Apžvelgiami klinikiniai tyrimai atlikti VU Imunologijos institute Molekulinės imunologijos laboratorijoje, bendradarbiaujant su Vilniaus universitetinės Greitosios pagalbos ligoninės Bendrosios chirurgijos centro Ginekologijos skyriumi bei „Vaisingumo klinika“. Tyrimams atlikti buvo gauti du Lietuvos Bioetikos komiteto leidimai. Darbo tikslas - nustatyti gimdos gleivinės imunines ląsteles bei įvertinti jų vaidmenį reprodukcijos procese. Medžiaga ir metodai. Tyrimuose dalyvavo vaisingos moterys (kontrolinė grupė) (n=142), nevaisingos moterys (121), moterys patyrusios savaiminį persileidimą (n=35) ir endometrioze sergančios moterys (n=181). Naudoti tyrimo metodai: ląstelių morfologiniam įvertinimui - citologinis, imuninių ląstelių fenotipo nustatymui - tėkmės citometrijos, tirpių medžiagų (citokinų, HLA-G molekulių) koncentracijos nustatymui – imunofermentinis metodas. Rezultatai ir išvados. Menstruacinio ciklo metu kito endometriumo imuninių ląstelių sudėtis, limfocitų ir makrofagų aktyvacijos molekulių ekspresija: priešmenstruaciniu periodu daugėjo makrofagų (proliferacijos fazėje – 7,3±2,8%, vėlyvos sekrecijos fazėje - 13,7±3,1%) ir NK ląstelių (proliferacijos fazėje – 18,3±5,8%, vėlyvos sekrecijos fazėje – 51,1±9,8%). Didžiausias skaičius aktyvuotų makrofagų gimdos gleivinėje, ekspresuojančių CD69 ir CD54 molekules, buvo randamas proliferacijos fazėje (14,3±5,6% ir 26,2±4,8% atitinkamai). Decidualiniame audinyje nustatėme ypatingo fenotipo intensyviai CD56... [toliau žr. visą tekstą] / Clinical tests performed in the Laboratory of Molecular Immunology of the Institute of Immunology of Vilnius University in cooperation with the Gynaecological Department of the General Surgery Center of Vilnius University Emergency Aid Hospital and Fertility Clinic, Vilnius are reviewed. Two permissions have been obtained from the Lithuanian Committee of Bioethics for carrying out the tests. The aim of the study was to determine endometrial immune cells and to evaluate their role in the reproduction process. Materials and methods. The following groups of women took part in our study: fertile women (they formed control group) (n=142), infertile women (n=121), women after miscarriage (n=35) and women with endometriosis (n=181). The following laboratory methods were used: cytological (to define the morphology of cells); flow cytometry (for detection of the phenotype of immune cells) and immunoenzyme assay – to quantify the concentration of soluble substances, such as cytokines and HLA-G molecules. Results and Conclusions. Composition of the endometrial immune cells, expression of lymphocyte and macrophage activation molecules has been changing during the menstrual cycle. In the pre-menstrual period the number of macrophages and NK cells has increased: in the stage of proliferation-7.3±2.8%; in the late stage of secretion – 13.7±3.1% and in the stage of proliferation – 18.3±5.8%, in the late stage of secretion – 51.1±9.8, respectively. The highest amount of activated macrophages... [to full text]

La réaction du greffon contre l’hôte (GvHD) est une complication majeure de la transplantation de cellules souches hématopoïétiques (HSCT). Les traitements de prophylaxie contre le développement de la GvHD reposent essentiellement sur l’utilisation d’agents immunosuppresseurs, ce qui contribue à ralentir la reconstitution immunitaire post-greffe et à prolonger la durée de l’état immunosupprimé des patients. Le développement de prophylaxie pour la GvHD à base d’agents immunomodulateurs est ainsi privilégié. À l’aide d’un modèle murin xénogénique chez les souris NOD/scid-IL2rγ-/- (NSG), on a étudié le potentiel immunomodulateur des immunoglobulines intraveineuses (IVIG) dans la prévention de la GvHD, ainsi que leurs effets sur la qualité et la cinétique de la reconstitution immunitaire. On a déterminé qu’un traitement hebdomadaire d’IVIG peut effectivement réduire l’incidence de la GvHD, ainsi que la mortalité qui y est reliée, avec une efficacité similaire à celle obtenue avec la cyclosporine A, un immunosuppresseur couramment utilisé dans la prophylaxie de la GvHD. Par ailleurs, on a déterminé que le mécanisme d’action des IVIG dans la réduction de la GvHD est distinct de celui des immunosuppresseurs. De plus, on a démontré que les IVIG induisent l’expansion et l’activation des cellules NK présentes au sein du greffon, lesquelles sont nécessaires pour l’obtention de l’effet protecteur des IVIG contre le développement de la GvHD, et sont dépendantes de la présence de lymphocytes T activés. Grâce à un modèle murin