Solid tumours are a hostile tissue environment in which the cells are exposed to many stresses including hypoxia. One consequence of hypoxic conditions is an increase in extracellular levels of the purine nucleoside adenosine, which enhances tumour cell migration. This is achieved in part through an increase in the levels of the chemokine receptor CXCR4, which along with its ligand CXCL12, is a key player in breast cancer metastasis. The cellular response to stress is mediated by a family of proteins known as heat-shock proteins (HSPs). The small heat shock protein 27 (HSP27) has been implicated in changes in cancer cell migration. I have therefore studied the regulation of HSP27 in human breast cancer cells by conditions that normally exist in the stressful tumor environment. My project specifically aimed to establish whether changes in HSP27 are linked to hypoxia, adenosine levels and alterations in the CXCL12-CXCR4 migratory pathway.

Waldenström's Macroglobulinemia (WM) is a rare, indolent, non-Hodgkin's lymphoma whose molecular pathology remains poorly understood. This disease is characterized by the accumulation of IgM-secreting lymphoplasmacytic cells in the bone marrow, and is often histopathologically indistinguishable from marginal zone lymphoma, IgM-secreting myeloma, and chronic lymphocytic leukemia with plasmacytic differentiation. To better understand the genomic landscape of this disease, whole genome sequencing was performed on bone marrow samples from thirty WM patients, ten of which were paired with germline tissue. This study identified two genes that are frequently mutated in WM: MYD88 and CXCR4. MYD88 was somatically mutated in 90% of WM samples, which displayed a single nucleotide variant resulting in a leucine to proline substitution at position 265. As prev iously demonstrated in activated B-cell subtype of diffuse large B-cell lymphoma, this mutation results in constitutive activation of the Toll-like receptor pathway and activation of nuclear factor kappa B (NF𝜅B). Highly sensitive allele specific polymerase chain reaction assays were developed to detect MYD88L265P in WM and related hematological malignancies. These studies demonstrated that MYD88L265P could be used to aid in the differential diagnosis, response assessment, and detection of minimal residual disease in WM. Moreover, MYD88L265P was observed in 50% of the precursor condition, IgM monoclonal gammopathy of undetermined significance, suggesting that it is an early event in the pathogenesis of WM. Blocking MYD88 dimerization or the use of downstream IRAK1/4 kinase inhibitors decreased the phosphorylation and nuclear localization of NF𝜅B. Somatic mutations in CXCR4 were only found in the regulatory C-terminal tail and were present in 29% of WM patients. These mutations were similar or identical to those found in the germline of patients with the autosomal dominant disease Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome. CXCR4 somatic WHIM-like mutations were found nearly exclusively in MYD88L265P mutated patients. These mutations impaired receptor internalization, increased signaling downstream of CXCR4, and instilled resistance to several WM directed therapeutics. WM patients who were wild type for both CXCR4 and MYD88 demonstrated inferior overall survival. These studies evidence highly recurring somatic events, and provide a genomic basis for the molecular pathogenesis of WM.

Dysregulation of the chemokine receptor 4 (CXCR4) and its primary ligand CXCL12 (SDF-1, stromal cell-derived factor-1), has been implicated in the progression of melanoma and the mechanisms by which the CXCR4/CXCL12 axis has been shown to activate cell cycle progression is via stimulation of the mitogen-activated protein kinase (MAPK) pathway. Given this, we sought to ascertain the potential cooperativity of CXCR4 with established histopathologic prognosticators including the BRAF status in primary cutaneous melanoma. In this IRB approved study, archived tissue samples with diagnosis of primary cutaneous melanoma were retrieved from the Skin Pathology Laboratory at BUSM, Boston, MA and a total of 107 cases identified as meeting criteria for inclusion. Protein expression of CXCR4 and CXCL12 were assessed using commercially available rabbit polyclonal antibodies (Ab2074 and, ab9797 respectively, Abcam, Cambridge, MA, USA). CXCR4 gene expression (mRNA) was measured by semiquantitative RT-PCR with appropriate