Natural killer (NK) cells are a type of innate immune cell that can identify and eliminate viral infected cells and cancer cells. NK cells express an array of inhibitory and activating receptors such as natural cytotoxicity receptors, the mouse Ly49 or human KIR family, and NKR-P1 family. The integration of signals that NK cells receive through these receptors controls their activation and ability to kill target cells. Both Ly49 and KIR recognize MHC-I molecules on healthy cells Ly49:MHC-I engagement is essential for functional NK cell development. In the absence of these interactions NK cell is consider as ‘uneducated’ or ‘unlicensed’. Ly49 receptor interactions with MHC-I are critical in an effective NK cell response against cancer. However, the role of unlicensed NK cells in NK-mediated control of viruses is poorly understood. Using NKCKD mice, we sought to determine how the loss of Ly49:MHC-I education, and the concomitant loss of inhibition via MHC-I, affected survival against influenza infection. In this study, we show that Ly49-deficient mice exhibit lower viral load and greater protection than WT mice when infected with influenza. However, this protection was lost when Ly49I was transgenically restored to these mice. Similarly, MHC-I-deficient mice, that also lack educated NK cells, were resistant to influenza infection, and lost this protection when NK cells were depleted before challenge. Based on the markedly reduced inflammation in the Ly49-deficient mice compared to the WT, we conclude that the Ly49-deficient NK cells are swifter and more effective in clearing influenza, resulting in less viral burden and consequentially less need for a dangerously aggressive inflammatory response. Furthermore, influenza infection enhanced MHC-I expression on lung epithelial cells, which could be responsible for inhibition of NK cells. Consequently, blockade of inhibitory Ly49C/I receptors protected WT mice from lethal influenza infection. Additionally, Perforin-deficient NKCKD succumbed to the infection demonstrating that NK cell directly eliminate influenza-infected cells. Collectively, these results confirm that influenza is capable of inhibiting NK cells through MHC-I engagement of KIR/Ly49, and suggests that blocking this interaction may provide a viable therapeutic avenue for severe influenza cases. these results challenge our understanding of basic NK cell function and suggest that, rather than subdividing NK cells into ‘licensed’ and ‘unlicensed’ based on their expression of self-specific Ly49 receptors, a more accurate depiction of these NK subsets would be ‘cancer-specialized’ and ‘pathogen-specialized’. While further work is required to fully test this paradigm of cancer- and pathogen-specialized NK cells, I hope that my findings will stimulate a new appreciation for the role of NK cells in virus control, and lead to a better understanding of this critical immune cell.

<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

<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.

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)

CD20 is a 35kDa surface antigen expressed on B cells from the early pre-B stage through the mature B stage. Moreover, the CD20 antigen is found on a majority of B cell malignancies. Rituximab is a chimeric anti-CD20 monoclonal antibody which has been extensively used alone or in combination in the treatment of CD20+ B cell malignancies including acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphomas (NHL) and chronic lymphocytic leukemia (CLL), as well as in the treatment of numerous autoimmune disorders. Despite its emerging use in the clinic, 30% to 50% of patients with low-grade NHL exhibit no clinical response to Rituximab. Previous work to elucidate the mechanisms of Rituximab resistance has established that antibody dependent cellular cytotoxicity (ADCC) is important as a predominant mechanism of lymphoma cell clearance and that Fcγ receptors (FcγRs) are critical for the in vivo actions of Rituximab in NHL. Natural killer group 2D, NKG2D is a major activating receptor on T lymphocytes and natural killer cells. The NKG2D–ligand (NKG2D-L) interaction triggers an activating signal which results in cytotoxic lysis of the cell expressing the ligand. One potential ligand for murine NKG2D is the retinoic acid early 1β (Rae-1β) protein which is expressed during cellular stress and has a high affinity for the NKG2D receptor in mice. We have recently shown that an anti-HER2-IgG3 fused to murine NKG2D Ligand, Rae1β inhibited HER2+ tumor growth significantly more than Herceptin alone. Similarly, our objective is to enhance the performance of anti-CD20 directed therapy through activation of NK cells by an anti-CD20 antibody encoding the same NK activation ligand. Previous results with anti-HER2-IgG3-Raelβ led us to hypothesize that a CD20 specific fusion protein will bind to CD20 expressing tumor cells and deliver an activation signal to local NKG2D receptors on effector cells triggering a non-FcγR dependent anti-tumor response. Here we show that anti-CD20-NKG2D-L can be synthesized and tested for its ability to bind human CD20 and activate NK cells through the NKG2D receptor in vitro.

