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Les cellules lymphoïdes innées (ILCs) sont des populations cellulaires d’identification récente, mais leur rôle in vivo chez l’homme reste mal connu. Dans une 1ère étude, nous avons pu montrer qu’un déficit sévère en NK au cours de déficits immunitaires communs variables est associé à un risque accru de manifestations non infectieuses et infectieuses bactériennes sévères, suggérant un rôle protecteur non redondant des cellules NK lorsque le système immunitaire adaptatif n’est pas fonctionnel. Dans une 2ème étude, nous avons montré que des patients atteints de déficits immunitaires combinés sévères ɣc et JAK3 déficients n’ont pas d’ILCs. Après allogreffe de moelle osseuse, le nombre d’ILCs circulantes reste indétectable, sans manifestation clinique notable associée. Ces résultats sont en faveur d’une redondance des fonctions des ILCs chez l’homme, lorsque les fonctions T et B sont conservées. Nous avons ensuite étudié les modifications phénotypiques et fonctionnelles des cellules NK au cours du purpura thrombopénique immunologique, et observé un défaut de production d’interféron-ɣ par les cellules NK circulantes et une augmentation de la cytotoxicité dépendante des anticorps des cellules NK spléniques. Une inhibition des fonctions des cellules NK par les immunoglobulines polyvalentes est également mise en évidence. Enfin, une étude des ILCs circulantes au cours de la maladie associée aux IgG4 ainsi qu’une revue de la littérature sur l’étude des ILCs au cours des pathologies inflammatoires sont rapportées. En conclusion, l’apparente redondance des ILCs chez l’homme ainsi que leur implication en pathologies inflammatoires en font de potentielles cibles thérapeutiques. / Innate lymphoid cells (ILCs) are recently identified components of the immune system, but their functions in vivo in humans are still elusive. In a first study, we show in patients with common variable immunodeficiency that non-infectious inflammatory complications and severe bacterial infections were more frequent in patients with severe NK cell lymphopenia, indicating potential non-redundant immune functions of NK cells when the adaptive immune response is not optimal. In a second study, we observe that in patients with ɣc and JAK3 severe combined immunodeficiencies, all ILC subsets are absent. After hematopoietic stem cell transplantation, ILCs remain indetectable with no susceptibility to disease, suggesting that ILCs might be redundant and dispensable in humans, if T and B cells functions are preserved. In the second part of this thesis, we study phenotypic and functional modifications of NK cell compartment in primary immune thrombocytopenia. Interferon gamma production by the peripheral blood NK cells of ITP patients is decreased. In contrast, splenic NK cells of ITP patients tend to be more efficient in antibody-dependent cell cytotoxicity. Intravenous polyvalent immunoglobulins lead to the inhibition of blood NK cell activation. Finally, we present the preliminary results of a study investigating the modifications of circulating ILCs in IgG4-related disease, and present an extensive litterature review concerning the role of ILCs in inflammatory diseases. In conclusion, the apparent redundancy of ILCs for protective immunity and their pathogenic role in inflammatory diseases make their targeting in humans for therapeutic purposes particularly promising.

S'ha establert un sistema experimental autòleg per a poder estudiar la resposta de les cèl.lules Natural Killer (NK) contra les cèl.lules dendrítiques derivades de monòcits (moDC), infectades pel Cytomegalovirus humà (HCMV). Els nostres resultats mostren que les cèl.lules NK responen contra les moDC infectades per HCMV, que presenten una expressió de les molècules MHC de classe I a superficie reduïda. Específicament, demostrem que la infecció per HCMV disminueix l'expressió en superficie d'HLA-E en les moDC, alliberant així la inhibició de les cèl.lules NK NKG2A+. Mostrem que els NKR anomenats NKp46 i DNAM-1 tenen un paper dominant en el reconeixement de les moDC infectades per HCMV i evidenciem la importància de la dinàmica dels mecanismes d'immunoevassió en la susceptibilitat a la resposta NK. Finalment, trobem que els interferons de tipus I i la IL-12 secretats en resposta a la infecció per HCMV, a més de participar en l'activació de la cèl.lula NK i en la secreció d'IFN-, inhibeixen l'expressió i la funció de NKG2D en les cèl.lules NK, com un mecanisme de regulació potencial per prevenir la reactivitat NK contra cèl.lules veïnes sanes. / Suitable experimental conditions have been established to dissect the role of NK cell receptors (NKR) and cytokines in the NK cell response against autologous human cytomegalovirus (HCMV) infected monocyte derived dendritic cells (moDC). Our results reveal that NK cells are capable of responding to HCMV infected moDC that have down-regulated surface MHC class I molecules. In particular, we prove that HCMV infection decreases surface HLA-E expression on moDC, thus releasing NKG2A+ NK cells from inhibition. We show that NKp46 and DNAM-1 NKR play a dominant role in the recognition of HCMV infected moDC and we provide evidences stressing the importance of the dynamics of viral immune evasion mechanisms in NK cell susceptibility. Finally, we find that type I interferons and IL-12 secreted in response to HCMV infection, beyond their participation in NK cell activation and IFN- secretion, transiently inhibit the expression and function of NKG2D in NK cells, thus providing a potential regulatory feedback mechanism to prevent NK cell reactivity against bystander healthy cells.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.