controls. For IHC, a semi-quantitative scoring (ranging from 0-3) was used and cases with a score of ≥2 (>10%) were considered positive. Molecular analysis for CXCR4 gene expression and BRAF exon 15 mutation status was performed using mRNA semi-quantitative RT-PCR and DNA Sanger sequencing respectively. Univariate analyses of CXCR4 mRNA expression revealed a statistically significant correlation between elevated CXCR4 expression (low ΔCt value) and presence of the BRAF mutation and absence of a host response (p=0.03 and p=0.0003 respectively). Univariate analyses revealed a significant correlation between elevated CXCR4 mRNA (low ΔCt value) and the following: presence of BRAF mutation and absence of a host response (p=0.03 and 0.0003 respectively). CXCR4 mRNA was significantly higher among both AJCC stage 1 and stage 3 compared to stage 2 (p=0.01). Compared with CXCR4 negative samples, univariate analyses of CXCR4 protein showed that the proportion of CXCR4 positives was significantly greater in melanomas with absence of mitoses (p<0.0001), ulceration (p=0.0008) and regression (p=0.02). Patients presenting at shallower stages (AJCC 1-2) exhibited a larger proportion of CXCR4 positives (76.9%, p<0.0001 and 69.0%, p=0.004), while those at deeper stages (AJCC 3-4) exhibited a larger proportion of negatives (75.0%, p=0.002 and 66.7%, p=0.10). In a multivariable analysis, lower odds of CXCR4 protein expression were associated with AJCC stage-3 (OR=0.16, p=0.01), stage-4 (OR=0.17, p=0.04), and mitoses (OR=0.21, p=0.01). Lack of correlation between CXCR4 mRNA and protein expression suggests that further study is required for a more precise understanding of mRNA-protein interaction for CXCR4 in order to identify factors contributing to the lack of concordance. CXCR4 protein appears to be associated with established prognosticators of good clinical outcome as its expression is less frequently observed in melanomas with mitoses, ulceration and depth >2 mm. The association between CXCR4 mRNA and a brisk host response suggests that it may serve as a biomarker for recruiting melanoma patients for immunotherapy. Higher CXCR4 mRNA in patients with a BRAF mutation suggests its utility as a putative therapeutic target.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23376号 / 医博第4745号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 佐藤 俊哉, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Die therapeutischen Möglichkeiten des metastasierten Prostatakarzinoms (Pca) haben sich durch die neuen Substanzen Docetaxel und Abirateron deutlich verbessert. Das prostataspezifische Membranantigen (PSMA) stellt für die Diagnose und Therapie des Pca´s einen vielversprechenden Angriffspunkt dar. PSMA wird in Prostatakarzinomzellen überexprimiert und dient als Zielstruktur für nicht-invasives bildgebendes Verfahren und Lutetium-177-PSMA-Radioligandentherapie als Therapieoption. Der CXCR4-Rezeptor wird an unterschiedlichen Zelltypen und Organen exprimiert. Seine Überexpression wird mit einer Metastasierung und schlechter Prognose assoziiert. Gallium-68-PSMA PET/CT liefert genaue Kenntnisse bezüglich Ausbreitung und Fortschreiten des Tumorgeschehens. Die vorliegende Arbeit untersucht die Zusammenhänge zwischen Expression von PSMA und CXCR4 in Verbindung mit etablierten Therapeutika und versucht Wege aufzuzeichnen, welche durch Erhöhung der PSMA-Expression zur verbesserten Sensitivität des PSMA PET/CT führen könnten, wodurch der personalisierte Therapieansatz weiter optimiert werden kann. / Novel therapeutic agents such as docetaxel and abiraterone have significantly improved treatment strategies for metastatic prostate cancer in recent years. Prostate-specific membrane antigen (PSMA) represents a promising target for diagnosis and therapy of prostate cancer. PSMA is over expressed in prostate cancer cells providing a target structure for non-invasive imaging and Lutetium-177-PSMA radioligand therapy. The CXCR4-receptor is expressed on different cell types and organs. Its over expression is associated with metastasis and poor prognosis. PET/CT imaging with Gallium-68-labelled PSMA ligands provide relevant information regarding tumor staging and progression. The present study investigates the interaction between expression of PSMA and CXCR4 considering established therapeutic agents to improve sensitivity of PSMA PET/CT imaging and optimize personalized cancer medicine.

No description available.