Τα φυσικά φονικά κύτταρα (NK) αποτελούν περίπου το 10% των κυκλοφορούντων λεμφοκυττάρων και παίζουν σημαντικό ρόλο στην πρώτη γραμμή άμυνας του ανοσοποιητικού συστήματος. Έχουν την ικανότητα να αναγνωρίζουν καρκινικά και μολυσμένα κύτταρα, τα οποία μπορούν να θανατώνουν απελευθερώνοντας κυτταροτοξικά κοκκία και κυτταροκίνες. Σκοπός της παρούσας εργασίας ήταν η μελέτη της έκκριση IFN-γ και TNF-α, από τα περιφερικά μονοπύρηνα (PBMCs) και ΝΚ κύτταρα, έπειτα από διέγερση με Gram θετικά και Gram αρνητικά βακτήρια (αναλογία ΝΚ κύτταρα προς βακτήρια 10:1), παρουσία ή απουσία εξωγενούς IL-2. Μικρή παραγωγή IFN-γ παρατηρήθηκε μόνο στα PBMCs, με μεγαλύτερη συγκέντρωση στις 20 ώρες έπειτα από διέγερση με L.monocytogenes, ενώ δεν παρατηρήθηκε παραγωγή TNF-α. Ο λόγος ΝΚ κύτταρα: βακτήρια (10:1) που χρησιμοποιήθηκε, η έλλειψη βοηθητικών κυττάρων και ο μικρός χρόνος διέγερσης, πιθανώς να ήταν και οι αιτίες που δεν παρήχθησαν καθόλου κυτταροκίνες από τα ΝΚ κύτταρα, ενώ η χαμηλή παραγωγή IFN-γ από τα PBMCs, πιθανώς να οφείλεται, είτε στην παραγωγή IL-10 από τα μακροφάγα είτε στο ότι το ερέθισμα διέγερσης δεν ήταν αρκετό, ώστε να ενεργοποιηθούν πλήρως. Περαιτέρω μελέτες χρειάζονται για την πλήρη κατανόηση της έκκρισης των κυτταροκινών από τα ΝΚ κύτταρα, παρουσία παθογόνων και δυνητικά παθογόνων μικροοργανισμών. / Natural killer (NK) cells comprise about 10% of all circulating lymphocytes and play crucial role to innate immune responses. NK cells can recognize tumor or infected cells and kill them by the release of cytotoxic molecules and cytokines. The aim of the study was to identify IFN-γ and TNF-α production from peripheral blood mononuclear cells (PBMCs) and purified ΝΚ cells, after stimulation with Gram positive and Gram negative bacteria (in ratio 10 NK:1 bacteria), in the presence or the absence of exogenous IL-2. Only PBMCs produced IFN-γ, with greater amounts after 20 hours stimulation with L.monocytogenes, while no production of TNF-α was observed. The low ratio, the absence of accessory cells and the short time of stimulation, probably, were responsible for the fact that no cytokine were produced from NK cells, whereas the low production of IFN-γ from PBMCs was, probably, due to the production of IL-10 from macrophages Alternatively, it is possible that the ratio and the time points were not enough for the full stimulation of PBMCs. Additional studies are needed to demonstrate the NK cytokine profile pattern, in the presence of pathogens and potential pathogen microorganisms.

Natural killer (NK) cells were so named for their uniqueness in killing certain tumor and virus-infected cells without prior sensitization unlike T lymphocytes. NK cells possess a myriad of activation receptors and cytokine receptors that allow them to recognize stress ligands on infected/tumor cells and respond to the cytokines produced during the inflammatory process. Upon activation, NK cells produce pro-inflammatory cytokines, cytotoxic granules and chemokines to recruit other cells which ultimately result in killing of target cells. These functions of NK cells are modulated in vivo by several immune mediators; IL-15 being the most potent in enabling NK cell homeostasis, maturation and activation. Indeed, IL-15 knockout mice have no detectable NK cells. During microbial infections, NK cells stimulated with IL-15 display enhanced cytokine responses. This priming effect has previously been shown with respect to increased IFN-γ production in NK cells upon IL-12 and IL-15/IL-2 co-stimulation. In this study, I explored if this effect of IL-15 priming can be extended to other cytokines and observed enhanced NK cell responses to stimulation with IFN-, IL-21, IL-2 and IL-4 in addition to IL-12. Notably, we also observed elevated IFN-γ production in primed NK cells upon stimulation through the Ly49H activation receptor. IL-15 treatments induced NK cell proliferation, enhanced NK cell responses to activating stimuli and equipped them with cytotoxic granules thereby “readying” them for battle against infections and tumors. Here, we try to understand the signaling mechanisms underlying IL-15 treatments that activate NK cells. Currently, the fundamental processes required for priming and whether these signaling pathways work collaboratively or independently for NK cell functions are poorly understood. To identify the key signaling events, we examined IL-15 priming on NK cells in which the pathways emanating from IL-15 receptor activation were blocked with specific inhibitors. Our results demonstrate that the PI3K-AKT-mTOR pathway is indispensable for cytokine responses in IL-15 primed NK cells. Furthermore, this pathway is also implicated in a broad range of IL-15 induced NK cell effector functions such as proliferation and cytotoxicity. Given that NK cells are critical for control of viral infections like murine cytomegalovirus (MCMV), we decided to analyze the consequences of blocking the PI3K-AKT-mTOR pathway in NK cells on its anti-viral responses. Likewise, NK cells from mice treated with rapamycin to block the mTOR pathway displayed defects in proliferation, IFN-γ and granzyme B production resulting in elevated viral burdens upon MCMV infection. Taken together, our data demonstrates the requirement of PI3K-mTOR pathway for enhanced NK cell functions by IL-15. It also shows that IL-15 primes NK cell responses to several cytokines and to Ly49H activation receptor stimulation. To our knowledge this is first report to demonstrate the requirement of mTOR activity in NK cells for efficient control of acute MCMV infections; thereby coupling the metabolic sensor mTOR to NK cell anti-viral responses.

