Impact of the chromatin remodeller SMARCAD1 on murine intestinal intraepithelial lymphocyte and white adipose tissue biology. Chromatin remodelling factors use the energy of ATP hydrolysis to drive the movement of and/or affect molecular changes to the nucleosome. One such factor, SMARCAD1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1), has been previously shown to restore heterochromatin at the replication fork in vitro. This project aimed to assess the impact of SMARCAD1 on mammalian biology, utilising an animal model in which the catalytic ATPase domain of murine SMARCAD1 had been deleted using Cre/lox technology. Preliminary results had implicated SMARCAD1 in adaptive-immunity and white adipose tissue biology, and SMARCAD1 expression in these tissues/cells was confirmed by tissue-panel western blot. This project therefore aimed to build on these results to understand better the impact of SMARCAD1 on adaptive immune development and white adipose tissue biology. In addition, fewer than expected viable Smarcad1-/- homozygous offspring were produced during Smarcad1+/- x +/- matings, which both confirmed the observation from a previous knockout model of Smarcad1, and limited the number of knockout animals available for this study. Investigation of systemic B- and T-cells in the bone marrow, thymus and spleen had previously suggested there was no significant defect in adaptive immune development in Smarcad1-/- mice, however a tissue-specific and age-related loss of intra-epithelial (IEL) T-lymphocytes was found in the small intestine by flow cytometry. Analysis by qPCR of duodenal RNA suggested that differentiation rather than inflammation may underpin any loss-of-IEL phenotype, although further examination of cell-proliferation and crypt/villus anatomy by EdU incorporation and immunofluorescence revealed no overt cell-anatomical or proliferative difference in the knockout mice. The requirement for large numbers of aged mice made further investigation of the intestinal IEL phenotype logistically prohibitive. The reduction of epididymal white adipose tissue (eWAT) size had also been observed in male Smarcad1-/- mice, and serum from these mice showed elevated triglyceride (TG) and free fatty acids (FFA) levels. Transcriptomic analysis by RNA-seq of whole-WAT revealed an elevation in macrophage-related markers in knockout mice, which was confirmed by flow cytometry. As a number of reports have implicated SMARCAD1 in stem cell biology, putative adipose stem cells were isolated from +/+ and -/- mice by FACS and used for adipogenic differentiation assays ex-vivo In parallel, mouse embryonic fibroblasts from +/- and -/- mice were also assayed for adipogenic differentiation. While no significant differences in adipogenesis were observed, Smarcad1-/- mice challenged with a (60%) high fat diet did show increased weight gain over +/+ mice, and measurements of adipocyte size and cell cycle/cell proliferation analysis suggested hyperplasia rather than defects in adipogenesis may drive any WAT-related pathology in these mice.

Embora existam vários mecanismos propostos, o papel das moléculas de histocompatibilidade na susceptibilidade ao DM1 ainda não está totalmente esclarecido. Existem várias evidências de que o número de células apresentadoras de antígenos e a densidade de expressão das moléculas de histocompatibilidade nessas células pode influenciar o resultado da resposta imune. Assim, neste estudo, foram avaliadas as porcentagens das células CD3+, CD4+, CD8+, CD19+ e CD14+, coexpressando as moléculas de histocompatibilidade de classe I e II, a densidade de expressão das moléculas de histocompatibilidade de classe I e II nessas populações linfomonocitárias e a correlação entre densidade de expressão dessas moléculas com o perfil imunogenético do DM1. Para esse fim, foram avaliados 20 pacientes com DM1, recentemente diagnosticados, metabolicamente compensados, sendo 12 do sexo masculino. Como controles, foram avaliados 20 indivíduos saudáveis, pareados com os pacientes em termos de idade, sexo e raça, procedentes da mesma região geográfica dos pacientes. A densidade de expressão das moléculas HLA nas diversas subpopulações linfomonocitárias foi avaliada por citometria de fluxo. Os marcadores imunogenéticos foram tipados utilizando-se iniciadores de oligonucleotídeos seqüência específicos. Os resultados foram analisados usando o teste não paramétrico de Mann Whitney U. Foi observado aumento da densidade de expressão das moléculas de histocompatibilidade de classe I em linfócitos T CD3+, CD4+ e CD8+ de pacientes com DM1 quando comparados aos controles. Em relação às moléculas HLA de classe II, o número e a porcentagem dos linfócitos T CD4+, coexpressando as moléculas HLA-DQ de pacientes estavam diminuídos em relação aos controles. Os