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Hypermutation somatique dans les cellules B normales et pathologiques : éléments cis-régulateurs et facteurs nucléaires impliqués / Hypermutation in B cells : cis and trans regulatory elements involvedMartin, Ophélie Alyssa 03 October 2018 (has links)
En introduisant fréquemment des mutations ponctuelles dans les régions variables des gènes d'immunoglobulines (Ig), le processus d'hypermutation somatique (SHM, initié par la déaminase AID) est essentiel pour augmenter l'affinité des anticorps. En marge de ses cibles physiologiques (les gênes d'Ig), AID peut induire des "dommages collatéraux" au niveau de cibles "illégitimes" qui sont appelées "off targets" (dont certains oncogènes, tel que Bcl6 fréquemment muté dans les lymphomes B). Le risque élevé de dommages collatéraux dans le génome des cellules B implique que les remaniements géniques soient précisément surveillés. Parmi les éléments cis-régulateurs impliqués dans cette surveillance, on compte l'activateur cEμ au locus des chaînes lourdes des Ig (IgH) et ses régions flanquantes d'attachement à la matrice nucléaire MARsEμ (étudiés en détails dans nos modèles de souris KO). Nous montrons que la délétion des régions MARsEμ diminue non seulement les mutations au locus des chaines lourdes des Ig (effet physiologique en cis) mais également au locus des chaines légères Ig situé sur un chromosome différent (effet de trans). A l'aide d'une outil bioinformatique (DeMinEr) que nous avons développé dans le but d'identifier des mutations rares, nous montrons également que les régions MARsEμ sont impliquées dans les dommages collatéraux infligés aux "off targets" des cellules B. Grâce à la technique de FISH 3D, nous proposons que les régions MARsEμ participent à la régulation de la SHM en influençant la position des cibles de AID dans le noyau des cellules B. Notre étude met en évidence un niveau de régulation spatiale du processus de SHM médié par les régions MARsEμ du locus IgH. / By introducing frequent point mutations into the variable regions of immunoglobulin (Ig) genes, somatic hypermutation (SHM, initiated by the AID deaminase) is a driving force for antibody affinity maturation. It is now admitted that AID-induces mutations in germinal centre B cells could affect in parallel to their Ig genes physiological targets, illegitimates targets (including oncogenes) so calles "off targets" (such as Bcl6 with frequent point mutation in B lymphomas). The high risk of "collateral damage" in the B cell genome implies that remodeling events are precisely surveyed. Among cisregulatory elements involved (transcriptional enhancers and chromatin isolators and anchors...), one best candidate is the intronic region including the cEμ enhancer and iths flanking MARsEμ regions that we have been studying extensively using mouse KO model. We recently showed that MARsEμ deletion decreases SHM not only at Ig Heavy chain locus IgH (physiological cis effect) but surprisingly also at the Ig Light chain Kappa locus Ig, located on a different chromosome (trans effect). To extend the study of this intriguing trans effect, we developed a bioinformatic tool called DeMinEr that unveiled that MARsEμ regions were also involved in AID-induced collateral damages to "off-targets". Using FISH 3D, we show that MARsEμ regions harboured the potential not only to locally recruit SHM but also to cause dynamic changes of nuclear structures. The surprising cis and trans effect of MARsEμ deletion, impacting simultaneously nuclear positioning and SHM, revealed an additional level of regulation for targeting mutations to Ig and "off-targets" genes.
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Characterization of Novel Lymphoid-Associated Genes Identified by Gene-Trapping: a DissertationJames, Pamela 25 April 2006 (has links)
The discovery of novel genes involved in hematopoietic development and lymphoid function is necessary for the understanding of these systems. To this end, we utilized transmembrane protein-specific gene trapping in embryonic stem (ES) cells, a method of forward genetics, to identify a novel, complex locus from which several splice variants arise. The trapped locus identified in the KST30 ES cell clone encodes several genes including outer membrane protein 25 (OMP25) and activin receptor interacting protein (ARIP2) and two novel genes, AK74 and AK88. AK74 is highly conserved between human and mouse with 85% identity at the amino acid level. The human homolog was cloned from CD34+ cord blood hematopoietic stem cell progenitors (HSCPs) implying that it may have a role in the hematopoietic system.
