<p>Natural killer (NK) cells are lymphocytes of the innate immune response with well-demonstrated activities against viral infections and tumors. Because of these abilities, we sought to glean insights into the mechanisms of NK cell activation so that they may be applied toward the design of new therapies.</p><p>NK cells are particularly critical for the control of poxviral infections. Vaccinia virus (VV) is the most-studied member of the poxviral family. It is robustly immunogenic and functions as the live vaccine responsible for the successful elimination of smallpox. VV infection provides a useful model for studying NK cell activation: NK cells play an important role in its clearance and the virus efficiently activates NK cells and recruits them to the site of infection. We had previously used this model to identify Toll-like receptor (TLR)-dependent and -independent mechanisms of NK cell activation to VV. One method of TLR-independent activation to VV requires the activation receptor NKG2D, which recognizes host ligands expressed upon viral infection by accessory cells such as dendritic cells (DCs) and macrophages.</p><p>In the first aim of this thesis, we sought to determine how the ligands for the NKG2D activation receptor become upregulated in the context of VV infection. Specifically, we asked whether interleukin-18 (IL-18), known to play a role in the innate immune response, could boost the expression of NKG2D ligands on DCs in response to viral infection. Using an in vivo infection model with IL-18R-deficient mice, our results confirmed an important role for IL-18 in NK cell activation to VV and viral control. We then made use of an NK-DC co-culture to show that IL-18 signaling on DCs, in addition to NK cells, is necessary to achieve efficient NK cell activation to viral infection. We further demonstrated in a cell-transfer experiment that cell-extrinsic IL-18 signaling is critical for NK cell activation in vivo. DC ablation via a mouse model designed to specifically ablate CD11c+ cells showed that DCs are also required for NK cell activation to VV in vivo. We finally showed how IL-18 can act on DCs in vivo and in vitro to boost the expression of Rae-1, an NKG2D ligand. Collectively, our data uncover a novel mechanism whereby NK cells become activated by IL-18 control of NKG2D ligand expression on DCs.</p><p>In the second aim of this project, we detailed how IL-18 signaling results in the upregulation of the NKG2D ligand Rae-1. Using an in vitro macrophage model, we showed how recombinant IL-18 was sufficient to upregulate Rae-1 expression. We compared IL-18 control of Rae-1 expression to LPS, a TLR ligand that also signals through the common adaptor MyD88 to govern Rae-1 expression. Using chemical inhibitors to cell signaling molecules, we then identified the importance of MyD88 signaling through PI3K. We then revealed that glycogen synthase kinase 3 (GSK-3) can act as a negative regulator of Rae-1 expression downstream of IL-18/TLR signaling. Specifically, we have shown that during inflammatory signaling, PI3K (acting downstream of MyD88) can inhibit GSK-3 to relieve its tonic suppression of Rae-1 expression and upregulate the NKG2D ligand. Finally, we showed that PI3K and GSK-3 signaling are also important to Rae-1 expression on DCs - the accessory cell where IL-18 signals to control Rae-1 expression to boost NK cell activation against VV.</p><p>In its entirety, this work seeks to address how NK cells become activated in the context of VV infection in order to identify new ways NK cells may be harnessed therapeutically.</p> / Dissertation