resultados referentes à correlação do perfil genotípico dos pacientes revelam que pacientes portadores dos alelos HLA-DQB1*02 apresentaram diminuição no número e porcentagem das células CD3+, CD4+, CD8+, CD19+ e CD14+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão da molécula HLA-DQ nas células CD19+, em relação aos pacientes sem esses alelos. Pacientes com o alelo HLA-DQB1*0302 apresentaram aumento do número de células CD14+ e CD19+ coexpressando as moléculas HLA-DQ, e ainda, o aumento da densidade de expressão dessas molécula nas células CD14+ em relação aos pacientes negativos para esse alelo. Além da instabilidade de ligação dos peptídeos com as moléculas de susceptibilidade ao DM1, este estudo reafirma a importância da densidade de expressão das moléculas de classe II na susceptibilidade ao DM1. / Althougth the role of MHC molecules in the susceptibility to DM1 has not been elucidated, the density of MHC molecules on cell surface may influence the outcome of the immune response. In this study, the number of CD3+, CD4+, CD8+, CD19+ and CD14+ cells coexpressing MHC class I and II, and the correlation between the density of MHC molecules and the immunogenetic profile of DM1 patients were studied. A total of 20 recently diagnosed patients (12 males) and 20 control individuals matched to the patients in terms of age, sex and race were studied. MHC molecules on cell sufaces were evaluated using flow cytometry. MHC aleles were typed using sequence specific probes. Statistical analysis was performed by the non-parametric Mann Whitney-U test. Increased expression of MHC class I molecules was observed in patients T CD3+, CD4+ and CD8+ cells in relation to controls. The number and porcentage of double-positive CD4+/HLA-DQ+ cells were significantly decreased in patients. Compared to DM1 patients who were not typed as HLA-DQB1*02, DM1 patients typed as HLA-DQB1*02 exhibited decreased numbers of CD3+, CD4+, CD8+, CD19+ and CD14+ cells expressing HLA-DQ molecules, whereas the density of HLA-molecules was increased in CD19+ cells. Compared to non-HLA-DQB1*0302 patients, those typed as HLA-DQB1*0302 presented increased number of CD14+ and CD19+ cells expressing HLA-DQ molecules. Besides the instability of peptide ligation with susceptibility molecules, this study stresses the relevance of the density of MHC class II molecules on the susceptibility to DM1.

Submitted to the faculty of the Bioinformatics Graduate Program in partial fulfillment of the requirements for the degree Master of Science in the School of Informatics, Indiana University September 2005 / Study of the transcriptional regulation of lymphocyte development has advanced greatly in the past 15 years. Owing to improved techniques and intense interest in the topic, a great many interactions between transcription factors and their target genes have been described. For these B and T cells, a more clear picture is beginning to emerge of how they start with a common progenitor cell, and progressively restrict potential to give many different types of terminally differentiated cells. As B and T cells develop they both follow a roughly similar path that involves early stepwise progression to later stages where multiple developmental options are available. To progress in the developmental regime they share requirements for proper anatomical location and successful rearrangements of the germ line DNA to give the plethora of antibodies and T cell receptors seen in the immune system. Because the amount of information is quickly becoming more than can be assimilated by researchers, a knowledge gap has opened between what is known about the transcription factor activities during this process and what any one individual can recall. To help fill this gap, we have created the LymphTF Database. This database holds interactions between individual transcription factors and their specific targets at a given developmental time. It is our hope that storing the interactions in developmental time will allow for elucidation of regulatory networks which guide the process. Work for this project also included construction of a custom data entry web page that automates many tasks associated with populating the database tables. These tables have also been related in multiple ways to allow for storage of incomplete information on transcription factor activity. This is done without having to replace existing records as details become available. The LymphTF DB is a relational MySQL database which can be accessed freely on the web at http://www.iupui.edu/~tfinterx/.