humanisé, on a également démontré que le traitement hebdomadaire d’IVIG induit un délai transitoire de la reconstitution humorale, ce qui n’affecte toutefois pas la qualité globale de la reconstitution immunitaire. Ces résultats mettent cependant en doute la pertinence de l’utilisation des IVIG dans les protocoles cliniques de prophylaxie de la GvHD, puisque les immunosuppresseurs seront toujours utilisés, et qu’on a démontré que les IVIG ont besoin de lymphocytes T activés afin de prévenir efficacement le développement de la GvHD. / Graft-versus-Host Disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT). Prophylactic treatments for the prevention of GvHD rely mostly on the use of immunosuppressors, which contribute to inhibit the patient’s immune reconstitution and prolong their immunosuppressed state. Development of immunomodulator-based prophylactic treatments is therefore preferred. Using NOD/scid-IL2rγ-/- mice, we developed a xenogeneic mouse model to assess the immunomodulatory potential of intravenous immunoglobulins (IVIG) for the prevention of GvHD, along with assessing their effect on the kinetics and the quality of the immune reconstitution in mice. We determined that weekly IVIG treatments reduced the incidence of GvHD and its related mortality. The effectiveness of IVIG for the prevention of GvHD was similar to that of cyclosporine A, an immunosuppressive drug routinely used for the prophylactic treatment of GvHD. Furthermore, we demonstrated that IVIG has a mechanism of action that is different from that of immunosuppressors. IVIG induce the expansion and activation of NK cells from the graft, which is mandatory for the preventive effect of IVIG on GvHD development. Furthermore, this IVIG-induced expansion and activation of NK cells require the presence of activated T lymphocytes. Using our humanized mouse model, we have also demonstrated that weekly IVIG treatments cause a transient delay of the humoral reconstitution, but do not affect the overall quality of the immune reconstitution. We have demonstrated that activated T lymphocytes are mandatory for the effective expansion and activation of NK cells, which in turn are essential to the IVIG-induced prevention of GvHD, and immunosuppressors will always be part of the prophylactic regimen of GvHD, therefore shining a doubt on the usefulness of adding IVIG to the prophylactic treatments for the prevention of GvHD.

Com o início da fortificação de farinhas com ácido fólico (AF) no Brasil, a partir 2004, a população passou a estar exposta de modo compulsório a maiores quantidades desta vitamina. Sabe-se que o AF na sua forma sintética pode não ser completamente convertido para formas metabolicamente ativas, levando ao aparecimento de uma fração não metabolizada no organismo. Este fato é mais preocupante nos indivíduos que além da fortificação, fazem uso terapêutico dessa vitamina, como em pacientes com anemias hemolíticas (esferocitose hereditária (EH), β-talassemia heterozigótica (β-TH), entre outras). O objetivo desse estudo foi avaliar se as concentrações séricas de AF não metabolizado (UMFA) afetam a metilação global do DNA; a expressão de RNAm de genes da DHFR, MTHFR, interferon-γ, TNF-α e interleucina (IL)-8; e a citotoxicidade de células NK. Foram incluídos 27 pacientes com EH, 50 indivíduos com β-talassemia heterozigótica (β-TH) e 136 indivíduos saudáveis. Outros 30 indivíduos saudáveis foram incluídos num estudo de intervenção com 5mg/dia de AF durante 90 dias. Foram realizadas as análises de: ácido fólico sérico, eritrocitário e UMFA, vitamina B12, homocisteína total; expressões de RNAm dos genes de MTHFR, DHFR, IFN-γ, TNF-α e IL-8; citotoxicidade das células NK; metilação global do DNA e citocinas séricas IL6, IL-8, IL10, IFN-γ e TNF-α. Resultados: Os pacientes EH apresentaram maiores concentrações de AF (sérico, eritrocitário e UMFA) que seus controles, sendo que 55,5% (método microbiológico) e 74,1% (método LC-MS/MS) apresentaram concentrações suprafisiológicas da vitamina, e 74,1% apresentaram concentrações aumentadas de UMFA. No grupo β-TH foi observado maiores concentrações de folato eritrocitário comparado ao controle e 22% dos indivíduos tinham concentrações suprafisiológicas de AF. No estudo de intervenção, após 45 e 90 dias de uso de AF as concentrações suprafisiológicas estavam presentes em 93,3% dos indivíduos e 100% deles apresentavam concentrações aumentadas de UMFA. Não foram observadas diferenças nas taxas de metilação global do DNA entre os grupos EH e β-TH quando comparados aos seus controles e