocolos de transferência genética em subpopulações T, aplicáveis em estudos clínicos
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Vávrová, Kateřina. « Adoptivní transfer tumor-specifických lymfocytů v imunoterapii nádorových onemocnění ». Doctoral thesis, 2020. http://www.nusl.cz/ntk/nusl-435278.
Texte intégralRésumé :
Prostate cancer is the second leading cause of cancer death in men in Europe and the US. In the context of previous preclinical experiments and clinical studies there are certain assumptions predicating successful application of immunotherapy in the treatment of patients with prostate cancer. Promising results have been achieved by a combination of different treatment modalities which provide a synergistic antitumor effect. One of these combinatorial options is the use of antitumor vaccines and adoptive T cell transfer. The topic of this thesis is to provide a fresh insight into the past and current trends following the long-term candidate's department program in the field of anti-tumor immunotherapy. The experimental part of this thesis revolves around our own results published in this field. The introductory chapter delivers a basic overview of cellular mechanisms of anti-tumor immunity and the role of individual immune components in these processes. Following chapters are dedicated to current immunotherapeutic approaches with emphasis on the adoptive T cell transfer and implication of this technology in the treatment of prostate cancer. The results section describes the establishment of our protocol for adoptive T cell transfer as well as the protocol for ex vivo enrichment of human T cell...
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Lin, Regina. « Targeting T Cells for the Immune-Modulation of Human Diseases ». Diss., 2015. http://hdl.handle.net/10161/9824.
Texte intégralRésumé :
Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell-mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune-modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and preclinically characterizing their mechanisms of action and in vivo therapeutic efficacy.
CD8+ cytotoxic T cells have potent antitumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo-expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR-23a was upregulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired antitumor potential of patient CD8+ T cells. We determined that tumor-derived TGF-β directly suppresses CD8+ T cell immune function by elevating miR-23a and downregulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGFβ-mediated tumor immune-evasion pathway.
Having established that miR-23a-inhibition can enhance the quality and functional-resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers ‒ such as TGFβ ‒ within the tumor microenvironment. Since TGFβ poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune-competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wildtype and miR-23a-deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wildtype EGFRvIII-CAR T cells were vulnerable to functional suppression by TGFβ, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGFβ. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a-deficient EGFRvIII-CAR T cells for GBM immunotherapy.
Lastly, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune-suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory IFNγ and IL-17 production by fully-differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.
Dissertation
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Zysk, Aneta. « Adoptive transfer of ex vivo expanded gamma delta T cells targeting osteolytic cancer in the bone ». Thesis, 2017. http://hdl.handle.net/2440/119272.
Texte intégralRésumé :
Bone metastases occur in more than 75% of patients with advanced breast cancer. Cancer in bone is associated with bone destruction and is responsible for high levels of morbidity and mortality but is notoriously difficult to treat. Bone destruction is also the primary cause of morbidity in patients with primary bone cancer, such as osteosarcoma, with metastatic spread to the lungs correlating with poor survival. Therefore, clearly new therapies are desperately required to target cancers in the bone. This study explored the therapeutic potential of gamma delta (Vγ9Vδ2) T cell based adoptive transfer using animal models of osteolytic breast cancer and osteosarcoma. Cytotoxic Vγ9Vδ2 T cells were expanded ex vivo from peripheral blood using IL-2 and zoledronic acid (ZOL). In vitro, expanded Vγ9Vδ2 T cells were cytotoxic against a panel of breast cancer and osteosarcoma cell lines and pre-treatment with ZOL sensitised all cancer cells to rapid killing by Vγ9Vδ2 T cells. Adoptive transfer of fluorescently labelled ex vivo expanded Vγ9Vδ2 T cells into NOD/SCID mice localised to cancer lesions in bone. Multiple infusions of Vγ9Vδ2 T cells reduced breast cancer growth, but had no effect on osteosarcoma growth in the bone marrow. However, in both cases, ZOL pre-treatment potentiated the anti-cancer efficacy of Vγ9Vδ2 T cells in bone, protected the bone from cancer-induced osteolysis and decreased the incidence of pulmonary metastases. Collectively these studies suggest this treatment regimen to be an effective immunotherapeutic approach for the treatment of primary and metastatic bone cancers.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2017
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Gary, Regina [Verfasser]. « Characterization and functional analysis of the transfer of cell components from human antigen-presenting cells onto T cells via antigen-specific trogocytosis / vorgelegt von Regina Gary ». 2010. http://d-nb.info/101213069X/34.
Texte intégral
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Monjezi, Razieh. « Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing ». Doctoral thesis, 2018. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-152521.