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19222号 / 医博第4021号 / 新制||医||1010(附属図書館) / 32221 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 長澤 丘司, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les lymphocytes T de type régulateur sont engagés dans la régulation de plusieurs réponses immunes physiologiques et pathologiques. Certains d’entre eux, comme les lymphocytes T régulateurs (Treg) et invariants Natural Killer T (iNKT), sont essentiels pour le maintien de la tolérance périphérique et la prévention de l’auto-immunité. Les patients atteints de la maladie de Graves ont des auto-anticorps activant le récepteur de la TSH exprimé sur les cellules de la glande thyroïde et sur les fibroblastes de l’orbite. En conséquence, les deux principales manifestations cliniques de la maladie sont une hyperthyroïdie ainsi qu’une orbitopathie. Lorsque l’hyperthyroïdie est traitée par l’iode radioactif (IR), une apparition ou une exacerbation de l’atteinte auto-immune ophtalmique peut survenir. L’irradiation de la glande cause une augmentation et une persistance temporelle des auto-anticorps dans la circulation sanguine qui ne sont pas observées suivant d’autres traitements de l’hyperthyroïdie tels que les médicaments antithyroïdiens. L’hypothèse proposée était que l’amplification de l’auto-immunité survenant après le traitement à l’IR résultait d’une perte de régulation des cellules T autoréactives par les lymphocytes T de type régulateur, puisque ceux-ci ont un rôle déterminant dans le contrôle de l’auto-immunité. L’objectif du projet de recherche était donc d’étudier les effets du traitement à l’IR sur les lymphocytes Treg et les lymphocytes iNKT des patients atteints de la maladie de Graves. Nous avons démontré que le traitement à l’IR induit un changement de la fréquence périphérique des lymphocytes Treg et des lymphocytes iNKT, ainsi que de la fonction des lymphocytes Treg. En effet, nous avons démontré une réduction de la fonction suppressive de ces lymphocytes, et ce, accompagnée d’une diminution de la production d’IL-10. Ainsi, la fenêtre temporelle suivant le traitement à l’IR s’est avérée un moment critique pour étudier une altération de ces lymphocytes qui corroborait l’amplification de l’auto-immunité décrite après le traitement. De plus, nous avons démontré qu’un prétraitement avec un antithyroïdien de synthèse prévient partiellement les altérations observées chez les lymphocytes Treg, supportant la propriété immunomodulatrice de ces médicaments. Ces résultats accentuent la perspective d’une régulation immunitaire déficiente suivant certains traitements de l’hyperthyroïdie auto-immune.

La reconnaissance des antigènes du soi et du non soi par les lymphocytes B et T est à l'origine des maladies auto-immunes et inflammatoires. Les anticorps et les auto-anticorps produits par les plasmocytes dérivés des lymphocytes B sont utiles à des fins diagnostiques, mais contribuent également à la physiopathologie de ces maladies. La déplétion des lymphocytes B au cours des maladies auto-immunes chez l'homme a recentré l'attention sur les rôles des lymphocytes B comme modulateurs de la fonction des lymphocytes T, en plus de leur rôle de cellules productrices d'anticorps. L'utilisation d'anticorps anti-CD20 chez la souris révèle le rôle fondamental des lymphocytes B comme adjuvants cellulaires pour l'activation des lymphocytes T CD4+ in vivo (lymphocytes B effecteurs) mais également le rôle régulateur inattendu d'une sous-population de lymphocytes B spléniques CD1dhighCD5+ qui produit de l'interleukine 10 (lymphocytes B10). Le rôle de ces lymphocytes B effecteurs est montré dans un modèle chez la souris NOD (Non Obese Diabetic) alors que le rôle des lymphocytes B régulateurs est montré dans un modèle murin d'eczéma de contact. Au cours de l'encéphalite auto-immune expérimentale, la déplétion précoce des lymphocytes B aggrave les signes neurologiques tandis que la déplétion tardive des lymphocytes B les améliore, reflétant la dualité fonctionnelle des lymphocytes B au cours des maladies inflammatoires / Autoimmunity results from abnormal B and T cell recognition of self antigens, which leads to autoantibody production in many cases. Autoantibodies produced by B cell-derived plasma cells provide diagnostic markers for autoimmunity, but also contribute significantly to disease pathogenesis. The therapeutic benefit of depleting B cells in mice and humans has refocused attention on B cells and their role in autoimmunity beyond autoantibody production. B cells specifically serveas cellular adjuvants for CD4+ T cell activation, while regulatory B cells, including those that produce IL-10 (B10 cells), function as negative regulators of inflammatory immune responses. The role of effector B cells is shown in the non obese diabetic mouse model (NOD) whereas the role of regulatory B cells is demonstrated in a contact dermatitis model. During experimental autoimmune encephalomyelitis, early B cell depletion makes the disease worst whereas B cell depletion after disease onset improves clinical symptoms, reflecting counter regulatory B cell functions during autoimmunity