Chronic Lymphocytic Leukaemia (CLL) remains the most abundant leukaemia in those aged over 65 years. It is characterised by the expansion of malignant monoclonal B-lymphocytes that were originally described as being functionally incompetent. Identified by immunophenotypic expression of monoclonal light chain restriction, it falls into the classification of chronic B-cell lymphoproliferative disorders (B-LPD). This thesis aims to demonstrate that CD160, an activating NK cell receptor, is aberrantly expressed in B-LPD and can function as a tumour specific antigen, which has clear translation roles within the clinical environment, aiding in the diagnosis of CLL and monitoring of minimal residual disease (MRD). More so, this study aims to provide an insight into the potential biological roles of CD160 within chronic B-cell malignancies. CD160 is an activating NK cell receptor whose major form is a glycosylphosphatidylinositol (GPI)-anchored cell surface molecule with a single immunoglobulin domain. In-vitro studies on a large cohort of B-LPD patients demonstrated that CD160 was primarily restricted to cases of CLL (98%) and Hairy Cell Leukaemia (HCL, 100%) with only a minor population of other B-LPDs expressing the antigen. More so, within the B-cell lineage, CD160 can be considered a tumour specific antigen (TSA) in that when looking for both transcript and protein, they were absent throughout the normal B-cell hierarchy. Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. Disease eradication has been shown to be prognostic. This study demonstrates the feasibility and clinical importance of MRD detection utilising CD160 as novel marker of residual disease. Subsequently, CD160 analysis by flow cytometry (CD160FCA) demonstrated to be as sensitive and specific as other methodologies, and independent of the type of therapy. Further to this the early detection of MRD was correlated with known biological prognostic risk groups. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and M), the time to detection of MRD or relapsing disease ß2using CD160FCA, was significantly shorter than those with a normal or good prognosis. Within normal NK and T lymphocytes, CD160 has a multifunctional role that upon triggering results in a unique profile of cytokine production via the recruitment of Phosphatidylinositol 3-kinase (PI3K). In CLL cells, CD160 stimulation resulted in the recapitulation of these observations including cell survival, an increase in Bcl-2 family antiapoptotic proteins, and cell cycle progression. This thesis has demonstrated that CD160 is aberrantly expressed in malignant B-cells, it has a clear clinical translation role in terms of diagnosis and MRD monitoring, and multiple biological functions which recapitulate those observed in NK-cells.