We generated mice from the gene trapped ES cells, called KST30 mice, to analyze the expression pattern of transcripts from the trapped locus in the hematopoietic system. Utilizing the gene trap LacZ reporter and RT-PCR, we found that AK88 and AK74 are expressed in hematopoietic stem cells and thymocytes and that AK88 and ARIP2 are dramatically up-regulated in activated Band T lymphocytes. In addition, we found restricted expression of the gene trap in most non-lymphoid tissues.
Interestingly, the expression pattern of the gene trap coincides with the expression of activin signaling components in many cell types including thymocytes, activated B cells, hematopoietic stem cells and the ductal cells of the pancreas. AK74, AK88 and ARIP2 share two exons that encode a 44 amino acid region. ARIP2 negatively regulates activin signaling through endocytosis of Activin type II receptors. The N-terminal PDZ domain associates with ActRII and mediates endocytosis via association with RalBP1. The region of ARIP2 that associates with RalBP1 encompasses the 44 amino acid region also found in AK74 and AK88, suggesting that these proteins may also associate with RalBP1, perhaps sequestering it from ARIP2. This possibility combined with the similarities between gene trap expression and expression of the components of activin signaling indicates a role of the trapped genes in activin signaling.
AK74 and AK88 have a signal sequence and transmembrane domain that are predicted to direct them to mitochondria. To confirm this prediction, we examined the subcellular localization of AK74 and found that it localizes to a punctate, perinuclear structure identified as mitochondria using a mitochondria specific dye. AK74 was not seen in the cytoplasm, nucleus or at the plasma membrane of cells.
To determine the function of these novel genes, AK74 was retrovirally over-expressed in a double positive thymoma cell line and examined the global expression profile using Affymetrix gene chip. AK74 changed the expression levels of 36 genes greater than 3-fold compared to vector alone. Of these genes, several are involved in cytoskeletal rearrangement, apoptosis or are regulated by calcium signaling. Using yeast two-hybrid, several candidate binding partners for AK74 were identified, one of which is the receptor for activated protein kinase C (RACK1). RACK1 was also identified as a potential binding partner for AK88. RACK1 is a WD40 domain-containing scaffolding protein that has been implicated in many pathways but most prominently in the protein kinase C signaling pathway. Association with RACK1 by either AK74 or AK88 suggests that they may be involved in RACK1 function. Both RACK1 and PKC are involved with Ca2+ signaling through different mechanisms. This, combined with global gene expression changes in AK74 over-expressing cells suggests a role for AK74, AK88 or ARIP2 in Ca2+ signaling.
When we examined the expression of the trapped genes in mice homozygous for the gene-trapped allele (KST30tr/tr) we found that insertion of the gene trap caused a severe decrease in AK88 and ARIP2 but not AK74 transcripts. Analysis of KST30tr/tr mice showed no abnormalities in conventional lymphoid populations and precursors, however, intraepithelial lymphocyte (IEL) populations were altered by the loss of AK88 and/or ARIP2. There was an approximate 2-fold decrease CD8αα+ T cells in the small intestine while CD8αβ+ T cells were largely unaltered.
Using gene trap technology, we have identified two novel, mitochondria-localized proteins. The cumulative findings described in this thesis, including the homology between AK74, AK88 and ARIP2, their expression pattern and the phenotype of KST30tr/tr mice, suggest possible roles of AK74 and AK88 in diverse pathways.
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Tolerance Induction to a Foreign Protein Antigen: Analysing the Role of B Cells in Establishing Peripheral ToleranceYuschenkoff, Victoria Nicole 14 September 1995 (has links)
Tolerance to self proteins is largely dependent upon the deletion of immature, self-specific T and B cells in the thymus and bone marrow. Although highly efficient, the elimination of these self-reactive lymphocytes is dependent on the expression of their target antigen in these primary lymphoid organs. Many proteins, however, such as hormones, are developmentally regulated and expressed at different stages of life, while other proteins are expressed outside the thymus and marrow. To ensure self-tolerance, other mechanisms must exist to inactivate or prevent the activation of mature, potentially self-reactive lymphocytes and maintain peripheral tolerance.