L’efficacité de la réponse immunitaire adaptative repose tout particulièrement sur la motilité des lymphocytes T naïfs entre la circulation sanguine et les organes lymphoïdes secondaires, leur permettant ainsi de rencontrer un antigène spécifique. De nombreuses voies de signalisation sont impliquées dans ce phénomène. En particulier, les Rho GTPases y jouent un rôle central, par leur capacité à moduler le cytosquelette d’actine. Nous avons identifié la protéine Fam65b comme nouveau régulateur de la circulation des lymphocytes T. En effet, nous avons montré que la diminution de l’expression de Fam65b dans des LT primaires humains induit une augmentation de leur polarisation, leur adhésion et leur migration in vitro. Afin d’étudier son rôle dans un contexte plus physiologique, nous avons développé au laboratoire une souris Fam65b-/-, dans laquelle l’expression de Fam65b est supprimée dans le lignage T. Les lymphocytes T issus de ces souris présentent un contenu global en F-actine réduit, une plus grande quantité de L-sélectine et d’intégrines actives à leur surface, et une migration moins rapide et moins rectiligne que leurs équivalents WT. Nous n’avons pu observer, avec nos méthodes, aucune différence significative de polarisation, de migration in vitro ou d’entrée dans les organes lymphoïdes secondaires pour les LT Fam65b-/-. Nous avons identifié les Rho GTPases comme médiateurs de ces effets de Fam65b. Nous avons observé, en cytométrie de flux, que les niveaux de RhoA-GTP et de Rac-GTP sont plus élevés dans les LT murins Fam65b-/-, et que cela est aussi vrai pour RhoA-GTP dans les LT humains exprimant de faibles niveaux de Fam65b. Nous avons identifié, dans des expériences in vitro, le mécanisme par lequel Fam65b inhibe l’activité de RhoA, puisqu’il ralentit sa charge en GTP par les protéines GEF. Nous avons montré, par des techniques de biochimie, que l’activation de RhoA en aval d’une stimulation chimiokine est permise par la dissociation de RhoA et de Fam65b, probable conséquence de la phosphorylation de Fam65b. Cette dissociation a aussi été observée pour Fam65b et Rac1, mais les mécanismes mis en jeu restent à déterminer. D’autre part, l’expression de Fam65b est sous le contrôle du facteur de transcription FOXO1, connu pour son rôle dans le contrôle de l’écotaxie (homing) via la régulation de l’expression de molécules permettant l’entrée dans les ganglions lymphatiques. Fam65b, régulateur atypique de l’activité des Rho GTPases, représente donc un lien inédit entre la voie PI3K/FOXO1 et les Rho GTPases. / The motility of naive T lymphocytes between the blood and secondary lymphoid organs is essential to the efficiency of the adaptative immune response, and allows those cells to meet their cognate antigen. Numerous signaling pathways are involved in this phenomenon, such as Rho GTPases, modulators of the actin cytoskeleton. We have identified Fam65b as a new regulator of T lymphocytes recirculation. We have shown that a decrease of Fam65b expression in human primary T cells increases the morphological polarization, the adhesion and the in vitro migration of those cells. Looking for a more physiological model, we developed, in the lab, a Fam65b KO (Knock-Out) mouse, specific to the T lineage. In those animals, T cells showed decreased levels of F-actin, an increase in the display of L-selectin and integrins, and a slower and less straight migration, compared to WT (Wild-Type) T cells. On the other hand, we weren't able to see any significant differences in the morphological polarisation, the in vitro migration or the homing capacity of the Fam65b KO T cells. We have identified Rho GTPases as mediators of the effects of Fam65b. We showed, in flow cytometry, that the amount of RhoA-GTP and Rac-GTP are increased in the Fam65b KO cells. The RhoA-GTP levels are also increased in human primary T cells expressing low levels of Fam65b. We have identified, in in vitro experiments, that Fam65b slows down RhoA loading with GTP by its GEF proteins, thus inhibiting RhoA activity. Moreover, we showed that Fam65b dissociates from RhoA after chemokine stimulation of T cells, thus allowing RhoA activation. The phosphorylation of Fam65b is a probable cause to this phenomenon. Fam65b also dissociates from Rac1 in these conditions, although no mechanism is yet known. Furthermore, the transcription factor FOXO1 controls the expression of Fam65b. FOXO1 is also known to control the homing capacity of T cells, since it controls the expression of molecules involved in the entry of lymphocytes in the lymph nodes. Fam65b, an atypical regulator of Rho GTPases activity, thus represents a new connection between the PI3K/FOXO1 and the Rho GTPases pathways.