Les cellules tueuses naturelles (NK pour Natural Killer) sont une population lymphoïde dotées d’une activité cytotoxique contre les cellules infectées ou les cellules cancéreuses. Les cellules NK ont un potentiel thérapeutique considérable en tant que thérapie cellulaire anti-tumorale, particulièrement dans le cadre des leucémies. Ces approches sont basées sur une amélioration de la production de cellules NK à partir de cellules souches hématopoïétiques (quantitative et qualitative en améliorant leur activité lytique), mais aussi sur une manipulation de la sensibilité des cellules leucémiques à la lyse par les cellules NK. L’amélioration de ces approches nécessite une compréhension plus approfondie des différents mécanismes de résistance leucémique et leur relation avec la sensibilité à la lyse. Dans ce contexte, nous avons étudié le rôle de HOXB4 dans la différenciation des cellules NK et leur fonction lytique. Nous avons montré que les cellules CD34+ différenciées en cellules NK en présence de HOXB4 ont un potentiel lytique plus important par rapport aux cellules différenciées en l’absence de HIOXB4. Cette augmentation est associée à une augmentation de la dégranulation des cellules NK en présence de cellules cibles. L’analyse transcriptionnelle globale basé sur un microréseau d'ADN montre une régulation positive de l’expression de granzyme B par HOXB4. Ces résultats démontrent que HOXB4 est un régulateur crucial dans la différenciation et la fonction des cellules NK. Ils soulignent également l’intérêt de son utilisation dans la production de cellules NK fonctionnelles dotées d’un plus grand potentiel lytique pour les stratégies d'immunothérapie anticancéreuse. Nous avons également essayé de comprendre comment l'acquisition de la résistance aux chimiothérapies par les cellules de leucémie aigüe myéloïde (LAM) influence leur reconnaissance et leur sensibilité aux cellules NK. Nous avons montré que l'acquisition de la résistance in vitro des cellules AML à la cytarabine induit une augmentation de leur susceptibilité à la cytotoxicité dépendante des cellules NK. Cette sensibilité accrue est en corrélation avec l’induction d’ULBP (UL-16 binding proteins) 1/2/3, ligands des récepteurs NKG2D, sur les cellules leucémiques résistantes. Cette induction est régulée par un mécanisme impliquant l'induction de c-Myc. Le test d’immunoprécipitation de la chromatine (ChIP) a révélé qu’ULBP1 et ULBP3 sont des cibles directes de c-Myc. L’utilisation de cellules AML primaires résistants à la chimiothérapie comme les cellules cibles, combinée à l'inhibition de c-Myc a entraîné une diminution de l'expression des ligands NKG2D et l'altération de la lyse par les cellules NK. Les propriétés d’alloréactivité des cellules NK pourraient être utilisées pour améliorer les résultats de la transplantation de cellules souches hématopoïétiques allogéniques chez les patients atteints d’AML. Cependant, la résistance croisée (chimiothérapie et NK) des blastes AML reste un problème majeur. Nous avons étudié la relation entre résistance des cellules leucémiques à la daunorubicine, la susceptibilité de ces cellules à la lyse par les cellules NK et l'expression putative des micro-RNAs. Nos résultats indiquent que l'acquisition de la résistance à la daunorubicine par les lignées de cellules parentales induit une résistance croisée à la cytotoxicité naturelle à médiation cellulaire. L'analyse des microréseaux de microRNAs a révélé que cette résistance croisée est associée à une diminution du miR-181a et une augmentation des gènes de la famille tyrosine kinase (MAP3K10 et MAP2K1) et de la famille Bcl-2 (Bcl-2 et Mcl-1). La surexpression de miR-181a dans les blastes AML entraîne l'atténuation de leur résistance à la daunorobucine et à la lyse par les cellules NK. / Natural Killer (NK) cells are a lymphoid population with potent cytotoxic activity against virus-infected or cancer cells, and which hold considerable potential for cell based therapies targeting human malignancies. Potential approaches include not only enhancing the generation of NK cells in number and improving their lytic activity, but also manipulating the susceptibility of blast cells to NK-mediated killing. Pursuing these approaches will require a more thorough understanding of the different mechanisms of resistance and their relationship with susceptibility to NK-mediated killing. In this context, we studied the role of HOXB4 in NK cells differentiation and lytic function. We showed that HOXB4 transduced MS-5 cells as compared with GFP-transduced MS-5 cells induced highly differentiated cytotoxic NK cells. This difference was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the upregulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy. We attempted to elucidate how acquisition of drug resistance in AML cells influences NK cell recognition and the killing of drug-resistant blasts. We showed that the in vitro acquisition of AML cell resistance to cytarabine resulted in an increase in their susceptibility to NK-mediated cell cytotoxicity. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML. Manipulating NK-cell alloreactivity might improve outcomes after hematopoietic stem-cell transplantation; however, cross-resistance among blasts remains a drawback. We attempted to investigate the relationship between AML to daunorubicin, the susceptibility to NK cellmediated cell lysis and the putative expression of miRs. Our results indicate that the acquisition of resistance to daunorubicin by the parental cell lines resulted in the acquisition of a cross-resistance to natural cell-mediated cytotoxicity. miR microarray analysis revealed that this cross-resistance was associated with miR-181a down regulation and the subsequent regulation of the tyrosine kinase (MAP3K10 and MAP2K1) and the BCL-2 (BCL-2 andMCL-1) families. Overexpression of miR-181a in AML blasts resulted in the attenuation of their resistance to daunorobucin and to NK-cell-mediated killing.

<p>The maternal immune system must be able to adjust during pregnancy and accept the foetus that expresses paternal antigens. These changes are found both in placenta and circulation, including a mild inflammatory response. NK cells are abundant during the early part of pregnancy in placenta and are thought to be important for placental development. During preeclampsia the placenta is poorly developed, together with an escalated pro-inflammatory profile noticed in both placenta and circulation. We wanted to study NK cells in placenta and circulation from preeclamptic cases as well as levels of cytokines. HMGB1, an alarmin involved in inflammation, was also measured in preeclamptic placentae.</p><p>When studying preeclamptic placentae in third trimester we found higher numbers of NK cells as well as a higher expression of CD94+ NK cells. We also found slightly elevated levels of HMGB1 together with significantly lower expression of IL-12 in preeclamptic placentae. Further, the NK cell activating cytokines IL-12/IL-23p40 and IL-15 in sera from preeclamptic women were increased compared to healthy pregnancies. The elevated levels of NK cell activating IL-12/IL-23p40 and IL-15 found in preeclamptic sera, made us investigate the circulating NK cells in preeclampsia. However, no differences were seen between healthy and preeclamptic pregnancies.</p><p>The main immunological alterations in third trimester preeclamptic pregnancies with regard to NK cells were found in placenta. Altered maternal cytokine levels in placenta could influence decidual NK cells in preeclampsia, noticed by their higher numbers and altered receptor expression. If these alterations also exist during early pregnancy it could result in a poorly developed and dysfunctional placenta.</p>