Children treated for acute lymphoblastic leukemia (ALL) are immunodeficient and therefore at an increased risk of infection and cancer recurrence. Natural killer (NK) cells are a subset of lymphocytes that are very efficient at combatting infections and cancer; however, children treated for ALL have impaired NK cell number and function. Exercise has the potential to bolster NK cell number and function, at least in healthy children and adults. Limited evidence suggests exercise may also have beneficial effects on NK cells in children treated for cancer. However, these previous exercise immunology studies in children with cancer have yielded low sample sizes. Therefore, the aim of this study was to assess the: 1a) feasibility, 1b) acceptability and 1c) safety of performing an exercise intervention in children with ALL. The secondary objectives were to assess the 2a) effects of acute exercise on NK cell number, function and receptor expression in children receiving maintenance therapy for ALL compared to healthy children, as well as to 2b) assess how the NK response changes over 4 months of therapy, and to 2c) assess the link between physical activity and NK cell number and function at rest in children receiving maintenance therapy for ALL. Children undergoing maintenance therapy for ALL (n=4) were recruited from McMaster Children’s Hospital, and healthy sex and pubertal-status matched children (n=4) were recruited from the Hamilton community. ALL patients completed a total of 3 exercise visits, occurring monthly after their regularly scheduled chemotherapy session. At each exercise visit, children were asked to complete 30 minutes of continuous biking, followed by 1 hour of rest. Blood samples were drawn at rest prior to exercise (PRE), immediately after exercise (POST) and 1 hour into recovery (REC). Healthy children only completed one exercise visit. During recovery, participants were asked to complete a physical activity enjoyment scale (PACES) questionnaire and a structured interview in order to assess exercise acceptability and to gauge participant feedback on study components, respectively. Participants were outfitted with an accelerometer to track physical activity levels between visits. Feasibility was assessed by tracking recruitment statistics, study completion rates and exercise completion rates. Acceptability of accelerometer wear was assessed by tracking accelerometer wear and log rates. Safety was assessed by tracking adverse events. All parameters were reported using descriptive statistics. We approached 22 patients to participate, and 4 children completed the study (100% completion rate) out of a goal of 15. Primary deterrents to participation were that patients and families did not want to extend time spent at the hospital or had time restrictions and that patients were uncomfortable with blood collection methods. Exercise was feasible (94% exercise completion rate), acceptable (4.2 ± 0.38 out of 5 PACES score), and safe. Accelerometer wear rates (61.9% (range 3.7-100.0%)) and log completion rates (69.0% (25.9-100.0)) were moderate. Exercise transiently increased NK cell number and function in healthy children and some children with ALL. There were no patterns in the change of the NK cell response to acute exercise over time. We were unable to assess the link between physical activity and NK cells due to a paucity of data. This study cautiously suggests that exercise is a feasible, acceptable and safe intervention that may increase NK cell number and function in children treated for ALL. / Thesis / Master of Science in Medical Sciences (MSMS) / Children treated for leukemia have weak immune systems, making them more susceptible to developing infections and cancer recurrence. Natural killer cells are a special immune cell that is very effective at combatting cancer and infections; however, children treated for leukemia have very low amounts of natural killer cells and they do not function well. Exercise is a simple way to boost the immune system in healthy adults and children, by increasing the number and function of natural killer cells. We don’t know what effect exercise has on natural killer cells in children with leukemia. Previous studies looking at the effects of exercise on the immune system of children with cancer have not been able to recruit enough children to participate. Therefore, it is also important to investigate why children with cancer may not want to participate in exercise studies looking at immune function. The main goals of this thesis were to assess how likely we are to recruit enough children being treated for leukemia to participate in a study looking at how exercise changes natural killer cells, if our participants enjoyed being part of this study, and how safe exercise is for children being treated for leukemia. We also wanted to learn about how natural killer cells respond to exercise in children being treated for leukemia. We found that most of the children and families that decided not to participate in our study felt they did not have time, and the second most common reason for not participating was because the children experienced anxiety surrounding blood draws for the study. The children that decided to participate in the study enjoyed the exercise and being in the study. We also found that the exercise was safe. Finally, we saw that exercise was able to increase natural killer cell numbers and function in some, but not all, children treated for leukemia. The results of this study suggest that exercise may be a realistic and safe way to improve immune function in some children with leukemia.