não foi verificada correlação entre metilação global e concentrações de UMFA. Tanto EH quanto β-TH apresentaram maior expressão de RNAm de IL-8, quando comparados aos seus controles. No grupo de intervenção houve maior expressão de IL-8 após 45 dias de uso de AF quando comparado ao período pré-intervenção. Foi mostrada uma correlação inversa entre as concentrações de folato eritrocitário com o número de células NK no grupo EH e com a capacidade citotóxica das células NK no grupo total (EH + controle). No grupo intervenção foi observado menor número e menor capacidade citotóxica das células NK após 90 dias de uso de AF. Conclusões: O uso de AF 5mg/dia foi associado com aumento expressivo das concentrações de folato sérico, eritrocitário, UMFA, na expressão de RNAm de genes da citocina inflamatória IL-8 e redução do número e da citotoxicidade das células NK. Dessa forma, altas doses de AF podem resultar em alterações de componentes do sistema imune podendo prejudicar os mecanismos de vigilância celular das células NK. Os nossos achados sugerem que é importante o acompanhamento terapêutico dos pacientes que fazem uso de AF, especialmente aqueles indivíduos que fazem uso crônico desta vitamina por longo tempo, como os pacientes com anemias hemolíticas, mulheres que desejam engravidar e gestantes. / With the beginning of the fortification of flour with folic acid (FA) in Brazil, since 2004, the population has been exposed compulsorily to larger amounts of this vitamin. It is known that FA in its synthetic form can not be completely converted to metabolically active forms, leading to the appearance of an unmetabolized fraction in the body. This fact is more worrying in subjects beyond fortification, make therapeutic use of this vitamin, such as patients with hemolytic anemia (hereditary spherocytosis (HS), β-thalassemia heterozygotic (β-TH), among others). The aim of this study was to evaluate if serum concentrations of unmetabolized FA (UMFA) affect the global DNA methylation; mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin (IL)-8 genes; and on cytotoxicity of NK cells. It was included 27 patients EH, 50 subjects β-TH and 136 healthy subjects. Another 30 healthy subjects were included in an intervention study with 5 mg/FA daily during 90 days. Analyzes performed were: serum and erythrocyte folate, and UMFA, vitamin B12, tHcy; mRNA expression of MTHFR, DHFR, IFN-γ, TNF-α and IL-8 genes; cytotoxicity of NK cells; global DNA methylation and serum cytokines IL6, IL-8, IL10, IFN-γ and TNF-α. Results: EH patients presented higher FA concentrations (serum, erythrocytes and UMFA) than controls ones, and 55.5% (microbiologic method) and 74.1% (LC-MS/MS method) showed supraphysiologic concentrations of vitamin, and 74.1% presented increased concentrations of UMFA. In β-TH group, it was observed higher erythrocyte folate concentrations compared with the control and 22% of subjects had supraphysiological concentrations of FA. In the intervention study, after 45 and 90 days of FA use, supraphysiological concentrations were present in 93.3% of subjects and 100% of them showed increased concentrations of UMFA. It was not observed differences in global methylation of DNA between EH and β-TH groups when compared to their controls and it was not observed significant correlation between global DNA methylation and UMFA levels. Both EH and β-TH showed higher mRNA expression of IL-8 gene, when compared to controls. In the intervention group, there was higher mRNA expression of IL-8 gene after 45 days of FA use when compared to pre-intervention period. It was demonstrated an inverse correlation between erythrocyte folate levels and the number of NK cells in EH group; and cytotoxic capacity of NK cells on total group (EH + control). In intervention group, it was observed fewer number and lower cytotoxic capacity of NK cells after 90 days of AF use. Conclusions: The use of AF 5mg daily was associated with a significant increase in serum and erythrocytes folate levels, accompanied by increase in UMFA levels, an increase in mRNA expression of IL-8 gene, and reduction of the number and the cytotoxicity capacity of NK cells. Thus, high doses of AF can result in modifications of the immune system components, which may damage the cell surveillance mechanisms of NK cells. Our findings suggest that is important the therapeutic follow up of patients that are using AF, especially those subjects with chronic use of this vitamin, such as patients with hemolytic anemia, women who wish to become pregnant and pregnant women.