The role of chemokines as chemotactic cytokines and their functions in the immune system and related pathologies are well defined. Recently, strong evidence supporting the hypothesis that chemokines can act as modulators of neuronal activity and influence neurotransmission has been reported. The chemokine CXCL12 is constitutively expressed in adult brain and expression of CXCL12 and its cognate receptor CXCR4 have been reported in regions of rat brain that construct dopamine (DA) and glutamate (GLU) pathways such as ventral tegmental area (VTA), substantia nigra (SN), and nucleus accumbens (NAc). In the central nervous system (CNS), activation of CXCR4 on dopaminergic neurons and astrocytes initiate cascade of events that leads to DA and GLU release and influence synaptic transmission. In vivo, intracerebroventricular (ICV) CXCL12 has been shown to potentiate cocaine-induced locomotor activity. Based on these evidences, the studies, as outlined in this dissertation, aimed to expand understanding of how CXCL12/CXCR4 interaction can affect cocaine-induced behavior and reinforcement, with special focus on mechanisms involving GLU. We first evaluated involvement of CXCR4 activation by CXCL12 on cocaine-induced locomotor activity using a selective CXCR4 antagonist AMD3100. Results demonstrated that AMD3100 (5, 10 mg/kg, IP) pretreatment dose-dependently attenuated cocaine-induced locomotor activity without affecting the baseline activity. Thereafter, effects of AMD3100 on cocaine’s reinforcing efficacy were tested using a biased conditioned place preference (CPP) paradigm. In all CPP experiments, saline pretreated controls established a significant preference for the cocaine-paired context following four pairings with cocaine (10 mg/kg, IP). Rats pretreated with 2.5 and 5 mg/kg AMD3100 prior to each pairing session showed significantly lower preference for the cocaine-paired side, whereas rats pretreated with 1 mg/kg AMD3100 showed similar preference for the cocaine-paired side as the saline controls when tested in the absence of the drug. Rats pretreated with AMD3100 (5 mg/kg, IP) just once prior to testing showed significantly lower preference for the cocaine-paired side. These results demonstrate that CXCR4 antagonism reduces development and expression of cocaine-induced CPP. Intravenous cocaine self-administration (SA) was performed to examine the effects of AMD3100 on the acquisition of cocaine-taking behavior and reinstatement to cocaine-seeking. Acquisition of cocaine SA was studied using three doses of cocaine (0.375, 0.5, 0.75 mg/kg/infusion) on a fixed-ratio 1 (FR-1) schedule of reinforcement. Two doses of AMD3100 (5, 10 mg/kg, IP) were tested. In all SA experiments, saline pretreated controls readily acquired cocaine self-administration. The lower and higher AMD3100 decreased the number of reinforcers earned during the two hour sessions compared to saline controls when tested against acquisition of 0.375 and 0.5 mg/kg/infusion cocaine. However, when tested against the 0.75 mg/kg/infusion cocaine, only 10 mg/kg of AMD3100 resulted in reduction in responding, but not the lower dose. When a dose response curve was plotted using all doses of cocaine and AMD3100 tested, the effects of AMD3100 were represented by a significant downward shift of the dose response curve. Effects of AMD3100 on cocaine-seeking were evaluated using the reinstatement model, in which rats that were extinguished from self-administration of 0.5 mg/kg/infusion cocaine underwent reinstatement testing with cue or cue + drug presentation. A compound cue (light and tone stimulus), that was used during the acquisition training, and submaximal dose of cocaine (5mg/kg, IP) were used. In both reinstatement conditions, rats pretreated with saline reinstated following the presentation of cue or cue + drug prime. Rats pretreated with AMD3100, 30 minutes prior to reinstatement session, showed significantly lower number of lever presses indicating drug-seeking was not as robust compared to the saline pretreated controls. Following the observations that cocaine-induced behaviors may be partially mediated by CXCL12/CXCR4 interaction, neurochemical changes were examined to elucidate the underlying mechanisms. CXCR4 immunoreactivity in prefrontal cortex (PFC) following withdrawal from 7 days of repeated cocaine (15 mg/kg, IP) was evaluated. Although positive CXCR4 immunoreactivity was observed in PFC, there were no significant differences in the intensity of CXCR4 expression compared to the saline treated controls at acute (2 hours, 2 and 10 days) or protracted (30 days) withdrawal time points. In contrast, CXCL12 protein levels in PFC and NAc were negatively influenced after protracted withdrawal from 7 days of repeated cocaine (15 mg/kg, IP) administration. Following assessment of regional expression of CXCR4, cellular expression was evaluated using triple labeling immunohistochemistry which revealed the positive CXCR4-immunoreactivity on cells staining positively for vesicular glutamate transporter 1 (vGlut1) and glial fibrillary acidic protein (GFAP) showing that the glutamatergic neurons and astrocytes in the PFC express CXCR4. To assess the effects of CXCL12 on GLU transmission, microdialysis of NAc was performed. Following unilateral injection of 50 ng CXCL12 into the lateral ventricle, increase in extracellular GLU was observed. In a follow up study, AMD3100 pretreatment (10 mg/kg, IP) attenuated