T cell activation requires direct recognition of a specific protein fragment, presented on the surface of an antigen presenting cell (APC), as well as the interaction between various T cell and APC surface molecules. In the absence of the costimulatory signals provided by these ligand-pair interactions and lymphokines, antigen recognition leads to T cell inactivation and tolerance to the protein. Since many autoimmune disorders appear to be based upon the aberrant activation of mature T lymphocytes, it is important to identify and understand the mechanisms of peripheral tolerance.
The obvious importance of the APC in initiating the T cell immune response has led our lab to examine one of the many antigen-processing cells, the B lymphocyte. Our studies have shown that B cells are highly efficient APC and can present antigen at very low doses to cultured T cell lines. In addition, we have found that we can induce tolerance, as measured by a reduced antibody response to an immunogenic form of the protein, in naive, normal mice by targeting a foreign protein to their B cells for antigen processing and presentation. Tolerance in the treated mice can be traced to a lesion in the T cell compartment of the animals, thus suggesting that B cells can act as tolerizing APC for peripherally expressed antigens. To further explore this idea and find more direct evidence for the role of B cells in establishing peripheral tolerance, we developed a model system that would more closely resemble in vivo conditions.
This thesis tests and provides additional evidence for the hypothesis that B cells are tolerizing antigen presenting cells for peripherally expressed protein antigens. Tolerance to the foreign protein human μ chain, is induced in normal recipient mice by the transfusion of splenocytes from transgenic mice that express the membrane-bound form of μ on their B cells. Tolerance is antigen-specific since the transfused recipients' antibody production to the irrelevant protein chicken IgG is not compromised. Only viable transgenic spleen cells are tolerogenic and even when human μ chain is accessible to other APCs for presentation, tolerance can be induced by the transfusion of live μ transgenic splenoctyes. These data suggested that the transfused μ chain-expressing B cells are the tolerizing APCs which was confirmed by experiments that compared the tolerizing abilities of purified B and T cells from the transgenic mice. Adoptive transfer experiments showed that the recipients' T cell response to human μ was impaired but an analysis of the isotypes produced by tolerized mice did not indicate that either helper T cell subset was specifically compromised. Splenocytes from human μ chain-secreting transgenic B cells also induce tolerance to human μ in nontransgenic mice. Although human μ chain-expressing B cells were not detected in transfused mice, the presence of measurable levels of human IgM in the sera of mice transfused with μ chain-secreting spleen cells suggests that the transfused transgenic B cells persist in their new host. In addition, the tolerizing ability of both resting and activated membrane-bound μ chain B cells was compared. Lipopolysaccharide (LPS)-activated transgenic spleen cells do not tolerize, nor do they prime for antibody to human μ, thus suggesting that the induction of costimulatory molecules on the transgenic B cells inhibits tolerance induction. To more specifically address this, human μ chain-expressing mice were bred to transgenic mice that express the costimulatory molecule, B7-1 (CD80), on their B cells. Double transgenic splenocytes, in which the B cells bear both human μ and B7-1, did not induce tolerance to human μ chain, a result that supports the idea that activated B cells are not tolerogenic. Together the data in this thesis show that resting B cells can process and present a foreign endogenous antigen in a tolerogenic manner to the immune system and suggest a role for the B cell in the maintenance of peripheral tolerance.
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Mechanisms of Establishment and Maintenance of RNA Virus Persistence in Primary Lymphocytes: a DissertationCabatingan, Mark S. 17 July 2001 (has links)
RNA virus persistence in lymphocytes has been studied extensively in vitro, but the influence of lymphocyte homeostatic mechanisms and antiviral immunity on persistence has not been well studied in an in vivo system. It is demonstrated here that vesicular stomatitis virus (VSV), a negative-strand RNA virus, is maintained in B lymphocytes in vivo despite the existence of homeostatic mechanisms that drive the cells to proliferate under conditions of B cell deficiency and a strong antibody response to the virus. It is also shown that antiviral antibodies inhibit VSV reactivation from persistently infected primary B cells in vitro. A model is proposed for virus persistence in vivo in which B cell homeostatic signals drive virus expression in some infected cells, resulting in an antibody response, which maintains virus persistence in B cells.