Dentre as enfermidades que causam alterações hematológicas em bovinos podemos citar a leucose enzoótica bovina (LEB). Podendo em alguns animais, determinar formações tumorais por infiltração de células mononucleares (linfócitos, pró-linfócitos e linfócitos atípicos), em diferentes tecidos. A doença possui quadro sintomático pleomórfico e alterações hematológicas, evidenciadas por leucocitose e linfocitose persistente, com aumento de formas linfocitárias atípicas. Desta forma o presente estudo tem como objetivo avaliar a atipia linfocitária em bovinos da raça Holandesa Preto e Branco em animais soropositivos com ou sem a presença de linfocitose persistente e animais soronegativos para LEB e correlacioná-la com a apoptose de células CD5+ e a proliferação linfocitária. Assim, foram selecionados 56 animais, divididos de acordo com o leucograma e o sorodiagnóstico da LEB pela imunodifusão em ágar gel e pelo ensaio imunoenzimático em: negativos (n = 25), alinfocitóticos (AL, n= 12) e com linfocitose persistente (LP, n=19). Destes, 15 animais tiveram suas amostras sanguíneas avaliadas quanto a proliferação de linfócitos e apoptose. Os resultados deste estudo mostraram que, a contagem de leucócitos totais e os valores absolutos e relativos de linfócitos atípicos foram maiores no grupo LP. Em relação aos linfócitos atípicos, os linfócitos sombra de Gümprecht, linfócito monocitóide e linfócito com núcleo duplo, foram encontrados nos três grupos de estudo, porém mais comumente em animais infectados pelo VLEB, predominantemente no grupo manifestando linfocitose persistente. Este trabalho demonstrou uma menor proliferação de linfócitos nos animais infectados pelo VLEB manifestando LP, associado a uma menor apoptose de células CD5+. Na análise da correlação entre a apoptose de células CD5+ e a porcentagem das populações linfocitárias entre os 15 animais, observou-se que a apoptose tem uma correlação positiva com os linfócitos com núcleo duplo, o que não ocorreu nas demais populações celulares. Na correlação entre a proliferação, foi observado nos 15 animais, que quanto maior a porcentagem de linfócitos menor é a proliferação e quanto maior a porcentagem de linfócitos monocitóides menor a proliferação, o que não ocorreu nas demais populações celulares. Portanto, a manifestação da LP em animais naturalmente infectados pelo VLEB, pode ser associada ao aumento de linfócitos atípicos do tipo sombra de Gümprecht, linfócito monocitóide e linfócito núcleo duplo e da menor apoptose das células CD5 + e da menor proliferação de linfócitos / Among the diseases that cause hematological changes in cattle can cite the enzootic bovine leukemia (EBL). And in some animals, to determine tumor formations by infiltration of mononuclear cells (lymphocytes, pro-lymphocytes and atypical lymphocytes) in different tissues. The disease has symptoms very pleomorphic and hematological changes, evidenced by leukocytosis, and persistent lymphocytosis, an increase of atypical lymphocyte forms. Thus this study aims to evaluate the atypical lymphocyte in Holstein Black and White in seropositive animals with or without the presence of persistent lymphocytosis and animals seronegative LEB and correlate them with apoptosis of cells CD5 + and lymphocyte proliferation. Thus, we selected 56 animals, divided according to the WBC and serodiagnosis of LEB by agar gel immunodiffusion and by enzyme immunoassay in: negative (n = 25), non- lymphocytic (AL, n = 12) and with persistent lymphocytosis (PL, n = 19). Of these, 15 animals had their blood samples evaluated for lymphocyte proliferation and apoptosis. The results of this study showed that in total leukocytes and absolute and relative values of atypical lymphocytes were higher in group LP. Regarding atypical lymphocytes, lymphocytes Gumprecht shadow, lymphocyte and monocytoid lymphocytes with dual core, were found in the three study groups, but more commonly in animals infected VLEB, predominantly in the group with persistent lymphocytosis. This study demonstrated a reduced proliferation of lymphocytes in animals infected VLEB expressing LP, associated with lower apoptosis of CD5 + cells. In analyzing the correlation between apoptosis of cells and the percentage of CD5 + lymphocyte populations among the 15 animals, we observed that apoptosis has a positive correlation with lymphocytes with dual core, which did not occur in other cell populations. Correlation between proliferation, was observed in 15 animals, the higher the percentage of lymphocyte proliferation and lower the higher the percentage of monocytoid lymphocytes less proliferation, which did not occur in other cell populations. Therefore, the expression of LP in animals naturally infected VLEB may be associated with increased atypical lymphocytes of the type of shadow Gumprecht, lymphocyte and lymphocyte monocytoid dual-core and reduced apoptosis of CD5 + cells and reduced lymphocyte proliferation