Chez les patients, la tumeur développe une immunosuppression qui compromet les capacités des effecteurs de l’immunité à réagir. Longtemps décriée, l’immunothérapie est considérée aujourd’hui comme une thérapie à part entière avec la chirurgie, la chimiothérapie et la radiothérapie. Parmi les premières immunothérapies développées dans les années 1990, l’IL-2 forte dose fut approuvée dans le traitement du mélanome et du cancer du rein métastatiques. Cependant, sa forte toxicité et la faible proportion de patients répondeurs ont limité son usage clinique. Une autre cytokine, l’IL-15, apparaît prometteuse dans le traitement des cancers pour son activité similaire à l’IL-2 sans ses facteurs limitants. Cependant, seule, son activité anti-tumorale n’est pas optimale du fait de sa courte demi-vie ou encore de l’affinité intermédiaire de liaison à son récepteur βγ. Différentes stratégies ont été mises au point pour améliorer l’efficacité de l’IL-15, notamment, sa stabilisation à travers l’association avec sa chaine de haute affinité IL-15Rα. Une molécule de fusion associant de manière covalente la partie sushi+ de l’IL-15Rα avec l’IL-15 humaine a été développée : le RLI. Au cours de cette thèse, nous avons étudié chez la souris, le potentiel thérapeutique de cette molécule dans le traitement des cancers solides. Nous avons ainsi mis en évidence une activité anti-tumorale du RLI et une association synergique avec un anticorps monoclonal anti-PD-1. / In cancer patients the immune system is compromised by the tumor and its microenvironment. In recent decades the role of the immune system in tumor control has been controversial, though today cancer treatments that modulate immunity are a reality. Immunotherapy is a unique therapy adding to pre-existing methods of cancer control: surgery, chemotherapy and radiotherapy. In the 1990’s, high dosing of IL-2 was the first immunotherapy approved by the FDA to treat metastatic melanoma and renal carcinoma, however high toxicities and low responder rates have limited its clinical use. IL-15 is another promising cytokine therapy. The similar properties with IL-2 and differences in terms of toxicities and Treg induction make IL-15 an attractive potential therapy. Nonetheless, the short half-life and intermediate affinity for its receptor βγ compromise its efficacy. Different strategies have been developed to facilitate IL-15 therapeutic bioactivity. IL-15Rα as a chaperon molecule allows stability of IL-15 in vivo. RLI is a fusion molecule that covalently links sushi+IL-15Rα, the binding domain of its high affinity receptor and a recombinant human IL-15. We have studied the therapeutic potential of RLI in mouse tumor models. Our results show anti-tumor activities of RLI and synergistic combination with anti-PD-1, a monoclonal antibody.

<p>Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca²⁺ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. </p><p>A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.</p>

Stimulation of the T cell receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins, leading to activation of the interleukin-2 (IL-2) gene in T lymphocytes. Shb is a ubiquitously expressed adapter protein, with the ability to associate with the T cell receptor and several signaling proteins in T cells, including: the TCR ζ-chain, LAT, PLC-γ1, Vav, SLP-76 and Gads. Jurkat T cells expressing Shb with a mutation in the SH2 domain, exhibited reduced phosphorylation of several proteins and abolished activation of the MAP kinases ERK1, ERK2 and JNK, upon CD3 stimulation. The TCR induced Ca2+ response in these cells was abolished, together with the activation of the IL-2 promoter via the transcription factor NFAT. Consequently, IL-2 production was also perturbed in these cells, compared to normal Jurkat T cells. Shb was also seen to associate with the β and γ chains of the IL-2 receptor, upon IL-2 stimulation, in T and NK cells. This association occurred between the Shb SH2 domain and Tyr-510 of the IL-2R β chain. The proline-rich domains of Shb were found to associate with the tyrosine kinases JAK1 and JAK3, which are important for STAT-mediated proliferation of T and NK cells upon IL-2 stimulation. Shb was also found to be involved in IL-2 mediated regulation of apoptosis. These findings indicate a dual role for Shb in T cells, where Shb is involved in both T cell receptor and IL-2 receptor signaling. A Shb homologue, Shf was identified, and seen to associate with the PDGF-α-receptor. Shf shares high sequence homology with Shb and a Shd (also of the Shb family) in the SH2 domain and in four motifs containing putative tyrosine phosphorylation sites. When Shf was overexpressed in fibroblasts, these cells displayed significantly lower rates of apoptosis than control cells in the presence of PDGF-AA. These findings suggest a role for the novel adapter Shf in PDGF-receptor signaling and regulation of apoptosis.