In the course of conducting experiments to define the homeostatic signals that might act on persistently infected B cells in vivo, it was found that a fraction of small, resting splenic B cells proliferates after adoptive transfer into B cell deficient hosts (sublethally irradiated, xid, or SCID). This process, termed homeostatic proliferation, is driven by B cell deficiency since proliferation is limited in B cell sufficient hosts. This reveals the existence of a mechanism by which B cells sense their own numbers. The proliferation is unique in that the replicating cells do not upregulate cell surface markers, such as CD25 and B7-2, associated with antigen or mitogen induced proliferation. They do, however, show transient increases in other activation markers (CD69, CD71), demonstrating the action of an inductive signal. Homeostatic proliferation is a property of both mature and immature B cells, but in competition experiments, only mature B cells inhibit proliferation. xid B cells express a defective form of Bruton's tyrosine kinase (Btk); as a result, these cells proliferate poorly in response to stimulation through a number of cell surface receptors including the BCR, IL-5R, IL-10R, the toll-like receptor RP-105, and CD38. Homeostatic proliferation is severely reduced in xid B cells; thus, this process is regulated by a Btk-dependent inductive signal, which is counterbalanced by an inhibitory signal provided by mature B cells. B cell homeostatic proliferation does not rely on transcription factors (c-rel and p50) critical for conventional proliferation induced by antigen or mitogen (c-rel), or for peripheral B cell survival (p50), suggesting that multiple signals drive this process and that survival and proliferation signals are not identical.
VSV persists in small, resting primary B cells for several weeks in vitro, and virus replication is restricted at multiple levels depending on the activation state of the cells. After adoptive transfer of infected B cells into B cell deficient (xid) recipients, viral RNA, but not infectious particles, can be detected by RT-PCR in recipient spleens for at least 72 days. RT-PCR analysis of FACS sorted donor cells stained with CFSE reveals that viral RNA is maintained in transferred B cells but can also found in recipient cells. Infected B cells can undergo homeostatic proliferation and an antibody response is generated to the virus, suggesting that homeostatic signals induce virus expression in some transferred cells. Virus persistence is maintained despite an active immune response to the virus. In fact, persistence may be maintained by antiviral antibody since in vitro treatment of infected primary B cells with anti-VSV antibody inhibits virus reactivation at multiple levels (transcription, protein synthesis, assembly/release of infectious particles). This inhibition is reversible upon antibody removal, demonstrating that functional virus is maintained in antibody treated cells. Antibody specific for a single viral protein (VSV G) is sufficient since inhibition is mediated by monoclonal antibodies specific for a VSV G; neutralizing activity is not required because inhibition occurs with non-neutralizing monoclonal antibodies to VSV G. It is proposed that antibody binding to VSV G on infected B cells generates inhibitory signal(s) that suppress signaling pathways required for virus replication in B cells. Finally, a model of RNA virus persistence in B cells is proposed in which lymphocyte homeostatic signals promote virus expression, leading to the production of antiviral antibodies, which suppress virus replication inside infected B cells and help to maintain persistence.