Le syndrome WHIM (SW) est un déficit immuno-hématologique rare causé principalement par des mutations autosomales dominantes du gène CXCR4 qui entrainent une troncation du domaine C-terminal (C-Ter) du récepteur. Les formes mutantes de CXCR4 associées au SW génèrent des altérations de la désensibilisation et de l’internalisation du récepteur en réponse à CXCL12, qui se traduisent par une hypersensibilité à l’action chimiotactique du ligand. CXCR4 est un récepteur de chimiokine exprimé sur les leucocytes dont le rôle dans l’hématopoïèse et le trafic leucocytaire à l’état basal suggère que la lympho-neutropénie des patients atteints du SW est due à des défauts de production et/ou de domiciliation périphérique des leucocytes causés par le gain de fonction de CXCR4. Néanmoins, la validation de cette hypothèse est difficile chez les patients. En générant une souche de souris (Cxcr4+/1013), porteuse d’une mutation rapportée chez une famille de patients par une stratégie de knock-in, nous avons mis en évidence le rôle du domaine C-Ter de Cxcr4 dans le développement, la domiciliation périphérique des lymphocytes et l’immunité adaptative à médiation humorale.Les principaux résultats issus de notre travail, obtenus en combinant des approches biochimiques, fonctionnelles, de reconstitution de l’hématopoïèse par compétition, de transferts adoptifs et d’injection d’anticorps anti-CD45 in vivo, sont : 1) La mutation Cxcr41013 tronquant le domaine C-Ter se comporte différemment en terme de signalisation, selon qu’elle soit présente à l’état hétérozygote ou homozygote, et perturbe respectivement les transitions double-négatif (DN) 2-DN3 et proB-preB de la lymphopoïèse dans le thymus et la moelle osseuse (MO). Au contraire, elle ne génère pas d’effets sur le développement des cellules NK et la myélopoïèse ; 2) La lymphopénie qui touche les lymphocytes B (LB) et T (LT) est un processus intrinsèque aux cellules porteuses de la mutation Cxcr41013 et suit un modèle allèle-dose-dépendant ; 3) Le défaut de désensibilisation de Cxcr41013 empêche le relargage des lymphocytes NK et B immatures de la MO et celui des LB et LT matures des ganglions lymphatiques dans le sang. A l’inverse, le gain de fonction exacerbe la migration des LB recirculants et LT matures et leur rétention dans le parenchyme médullaire ; et 4) malgré l’absence de follicules primaires dans les ganglions lymphatiques, les souris mutantes sont capables de mettre en place une réponse immunitaire humorale efficace et spécifique d’un antigène T-dépendant, comme en témoigne l’augmentation des LB du centre germinatif et des plasmocytes ayant effectué une commutation isotypique. En conclusion, nous démontrons que la signalisation fine médiée par Cxcr4 est nécessaire pour le développement, la compartimentalisation périphérique et la fonction des lymphocytes. / The WHIM Syndrome (WS) is a rare combined immuno-hematological disorder caused by inherited heterozygous autosomal dominant mutations in CXCR4, which result in most cases in the distal truncation of the receptor’s Carboxyl-terminal tail (C-Tail). Mutants of CXCR4 associated with WS display impaired desensitization and internalization of the receptor upon CXCL12 exposure, leading to enhanced migratory response. Because CXCR4 is expressed on leukocytes, we hypothesized that circulating pan-leukopenia could arise from altered CXCR4-mediated signalling that would skew tissue distribution and differentiation of leukocytes. This assumption was obviously difficult to address in patients. By generating a knock-in mouse strain (Cxcr4+/1013) that harbors a WS-linked gain-of-Cxcr4-function mutation, we establish that the C-tail domain in Cxcr4-mediated signalling is a pivotal regulator of lymphocyte development, peripheral trafficking and humoral immunity. The essential findings of our work, obtained by combining biochemical, bone marrow (BM)-mixed chimeras, in vivo labelling, adoptive co-transfers and functional approaches, are: 1) the C-tail truncating Cxcr41013 mutation caused differential signalling capacities depending on its heterozygous versus homozygous status and inhibited double-negative (DN) 2-to-DN3 and pro-B-to-pre-B developmental transitions during lymphopoiesis. In contrast, it had no effect on NK lymphopoiesis and granulopoiesis; 2) the resulting circulating B and T lymphopenia was due to hematopoietic cell-intrinsic defects and followed a mutated allele dose-dependent pattern; 3) impaired Cxcr41013 desensitization prevented the release of immature BM NK and B cells and mature lymph node (LN) B and T lymphocytes into the blood. Conversely, it forced homing and retention of mature recirculating B and T cells in the BM parenchyma; and 4) despite the absence of primary B-cell follicles in LNs, mutant mice produced efficient humoral responses upon immunization as illustrated by increased antigen-specific germinal center B cells and isotype-switched plasma cells. Collectively, our findings demonstrate that fine-tuning of Cxcr4 signal strength is required for optimal trafficking, egress and fitness of lymphocytes.