cocaine-induced increase in extracellular GLU in the NAc. Since the glutamate transporter subtype-1 (GLT-1) is a major regulator of extracellular GLU and upregulation of GLT-1 expression and function has been shown to attenuate reinstatement to cocaine-seeking, its expression was evaluated using immunoblot analysis of the NAc and PFC of rats that self-administered cocaine. The results revealed that AMD3100 (10 mg/k, IP; daily injection 30 min prior to SA session) pretreatment upregulated GLT-1 levels in the NAc but not PFC. In summary, results of the present study show that CXCL12/CXCR4 interaction may modulate cocaine-induced behavioral effects including reinforcement and reinstatement to cocaine-seeking. Neurochemical assessments revealed the presence of CXCR4-expressing glutamatergic neurons in the PFC and AMD3100-induced up-regulation of GLT-1. Most importantly, we provided direct evidence of CXCL12/CXCR4 mediated GLU transmission in NAc. Together, these results expand our understanding of chemokine’s role as neuromodulators and identify CXCR4 as a novel target for development of new pharmacotherapies for the treatment of cocaine addiction. / Pharmacology

Macrophage migration inhibitory factor (MIF) has been implicated in mediating both innate and adaptive immune responses in inflammatory and infectious diseases. The sequence and structure of MIF is highly conserved across the avian phylogeny, which underlies high sequence homology and functional similarities between turkey and chicken MIFs. Turkey MIF (TkMIF) inhibited cell migration and promoted cell proliferation with production of inflammatory mediators, comparable to the biological properties of chicken MIF (ChMIF), thus indicating the biological cross-reactivity between turkey and chicken MIFs. This study identified the cell surface receptor(s) that could bind ChMIF and the biological roles triggered by such interactions. In addition to CD74, a previously identified receptor, CXCR4 also interacts with ChMIF. Moreover, the formation of receptor complexes was shown between CXCR4 and CD74. MIF signaling through CXCR4 and CD74 led to cell chemotaxis and proliferation activity as well as intracellular calcium influx. Intriguingly, Eimeria MIF (EMIF), a homologue secreted following parasitic infection, also interacted with CD74 leading to comparable biological functions to those of ChMIF. Given such observations, we hypothesized that CXCR4 and CD74 are receptors for ChMIF leading to the functional consequences similarly manifested by EMIF interaction with the corresponding receptors. EMIF, predominantly secreted from the invasive merozoite stage, may help the parasite exploit the host immune response by interacting with common ChMIF receptors. This may lead to functional mimicry thus provoking the question of whether EMIF would modulate the biological functions of ChMIF to manipulate the host defense that allows more efficient invasion of the host. To evaluate this concept, a transgenic E. tenella lacking MIF was generated by in vivo passage of E. tenella transfected with a CRISPR plasmid targeting EMIF. Although not fully disrupted, reduction of EMIF expression was observed in the transgenic E. tenella itself as well as in inoculated cells, which resulted in enhanced survival of host cells. Herein, we achieved a better characterization of the functional roles of both avian and parasite MIFs underlying the interaction with common host receptors, along with the essential role of parasite MIF promoting host cell death during parasitic infection. / PHD / When animals get infected or injured, their immune system senses invading pathogens or damaged tissues as danger signals, which often elicits the production of inflammatory mediators. These are chemical messengers secreted mostly by immune cells that initiate cellular communication and infiltration of immune cells to the infection/damaged site leading to inflammatory responses to eliminate the infectious agents and repair damaged tissues. Among many inflammatory mediators, macrophage migration inhibitory factor (MIF) is involved in inflammatory and immune response by regulating cell migration. Interestingly, MIF is secreted by Eimeria parasites (that cause the costly coccidiosis disease in poultry) as well as by chickens (host animal) after infection with this pathogen. Toward a better understanding of the impacts of both avian and parasite MIFs on the host immune response, three specific studies were completed. First, MIF displayed high degree of gene sequence identity and functional similarity between chicken and turkey, supporting the evolutionarily conservation of MIF across birds. The second study identified the MIF receptors and their complexes, which engage in the biological functions of chicken MIF. Through binding to these cell surface receptors, chicken MIF can regulate cell migration and proliferation with calcium release. Intriguingly, Eimeria MIF secreted after parasitic infection is able to bind the same receptors leading to comparable biological functions to those of chicken MIF. Lastly, the role of Eimeria MIF was further evaluated by disrupting its gene in the parasite. Although not fully disrupted in the transgenic parasites, its expression was decreased resulting in enhanced survival of host cells, thus suggesting a deleterious effect of Eimeria MIF on the host, as well as its potential as a therapeutic target to control coccidiosis in poultry.