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Die Wirkung des Chemokins "Pulmonary and activation-regulated chemokine" (PARC)auf B-CLL-Zellen und B-Zell-Linien, sowie Untersuchungen zur Expression und Signaltransduktion eines potentiellen PARC-RezeptorsStadler, Maike 27 October 2009 (has links)
Bis heute sind über 50 Chemokine und fast 20 Chemokinrezeptoren identifiziert. Dennoch gibt es Chemokine und Chemokinrezeptoren, deren zugehörige Rezeptoren bzw. Liganden noch nicht bekannt sind. PARC (=CCL18) ist ein ausschließlich in Primaten nachgewiesenes, bisher nur wenig charakterisiertes, im Organismus jedoch weit verbreitetes Chemokin, für das bisher noch kein Rezeptor beschrieben wurde. Die Wirkung dieses Chemokins wurde bisher vor allem an T Lymphozyten nachgewiesen. Die vorliegende Arbeit untersucht die Wirkung von PARC auf B-Lymphozyten und das Vorkommen des putativen PARC-Rezeptors DRY12. Dabei wurden B-Zellen von CLL Patienten sowie mehrere standardisierte B-Zelllinien als Untersuchungsgut verwendet. In funktionellen Assays (Kalziummobilisation, Aktinpolymerisation und Chemotaxis) wurde die Wirkung von PARC auf diese Zellen charakterisiert. Untersuchungen zu beteiligten Signalkaskaden wurden durch Einsatz von spezifischen Inhibitoren (Pertussis-Toxin) und mittels Western Blot durchgeführt. Weiterhin wurde das Vorkommen des putativen PARC-Rezeptors DRY12 bei den verschiedenen B Lymphozyten mittels Antikörperfärbung und RT-PCR sowohl auf Protein- als auch auf mRNA-Ebene nachgewiesen. Der Nachweis der genauen Lokalisation des Rezeptors in der Zelle erfolgte mittels Immunfluoreszenzcytologie. Abschließend wurde vergleichend das Vorkommen des DRY12 im Lymphknoten von CLL-Patienten und gesunden Spendern untersucht. PARC löst bei den B-Zellen der CLL-Patienten die Polymerisation von Aktin aus. Es induziert jedoch keine gerichtete Migration der Zellen. PARC wirkt auf die in dieser Arbeit untersuchten B-Zellen also nicht als Chemokin im klassischen Sinne. Seine Wirkung besteht möglicherweise in einem synergistischen Effekt, indem es im Zusammenspiel mit anderen Faktoren die Migration der Zellen beeinflusst. Weiterhin wäre denkbar, dass PARC das Verhalten von hämatopoetischen Stamm- und Vorläuferzellen beeinflusst. Die beteiligte Signalkaskade beinhaltet ein Pertussis-Toxin-sensitives Gi-Protein und die Aktivierung der p42/44-MAP Kinase. Ein intrazellulärer Einstrom von Ca2+ spielt bei der Wirkungsvermittlung von PARC keine Rolle. Der putative PARC-Rezeptor DRY12 konnte bei verschiedenen B-Zellen in unterschiedlicher Intensität nachgewiesen werden. Die Expression des DRY12 scheint sowohl auf Ebene der mRNA als auch auf Proteinebene durch multiple Faktoren reguliert zu sein. Dazu gehören z.B. der Reifungs- und Aktivierungszustand der Zellen oder die Kultivierungsdauer nach dem Auftauen der Zellen bis zur Durchführung des Versuchs. Bisher konnten jedoch keine entsprechenden Zusammenhänge nachgewiesen werden. Der DRY12 ist demnach kein konstitutiv exprimierter Rezeptor. Durch Immunfluoreszenzcytologie konnte die Lokalisation des Rezeptormoleküls auf der Zelloberfläche gezeigt werden. Im Lymphknoten wird DRY12 v.a. von Lymphozyten exprimiert. Bei Makrophagen konnte das Rezeptorprotein nicht nachgewiesen werden. In den Lymphknoten von CLL-Patienten exprimieren die Lymphozyten deutlich mehr DRY12 als Lymphozyten im Gewebe gesunder Individuen. Ein direkter Zusammenhang zwischen Rezeptorexpression und Reaktion auf PARC konnte nicht sicher aufgezeigt werden. Die Ergebnisse dieser Arbeit schließen aber auch nicht aus, dass PARC ein möglicher Bindungspartner von DRY12 ist. Bei der Wirkungsvermittlung spielen vermutlich auch andere Botenstoffe und weitere Faktoren eine Rolle, indem sie die Reaktionsfähigkeit der Zellen gegenüber PARC bzw. die Rezeptorexpression des DRY12 beeinflussen. Hinsichtlich der Frage, ob es sich bei DRY12 um einen Rezeptor für PARC handelt, kann diese Untersuchung zu keinem abschließenden Ergebnis gelangen, so dass dieser Aspekt in weiterführenden Analysen eingehender betrachtet werden sollte. / Today there are more than 50 chemokines and almost 20 chemokine receptors described. Despite growing knowledge, the ligands for some orphan chemokine receptors have not been identified and for several chemokines the receptor has not been discovered. PARC (=CCL18) is one of these chemokines for which the receptor has not been recognized. It has been detected in primates only and, despite being widely spread in the organism, it is still poorly characterized. Up to now, the effects of PARC were mainly shown on T-lymphocytes. Therefore, the objective of this study was to investigate the function of PARC and the expression of the putative PARC-receptor DRY12 in B-lymphocytes. For the purpose of the present study, B-CLL-cells and several lymphocytic B-cell-lines served as models to cover different stages of B-cell maturation. In order to characterize the effect of PARC, several functional assays (calciummobilisation, actinpolymerisation and chemotaxis), specific inhibitors (pertussis toxin) and Western Blotting were used. Expression analyses of the DRY12-receptor were performed by FACS-analysis, RT-PCR and immunofluorescence cytochemistry. In addition, lymph nodes from patients with CLL and healthy donors were stained immunohistochemically. In B-CLL-cells, PARC stimulation leads to phosphorylation of p42/44-MAP-Kinase and polymerization of actin, which can be inhibited by pertussis toxin, but does not induce calcium signaling or chemotactic migration. In this case, PARC is no classical chemokine but may act as synergist to potentiate the effect of other chemokines or may influence the behavior of hematopoetic stemm-cells. The results of the study show expression of the putative PARC-receptor DRY12 present on several subsets of B lymphocytes. As they showed different intensity of expression, DRY12 may be regulated by different factors in translation as well as transduction. Among these factors might be their current state of maturation and activation and the time period from revitalization to the start of the experiments. The reasons for these differences are still unknown. According to these findings, the receptor is not constitutively expressed, but may be itself regulated by several chemokines and other factors. DRY12 is located at the surface of the cell, as shown by immunocytochemistry. In lymph nodes, particularly lymphocytes but not macrophages express DRY12. In lymph nodes of CLL-patients lymphocytes express much more DRY12 than in healthy samples. However, it could not be proved that DRY12 is the agonistic receptor for PARC, as the expression of DRY12 did not completely correlate with the effects on PARC stimulation. But results of this study do not exclude this possibility either, as different factors are considered to influence the effect of PARC and the expression of DRY12 in B-cells. Although there are hints to it, from this study we can not conclude that DRY12 is the agonistic receptor for PARC. Therefore, further investigation is necessary to find the answer to this question.
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A Study of the Distal Molecular Mechanism by which Beta-2 Adrenergic Receptor Stimulation on a B Cell Regulates IgE ProductionPadro, Caroline Jeannette January 2013 (has links)
No description available.
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Role for cyclic adenosine monophosphate (cAMP) response element binding proteins in B lymphocyte development and functional maturationChen, Hui-Chen 17 October 2003 (has links)
No description available.
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Role of Ets-2 in lymphocyte development, function, and survivalFisher, Ian Bradford 22 December 2004 (has links)
No description available.
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New formats for affinity selection of human cellsSutar, Tina January 2015 (has links)
Despite recent advances in stem cell biology, immunotherapy and transplantation, substantial barriers still exist in the large-scale specific separation of a discrete population of human therapeutic cells from a cell suspension. The ideal purification technique should combine high cell purity, yield and function, with fast processing and affordability. Currently, fluorescence-activated cell sorting with flow cytometry (FACS) and magnetic activated cell sorting (MACS®) are the most used methods for cell separation and purification and have been employed extensively in molecular biology, diagnostic and cell sorting applications, because they are considered to be gentle, fast and scalable. However, these methods have several key disadvantages; they are invariably expensive, yield low log cell reduction (LCR) rates, and suffer from drawbacks when applied to niche cell populations, such as those requiring multiple tandem separation steps and/or involving combined positive and negative cell selection steps. To address this challenge, a new cell affinity selection system was developed. The selectivity is based on the reversible monomeric avidin biotin interaction and it is primary designed for positive selection. The initial studies were performed on flat, nonporous, glass coverslips and the technology was then successfully transferred on high grade smooth non-porous glass beads (with a diameter of 79.12 to 118.59 μm). The multi-step layer-by-layer deposition procedure culminating in dextran-coated supports bearing monomeric avidin was rigorously characterized and subsequently employed in packed bed chromatography experiments with human erythrocytes isolated from cord blood and B lymphocytes from cell lines. The developed affinity selection platform was highly selective, efficient and, most importantly, resulted in high yields, cell purity and viability comparable with MACS® technology. Additionally scale up is possible and could be easily transferred to another chromatographic matrix with the appropriate structure.