Today,   most traditional method used in the research of immune cells, such as flow   cytometry and microscopy, are based on average values of cell responses.   However, immune cells are heterogeneous and respond differently to a given   stimuli. There is also a risk that important, but rare, behaviors of   individual cells are missed when a larger population of immune cells is   analyzed. Also, flow cytometry and microscopy do not allow long-term survival   of cells; these methods lack the ability to do dynamic long-term analysis of   motile immune cells, i.e. studies of cell-cell interactions, morphology and proliferation.   In a   patient who is affected by cancer, the cell heterogeneity contributes to the   ability to battle various types of cancer or virus infections. In an   outbreak, immune cells recognize and kill tumor cells. However, the number of   specific immune cells is sometimes too few to kill all the tumor cells in a   successful way. One way to help these patients is to isolate, select out and   cultivate the active immune cells with capacity to kill tumor cells.   The   Cell Physic Laboratory (a part of the department of Applied Physics) at the   Royal Institute of Technology (KTH) has developed a method for single-cell   analysis where the immune cells are trapped in microwells in a silicon chip.   The immune cells are then studied by using fluorescence microscopy in an   inverted setup. The method enables high-throughput experiments due to the   parallelization. Furthermore, since the immune cells survive long periods in   the chip, the cells can be analyzed over several days up to weeks. The   research group has also developed a semi-automatic ‘cell-picker’. The   cell-picker will be used in combination with the developed method for   single-cell analysis, which enables picking of cells of interest. In this report, experiments for the characterization and evaluation of the biocompatibility of two generations of the cell-picker will be presented. The experiments include development of a protocol for the cell-picking process, studies of the survival time of transferred cells for both generation of the cell-picker and studies of surface coating in the chip in order to increase the biocompatibility. The preliminary results indicate that the cell-picker has potential to be used as a selection tool for immune cells of interest.

No description available.

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

L’infection par le VIH-1 est caractérisée par une activation chronique du système immunitaire et par une réduction graduelle du nombre de lymphocytes TCD4+, qui contribuent à une détérioration lente du système immunitaire menant à la phase SIDA. Paradoxalement, ce sont majoritairement des lymphocytes T CD4+ non infectés qui sont détruits et la cause de ce phénomène reste encore inconnue. Certaines protéines virales, dont la protéine accessoire Vpr, sont soupçonnées de jouer un rôle dans ce processus. Synthétisée tardivement, Vpr est incorporée à l’intérieur des virions, en plus d’être relâchée sous forme soluble dans le milieu extracellulaire. La principale fonction biologique de Vpr est l’induction d’un arrêt de cycle en phase G2/M, via le recrutement du complexe d’ubiquitine E3 ligase CUL4A-DDB1VprBP et l’activation de la voie de dommage à l’ADN contrôlée par la kinase ATR. Une étude démontre que l’activation des voies de dommages à l’ADN conduit à l’expression de ligands du récepteur activateur NKG2D, exprimés par les cellules NK, déclenchant leurs fonctions cytolytiques. Chose intéressante, plusieurs études suggèrent que le VIH-1 régule positivement l’expression des ligands de NKG2D à la surface des lymphocytes T CD4+ infectés. Cependant, le facteur viral impliqué dans ce processus reste encore indéfini. Le but de cette thèse était d’évaluer le rôle de Vpr dans la modulation des fonctions cytolytiques des cellules NK et son implication potentielle dans la destruction des lymphocytes T CD4+. Nos travaux ont permis de démontrer que l’expression de Vpr, seule ou dans le contexte de l’infection, est suffisante afin d’augmenter spécifiquement l’expression du ligand de NKG2D, ULBP2, au niveau de lymphocytes T CD4+ primaires. Conséquemment, Vpr augmente ainsi la susceptibilité de ces cellules à une lyse par des cellules NK autologues. Nous