Les récepteurs couplés aux protéines G (RCPG) sont la famille de récepteurs membranaires la plus représentée chez les vertébrés, et la plus grande cible thérapeutique chez l'Homme. L'évolution du paradigme initial qui énonçait une stœchiométrie récepteur : protéine G : effecteur de 1 :1 :1 sera présentée sur le modèle des récepteurs aux chimiokines CXCR4 et CCR2. Grâce à la technique de transfert d'énergie par bioluminescence (BRET), les travaux réalisés durant cette thèse montrent (1) que c'est par un couplage alternatif de CXCR4 à Gα13 au lieu de la voie classique Gαi que les cellules de cancer du sein migrent pour former des métastases, (2) que la désensibilisation de CXCR4 implique le recrutement d'une combinaison définie de protéines (GRK et arrestines) permettant l'arrêt sélectif des multiples voies engagées en réponse à l'agoniste, et (3) que le protomère CXCR4 a un rôle déterminant dans l'engagement de la protéine Gαi et le recrutement de la β-arrestine par l'hétéro-oligomère CXCR4/CCR2 lorsque CCR2 est activé. Dans cette dernière et principale étude, les résultats montrent également que le dimère CCR2 peut s' assembler au dimère CXCR4 pour former un tétramère, et que l'activation de CCR2 influence la conformation du dimère CXCR4. Les phénomènes de coopérativité et d'activation asymétrique déjà rapportés pour cet hétérodimère pourraient donc impliquer l'interaction de quatre protomères. En conclusion les travaux effectués durant cette thèse démontrent une régulation supplémentaire de l'activité des récepteurs chimiokines au niveau de leur structure quaternaire, de leur signalisation, et de l'arrêt de cette signalisation. / G protein coupled receptors (GPCR) are the most represented cell surface receptors among vertebrates, and the major therapeutic target in humans. The initial paradigm stating a 1 :1 :1 stoichiometry for receptor :G protein :effector has evolved to a more complex model, as illustrated here with the example of the chemokine receptors CXCR4 and CCR2. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that (1) CXCR4 is able to couple Gα13 instead of Gαi to promote breast cancer metastasis, (2) the multiple pathways engaged by stimulation of CXCR4 are selectively desensitized by the specific recruitment of a defined combination of proteins (GRKs and arrestins) and (3) the CXCR4 protomer plays a crucial role during Gαi engagement and β-arrestin recruitment by the CXCR4/CCR2 heterodimer upon CCR2 activation. In this last and main study, the results shown also demonstrate that CCR2 dimers could assemble with CX CR4 dimers into hetero-tetramers, and that CCR2 activation leads to a conformational change in the CXCR4 dimer. Former results showing cooperativity and asymmetric activation of a simple CXCR4/CCR2 heterodimer could then be applied to a tetramer. To conclude, the work done during this thesis demonstrates a more sophisticated regulation of chemokine receptors than previously suspected at 3 different levels: quaternary structure of the protomers, G protein signalling, and signalling termination

CCR5 et CXCR4 sont les corécepteurs d'entrée du VIH utilisés par le virus in vivo en plus du récepteur principal CD4 pour infecter les cellules. Au début et tout le long de l'infection, on retrouve chez les patients infectés par le VIH, des virions R5 utilisant le corécepteur CCR5 pour infecter les cellules. Dans les stades tardifs de l'infection et chez environ la moitié des personnes infectées par le VIH, on observe en plus de ces souches R5, l'apparition de souches X4, utilisant le corécepteur CXCR4 pour infecter les cellules. Cette apparition de souches X4 est un facteur d'aggravation de la maladie. Les causes de cette commutation de R5 vers X4 sont mal définies. Le but de mon travail de thèse a été de trouver de nouvelles stratégies thérapeutiques visant l'un ou l'autre de ces corécepteurs. La première partie de mon travail compare les deux isoformes de CXCR4 en tant que corécepteurs du VIH. Ces deux isoformes, CXCR4-A et CXCR4-B, diffèrent de 9 acides aminés en NH2 