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Etude des mécanismes de régulation après injection de cellules dendritiques allogéniques OX62+ associées à un anticorps anti-CD4 non déplétant dans un modèle de rejet chronique chez le ratAlawieh, Mohamad 09 May 2012 (has links)
La manipulation du système immunitaire du receveur avant la transplantation pourrait permettre d’obtenir la tolérance de transplants allogéniques vascularisés en évitant la réaction de rejet à médiation lymphocytaire et humorale et l’utilisation permanente de traitement immunosuppresseur. Chez l’animal, l’injection au receveur, avant la transplantation, de cellules du donneur sous couvert d’anticorps bloquant le premier ou le second signal permet l’expansion périphérique d’une sous population de lymphocytes T CD4+CD25+FOXP3+, à activité suppressive (Treg).Dans ce travail de thèse, j’ai montré, dans un modèle murin utilisant des rats Fischer F344 comme donneur et des rats Lewis comme receveur, que l’injection au receveur avant la transplantation de cellules dendritiques spléniques OX62+ du donneur sous couvert d’anticorps monoclonal anti-CD4 W3/25 non dépletant permet d’obtenir la tolérance de transplants allogéniques cutanés et cardiaques à condition que ce protocole soit appliqué 28 jours avant la transplantation. La tolérance est due en partie à l’expansion de Treg inductibles spécifiques des alloantigènes. Elle n’est associée à aucune lésion d’artériopathie oblitérante observé au cours du rejet chronique de transplants vascularisés. Ces Treg ont aussi la capacité d’induire la tolérance de transplants allogéniques lorsqu’ils sont injectés au receveur la veille de la transplantation. Le blocage partiel de la production d’anticorps spécifiques du donneur chez les animaux tolérants suggère que les Treg interagissent avec les lymphocytes B en empêchant, soit la maturation des lymphocytes B en plasmocytes soit en favorisant l’expansion de lymphocytes B régulateurs. Ces résultats confirment que les Treg inductibles sont capables de prévenir les lésions vasculaires du rejet chronique. Ils soulignent également que les anticorps spécifiques du donneur apparaissant après la transplantation n’ont pas toujours un effet délétère. / The manipulation of recipient’s immune system before transplantation could lead to tolerance to allogenic vascularised transplants without lymphocyte and humoral-mediated rejection and the need for permanent immunosuppressive drugs. In rodents, the pre-transplantation injection into the recipient of donor cells under the cover of monoclonal antibodies blocking the primary or second signal allows the expansion of CD4+CD25+FOXP3+ T cells with suppressive activity (Treg) in the periphery.In this thesis, I demonstrated, in a rat model using Fischer F344 as donor and Lewis as recipients that the pre-transplantation injection into the recipient of donor-splenic OX62+ dendritic cells with anti-CD4 (W3/25) non-depleting monoclonal antibodies led to tolerance to skin and cardiac allogenic transplants provided that the protocol is done 28 days before transplantation. Tolerance is due in part to expansion of inducible Treg specific to allo-antigens. Sensitized Treg were also capable of inducing tolerance to allogenic transplants after adoptive transfer to the recipients, the day before transplantation. Tolerant grafts did not show any lesions of obliterant arteriopathy, usually associated with chronic rejection of vascularised transplants. Partial blockade of donor-specific antibody production (DSA) in tolerant animals suggests that Treg interact with B lymphocytes inhibiting the maturation of B cells in plasma cells, or favouring the expansion of regulatory B lymphocytes. These results confirm that inducible Treg are able to prevent vascular lesions of chronic rejection. They also underline the fact that DSA occurring after transplantation are not always deleterious to the graft.
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