démontrons que cette régulation positive d’ULBP2 repose sur la capacité de Vpr de recruter le complexe d’ubiquitine E3 ligase DDB1-CUL4AVprBP et l’activation de la voie de dommage à l’ADN ATR. Plus important encore, nous apportons des preuves que Vpr augmente également l’expression d’ULBP2 au niveau des cellules non infectées lors d’une infection de lymphocytes TCD4+ par le VIH-1. À cet effet, nous montrons que l’acheminement de Vpr au niveau de lymphocytes T CD4+ non infectés via des particules virales défectives est suffisant afin de réguler positivement ULBP2 et d’augmenter leur lyse par des cellules NK autologues. De plus, nous décrivons pour la première fois que Vpr, sous forme soluble, a la capacité d’induire des dommages à l’ADN et de réguler positivement ULBP2 suite à la transduction de différents types cellulaires, incluant des cellules T. Globalement, nos résultats démontrent que Vpr est un facteur viral clé impliqué dans la régulation positive des ligands de NKG2D induite par le VIH-1. Cette régulation positive d’ULBP2 pourrait alors contribuer à la destruction des lymphocytes T CD4+ infectés et non infectés via l’activation des fonctions cytolytiques des cellules NK. Une meilleure compréhension de la contribution de cette activité de Vpr dans la pathogenèse du VIH-1 a le potentiel de permettre le développement de nouvelles cibles ou stratégies thérapeutiques contre le VIH-1. / Chronic immune activation and gradual depletion of CD4+ T cells are hallmarks of HIV-1 infection, which are thought to contribute to the progressive deterioration of the host’s immune response that ultimately leads to AIDS. Paradoxically, the majority of CD4+ T cells that are destroyed are uninfected and causes for this bystander effect of infection on CD4+ T cells remains unclear. Some HIV-1 proteins, including the accessory protein Vpr, are suspected to play a role in this process. Vpr, expressed late during HIV-1 infection, is shown to be incorporated within the budding virions as well as secreted as soluble protein in the extracellular medium from the infected cells. The main biological function of Vpr is the induction of a G2/M cell-cycle arrest through the recruitment of the E3 ubiquitin ligase complex DDB1-CUL4AVprBP and activation of the ATR-mediated DNA damage pathway. One study showed that activation of DNA damage pathways leads to the expression of specific ligands for the activating receptor NKG2D expressed on NK cells, thus triggering NK cell cytolytic function. Interestingly, several evidences suggest that HIV-1 upregulates expression of specific NKG2D ligands on infected CD4+ T cells. However, the viral factor involved in this process remains undefined. The aim of this thesis was to evaluate the role of Vpr in modulating NK cell cytolytic function and its potential involvement in CD4+ T cells depletion. Our work demonstrated that the expression of Vpr, alone or in the context of HIV-1 infection, is sufficient to specifically increase expression of the NKG2D ligand, ULBP2, on primary CD4+ T cells. Consequently, these CD4 T cells become more susceptible to autologuous NK cell-mediated lysis. Our studies have shown that this Vpr-mediated ULBP2 upregulation requires the recruitment of the E3 ubiquitin ligase complex DDB1-CUL4AVprBP and the activation of the ATR-mediated DNA damage pathway. More importantly, we provide evidence that Vpr augments ULBP2 expression on both infected and uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. In that context, we show that delivery of Vpr into uninfected cells via defective viral particles is sufficient to upregulate ULBP2 and increase their susceptibility to autologuous NK cell-mediated killing. In addition, we describe for the first time that soluble Vpr has the ability to induce DNA damages and upregulate ULBP2 upon transducing target cells, including T cells. Overall, our results show that Vpr is a key HIV-1 factor involved in the upregulation of NKG2D ligands induced by HIV-1. This upregulation of UBP2 might contribute to depletion of infected and uninfected CD4 + T cells through activation of NK cell cytolytic functions. A better understanding of the contribution of this new activity of Vpr in HIV-1 pathogenesis has the potential to enable the development of new therapeutic targets or therapeutic strategies against HIV-1.