L’ATP extracellulaire, à travers l’activation du récepteur P2X7, joue un rôle important dans l’immunité inné comme signal de danger responsable de l’assemblage de l’inflammasome, de la migration des cellules immunitaires et de la mort cellulaire. Bien que le rôle de la voie ATP/P2X7 dans l’immunité adaptative reste sous-estimé, il a été rapporté que le récepteur P2X7 participe aux mécanismes de signalisation impliqués dans l’activation des lymphocytes T, leur prolifération et leur différentiation. Notre laboratoire a récemment montré que les lymphocytes T effecteurs (CD4+ ou CD8+) en fin de réponse immunitaire secondaire, exprimant à la membrane la tyrosine phosphatase de membrane B220, sont totalement résistant à l’activation du récepteur P2X7 à cause d’une perte d’adressage de ce récepteur à la membrane. Le but de ce travail de thèse est d’étudier la sensibilité des lymphocytes T, à différents stades d’activation, aux activités cellulaires induites par l’ATP, notamment le clivage de la molécule de homing CD62L ou L-sélectine, l’ouverture du canal ionique, la formation du pore et l’externalisation de la PS. Mes principaux résultats montrent que les activités cellulaires dépendantes du récepteur P2X7 sont dissociées. Les lymphocytes T au stade effecteur/mémoire sont moins sensibles au clivage de la molécule CD62L que les lymphocytes T au stade naïf et récemment activé. Les lymphocytes naïfs T récemment activé en réponse immunitaire primaire sont les plus sensibles à la formation du pore. De plus, les lymphocytes T récemment activés, aussi bien en réponse immunitaire primaire que secondaire, sont les plus sensibles à l’externalisation de la PS. Enfin, dans les lymphocytes T récemment activé, les activités de pore et d’externalisation de PS, mais pas le clivage de CD62L, sont dépendantes du taux de calcium. / Extracellular ATP through the receptor P2X7 (P2X7R) plays a key role in innate immunity as a danger signal that causes the activation of the inflammasome, enhancement of immune cell migration and cell death. Although the role of the ATP/P2X7R pathway in adaptative immunity remains underestimated, it has been reported that P2X7R regulates signaling events involved in T-cell activation, proliferation, and differentiation into effector lineages. Moreover, we have previously shown that effector T lymphocytes (either CD4+ or CD8+) that express the B220 isoform of CD45 at the plasma membrane at the end of the secondary immune response are totally resistant to ATP stimulation due to loss of P2X7R membrane expression. In the present study, we compared the sensitivity of T lymphocytes to cellular activities trigerred by P2X7R according to their stage of activation. Interestingly, our results showed that P2X7-dependent cellular activities are dissociated. T lymphocytes at effector/memory stage are less sensitive to CD62L shedding than naïve or recently activated T lymphocyte during primary immune response. Naive T lymphocytes recently activated during primary immune response are the most sensitive to pore formation. Furthermore, recently activated T lymphocytes at both primary and secondary immune responses are the most sensitive to PS externalization. Finally, pore formation, PS externalization but not CD62L shedding, are dependent on calcium signaling.