The maternal immune system must be able to adjust during pregnancy and accept the foetus that expresses paternal antigens. These changes are found both in placenta and circulation, including a mild inflammatory response. NK cells are abundant during the early part of pregnancy in placenta and are thought to be important for placental development. During preeclampsia the placenta is poorly developed, together with an escalated pro-inflammatory profile noticed in both placenta and circulation. We wanted to study NK cells in placenta and circulation from preeclamptic cases as well as levels of cytokines. HMGB1, an alarmin involved in inflammation, was also measured in preeclamptic placentae. When studying preeclamptic placentae in third trimester we found higher numbers of NK cells as well as a higher expression of CD94+ NK cells. We also found slightly elevated levels of HMGB1 together with significantly lower expression of IL-12 in preeclamptic placentae. Further, the NK cell activating cytokines IL-12/IL-23p40 and IL-15 in sera from preeclamptic women were increased compared to healthy pregnancies. The elevated levels of NK cell activating IL-12/IL-23p40 and IL-15 found in preeclamptic sera, made us investigate the circulating NK cells in preeclampsia. However, no differences were seen between healthy and preeclamptic pregnancies. The main immunological alterations in third trimester preeclamptic pregnancies with regard to NK cells were found in placenta. Altered maternal cytokine levels in placenta could influence decidual NK cells in preeclampsia, noticed by their higher numbers and altered receptor expression. If these alterations also exist during early pregnancy it could result in a poorly developed and dysfunctional placenta.

No description available.

Thèse de doctorat : Immunologie : Paris 12 : 2006. / Bibliogr. : 199 réf.

The WHO predicts that by the year 2035 the world will be facing a “cancer tidal wave”. This has spurred on the development of many cancer immunotherapies. The adoptive transfer of ex vivo expanded NK cells is one such therapy that could have high efficacy and target specificity. However, the adoptive transfer of NK cells has some negative side effects. Fortunately, these are not due to the direct effects of the NK cells. Instead, toxicity arises from the systemic administration of IL-2 which supports NK cell function. To sidestep the need for IL-2 injections our project investigated the effect of stimulating NK cells with interleukin 12, 15, and 18 in vitro. Our hope is that one-day pre-stimulation of NK cells with these cytokines in vitro before their adoptive transfer will maintain NK cell activation and survival in vivo. Our research has revealed that ex vivo expanded NK cells stimulated with IL-12 and IL-18 +/- IL-15 significantly upregulates the expression of IFN-γ, TNF-α, CXCL-8, CCL3L1, and LTA. Furthermore, production of these cytokines can continue up to 72 hours post stimulation in vitro. If the production of these cytokines continues after adoptive transfer of NK cells into cancer patients it could drastically alter the anti-inflammatory milieu of the cancer patient. Our attention was then turned to elucidating the factors responsible for the long term activation of the NK cells in the IL-12 and IL-18 +/- IL-15 conditions. We have determined that the increase in production of proinflammatory cytokines is due to direct increases in IFN-γ transcription. The results of these trials will direct the future use of NK cells in clinical trials. Specifically, there is great potential for this research to be used to predict potential negative side effects of using ex vivo expanded and stimulated NK cells as a cancer immunotherapy. / Thesis / Master of Science (MSc)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.