Texte intégralRésumé :
The advances in genetic engineering have enabled us to confer T cells new desired functions or delete their specific undesired endogenous properties for improving their antitumor function. Due to their efficient gene delivery, viral vectors have been successfully used in T-cell engineering to provide gene transfer medicinal products for the treatment of human disease. One example is adoptive cell therapy with T cells that were genetically modified with gamma-retroviral and lentiviral (LV) delivery vectors to express a CD19-specific chimeric antigen receptor (CAR) for cancer treatment. This therapeutic approach has shown remarkable results against B-cell malignancies in pilot clinical trials. Consequently, there is a strong desire to make CAR T cell therapy scalable and globally available to patients. However, there are persistent concerns and limitations with the use of viral vectors for CAR T cell generation with regard to safety, cost and scale of vector production. In order to address these concerns, we aimed to improve non-viral gene transfer and genome editing tools as an effective, safe and broadly applicable alternative to viral delivery methods for T-cell engineering. In the first part of the study, we engineered CAR T cells through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles rather than conventional SB plasmids. This novel approach dramatically increased stable gene transfer rate and cell viability and resulted in higher yield of CAR+ T cells without the need of long ex vivo expansion to generate therapeutic doses of CAR+ T cells. Importantly, CD19-CAR T cells modified by MC-based SB transposition were equally effective as LV transduced CD19-CAR T cells in vitro and in a murine xenograft model (NSG/Raji-ffLuc), where a single administration of CD8+ and CD4+ CAR T cells led to complete eradication of lymphoma and memory formation of CAR T cells after lymphoma clearance. To characterize the biosafety profile of the CAR T cell products, we did the most comprehensive genomic insertion site analysis performed so far in T cells modified with SB. The data showed a close-to-random integration profile of the SB transposon with a higher number of insertions in genomic safe harbors compared to LV integrants. We developed a droplet digital PCR assay that enables rapid determination of CAR copy numbers for clinical applications. In the second part of the study, we ablated expression of PD-1, a checkpoint and negative regulator of T cell function to improve the therapeutic index of CAR T cells. This was accomplished using non-viral CRISPR/Cas9 via pre-assemble Cas9 protein and in vitro-transcribed sgRNA (Cas9 RNP). Finally, we combined our developed Cas9 RNP tool with CAR transposition from MC vectors into a single-step protocol and successfully generated PD-1 knockout CAR+ T cells. Based on the promising results achieved from antibody-mediated PD-1 blockade in the treatment of hematological and solid tumors, we are confident that PD-1 knockout CAR T cells enhance the potency of CAR T cell therapies for treatment of cancers without the side effects of antibody-based therapies. In conclusion, we provide a novel platform for virus-free genetic engineering of CAR T cells that can be broadly applied in T-cell cancer therapy. The high level of gene transfer rate and efficient genome editing, superior safety profile as well as ease-of-handling and production of non-viral MC vectors and Cas9 RNP position our developed non-viral strategies to become preferred approaches in advanced cellular and gene-therapyDie Fortschritte des genetischen Engineerings erlauben uns, T-Zellen neue, erwünschte Funktionen zu verleihen oder ihnen bestimmte, unerwünschte endogenen Eigenschaften zu nehmen, um ihre Antitumorfunktion zu verbessern. Aufgrund ihrer Effizienz im Gentransport, werden virale Vektoren für das TZellengineering verwendet, um gentransferierte, medizinische Produkte zur Behandlung humaner Krankheiten herzustellen. Ein Beispiel hierfür ist die adoptive Zelltherapie mit T-Zellen, die mit gamma-retroviralen und lentiviralen (LV) Vektoren genetisch modifiziert wurden, so dass sie einen CD19-spezifischen chimären Antigenrezeptor (CAR) exprimieren. In klinischen Pilotstudien zu B-Zellerkrankungen zeigte dieser therapeutische Ansatz bereits beachtliche Erfolge. Hieraus resultiert das Bestreben, die CAR-T-Zelltherapie für Patienten skalierbar und weltweit zugänglich zu machen. Aufgrund gesundheitlicher Risiken, finanzieller Kosten und dem Umfang der Vektorenproduktion bestehen jedoch anhaltende Bedenken und Grenzen bezüglich der Verwendung viraler Vektoren für die Herstellung von CAR-T-Zellen. Um diese Problematiken zu umgehen, beabsichtigten wir, den nicht-viralen Gentransfer sowie genomverändernde Techniken soweit zu verbessern, dass sie als eine effiziente, sichere und umfassend einsetzbare Alternative zum virusbasierten T-Zellengineering verwendet werden können. Im ersten Teil dieser Arbeit stellten wir durch die Sleeping Beauty (SB) Transposition von CAR-Genen auf minimalistischen