terminal suite à un épissage alternatif. Nous avons montré que l'isoforme CXCR4-B est la plus performante en tant que corécepteur du VIH mais que ces deux variants sont équivalents pour la migration vers leur ligand commun SDF-1. Ainsi, nous proposons qu'en ciblant exclusivement l'isoforme B qui est la plus favorable à l'infection, via par exemple des siRNA, il serait possible de limiter les infections par des souches X4 tout en gardant une partie des fonctions essentielles de ce récepteur dans l'organisme, assurées par l'isoforme A. Nos résultats suggèrent également que l'infection par des souches R5 augmente le ratio en ARNm CXCR4-B / CXCR4-A dans des PBMC, et que ce ratio est en partie responsable de la commutation de R5 vers X4 associée à une aggravation de la maladie. Cibler cette isoforme CXCR4-B pourrait donc se révéler bénéfique. La deuxième partie de cette thèse étudie la modulation de la fonction de corécepteur du VIH de CCR5 par S1P1, un autre membre de la famille des récepteurs couplés aux protéines G qui permet la remise en circulation des lymphocytes après leur séjour dans les organes lymphoïdes secondaires par chimiotactisme vers son ligand S1P, abondant dans le sang. Nous montrons que S1P1 interagit physiquement avec CCR5 et gêne l'entrée des virus R5 dans la cellule-hôte. A l'inverse, S1P1 active les étapes post-entrée du cycle viral, notamment l'expression génique virale. La résultante de ces effets opposés est une augmentation de la production virale par des cellules infectées in vitro. Ce projet a également montré que l'utilisation de FTY720, un antagoniste fonctionnel de S1P1, diminue l'infection de cellules dendritiques par des virus HIV-R5 in vitro, ainsi que la virémie dans un modèle de souris SCID infectées après reconstitution immunologique. La mise en évidence des interactions entre CCR5 et S1P1 ouvre donc des perspectives thérapeutiques. / CCR5 and CXCR4 are the two HIV entry coreceptors used by the virus in addition to the main receptor CD4 in vivo to infect cells. R5 virions, that use CCR5 as a coreceptor to infect cells, are detected in most HIV patients. At late stages of infection and in about half of HIV infected persons, there is an emergence of X4 virions that use CXCR4 as a coreceptor, in addition to R5 virions. This emergence is associated with an increase in disease progression. The reasons for this R5 to X4 switch are poorly understood. The goal of my PhD work was to find new therapeutic strategies that target these coreceptors.The first part of this work compares the two CXCR4 isoforms as HIV coreceptors. Those two isoforms, CXCR4-A and CXCR4-B, differ by 9 amino acids at their NH2 terminal extremity as a consequence of an alternative splicing. We have shown that CXCR4-B isoform is more efficient as an HIV coreceptor but that those two variants are equivalent in terms of chemotaxis toward their common ligand SDF-1. Thus, we propose that by targeting specifically the B isoform that supports infection, via siRNA by example, it is possible to limit X4 development while keeping essential functions of this receptor. Our results also suggest that R5 infection increases CXCR4-B / CXCR4-A mRNA ratio in PBMC and that this ratio is in part responsible for R5 to X4 switch. Thus, targeting CXCR4-B isoform could be beneficial.The second part of this PhD thesis studies the effect on CCR5 coreceptor function of S1P1, another G protein-coupled receptor that enables lymphocytes egress from lymph nodes by chemotaxis toward its ligand S1P that is abundant in blood. We have shown that S1P1 physically interacts with CCR5 and blocks R5 virus entry. On the other hand, S1P1 activates post-entry steps of the viral cycle, in particular gene expression. The resulting effect is an increase in viral production by infected cells in vitro. We also showed that the use of FTY720, a S1P1 functional antagonist, decreases dendritic cell infection by R5 viruses in vitro, and in vivo infection in a SCID mouse model. The emphasis of CCR5 and S1P1 interactions opens new therapeutic strategies.