DNA Vektoren (sogenannten Minicircles) CART-Zellen her. Die Minicircles wurden anstelle von konventionellen SB Plasmiden verwendet. Mithilfe dieser neuen Vorgehensweise wurden die Rate des stabilen Gentransfers sowie das Überleben der Zellen drastisch erhöht und führte zu einer gesteigerten Rate an CAR+ T-Zellen, ohne dass eine langwierige ex vivo Expansion zur Herstellung therapeutisch relevanter CAR-T-Zelldosen nötig wurde. CD19-CART-Zellen, die mit MC-basierter SB-Transposition modifiziert wurden, zeigten in vitro und in einem murinen Xenograftmodell (NSG/Raji-ffLuc) eine vergleichbar hohe Effizienz, wie LV-transduzierte CD19-CAR-T-Zellen. Hierbei genügte eine einzige Verabreichung von CD4+ und CD8+ CAR-T-Zellen für eine komplette Eliminierung des Lymphoms und der anschließenden Gedächtnisbildung von CAR-T-Zellen. Um die Biosicherheit der CAR-T-Zellprodukte zu charakterisieren, führten wir die bislang umfassendste vergleichende Analyse von Genominsertionsstellen nach SB-basierter Modifikation von T-Zellen durch. Im Vergleich zur LV Integration zeigten diese Daten ein beinahe zufälliges Integrationsmuster des SB Transposons mit höheren Integrationsraten in genomisch „sicheren Häfen“. Wir entwickelten eine Analyse basierend auf digitaler Tröpfchen-PCR, um eine rasche Ermittlung der Anzahl an CAR-Genkopien in klinischen Anwendungen zu ermöglichen. Im zweiten Teil der Arbeit verminderten wir die Expression von PD-1, einer Prüfstelle und negativen Regulator der T-Zellfunktion, um den therapeutischen Index der CART- Zellen zu verbessern. Dies wurde durch die Verwendung eines nicht-viralen CRISPR/Cas9, durch das Zusammensetzen von Cas9 Protein und in vitrotranskribierter sgRNA (Cas9 RNP), erzielt. Schließlich verwendeten wir unsere entwickelte Cas9 RNP-Technik in Kombination mit CAR-Transposition von MCVektoren, um PD-1-knock out, CAR-positive T-Zellen herzustellen. Da die antikörperbasierte PD-1-Blockade in der Behandlung hämatologischer und solider Tumore vielversprechende Ergebnisse zeigt, sind wir zuversichtlich, dass PD-1-knock out CAR-T-Zellen die Effizienz von CAR-T-Zelltherapien verschiedener Krebsarten verbessern können und dabei die Nebenwirkungen der antikörperbasierten Therapien umgehen. Wir zeigen in der vorliegenden Arbeit Möglichkeiten mit virusfreien, gentechnischen Methoden CAR-T-Zellen herzustellen, die in der T-Zellkrebstherapie umfassend Anwendung finden können. Das hohe Level der Gentransferraten und der effizienten Genomeditierung, ein zu bevorzugendes Sicherheitsprofil sowie die einfache Handhabung und Produktion nichtviraler MC-Vektoren und Cas9 RNP machen es möglich, dass unser neuentwickelter, nichtviraler Ansatz zu einer bevorzugten Herangehensweise in der künftigen Zell- und Gentherapie werden kann
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Wang, Ting. « Transfer of intracellular HIV Nef to endothelium causes endothelial dysfunction ». Thesis, 2014. http://hdl.handle.net/1805/5584.
Texte intégralRésumé :
Indiana University-Purdue University Indianapolis (IUPUI)With effective antiretroviral therapy (ART), cardiovascular diseases (CVD), are emerging as a major cause of morbidity and death in the aging population with HIV infection. Although this increase in CVD could be partially explained by the toxic effects of combined anti-retroviral therapy (ART), more recently, HIV infection has emerged as an independent risk factor for CVD. However, it is unclear how HIV can contribute to CVD in patients on ART, when viral titers are low or non-detectable. Here, we provide several lines of evidence that HIV-Nef, produced in infected cells even when virus production is halted by ART, can lead to endothelial activation and dysfunction, and thus may be involved in CVD. We demonstrate that HIV-infected T cell-induced endothelial cell activation requires direct contact as well as functional HIV-Nef. Nef protein from either HIV-infected or Nef-transfected T cells rapidly transfers to endothelial cells while inducing nanotube-like conduits connecting T cells to endothelial cells. This transfer or transfection of endothelial cells results in endothelial apoptosis, ROS generation and release of monocyte attractant protein-1 (MCP-1). A Nef SH3 binding site mutant abolishes Nef-induced apoptosis and ROS formation and reduces MCP-1 production in endothelial cells, suggesting that the Nef SH3 binding site is critical for Nef effects on endothelial cells. Nef induces apoptosis of endothelial cells through both NADPH oxidase- and ROS-dependent mechanisms, while Nef-induced MCP-1 production is NF-kB dependent. Importantly, Nef can be found in CD4 positive and bystander circulating blood cells in patients receiving virally suppressive ART, and in the endothelium of chimeric SIV-infected macaques. Together, these data indicate that Nef could exert pro-atherogenic effects on the endothelium even when HIV infection is controlled and that inhibition of Nef-associated pathways may be promising new therapeutic targets for reducing the risk for cardiovascular disease in the HIV-infected population.