LIST OF ABBREVIATIONS AND ACRONYMS 7-AAD : 7-aminoac)nomycine ALL: Acute lymphoblas)c leukemia AML: Acute myeloid leukemia APAF-1: Apopto)c protease ac)va)ng factor 1 APC : Allophycocyanine

BCA: Bicinchoninic acid B-EBV LCL: Epstein-Barr virus-transformed human B-lymphoblastoid cell

ACKNOWLEDGEMENTS

The study that is presented here is a synthesis of a story that started in August 1 st , 2010, when I first arrived in Canada. Since then, many different people contributed to make it more than a research work.

I want to thank Élie Haddad for such incredible relaNonships. In my work, he made me learn experimental controls, rigor, experimental controls, creaNvity, experimental controls, analogies, experimental controls, and so much more. More than that, I believe I have been very lucky to meet a pediatrician, physician, with a very simple and direct contact. Someone that, as a paNent or a family, I know I would like to meet. Un immense merci Élie, ce travail avec vous a été simplement génial et le meilleur moment de ma vie.

Canada has been an amazingly welcoming country. The very beginning was a complicated start, arriving alone in an unknown land. But I was lucky enough to meet people that I definitely keep in my mind and in my heart.

I want to thank: Silvia Selleri, the Italian source of inspiraNon to whom I owe many knowledges in science and immunology; Mame Massar Dieng and Vimal Krishnan, incredible friends, a family; Françoise Le Deist, for her help, her rigor, her mentorship, many thanks to her; Isabelle Louis, Panojot Bifsha, François Fontaine, Kathie Béland, Renée Dicaire, Ludovic Durrieu, Joëlle Grégoire-Gauthier, Arnaud Duval, Yuanyi Li, Mark Abramovitz, Zoulfia Allakhverdieva, Didier Doranger, Aurélien ColamarNno, Chloé Colas, Hugo Roméro, in my lab.

Et quel laboratoire! I also need to thank my roommates: Ma)hew Morantz, actually as a part of my family; Marigona Uka, a sweet friend. I wish them both the best. And George Fisher as well.

Looking at these names, you understand Canada and its strength. I'm proud to have been part of it. Back to France, I have to thank Olivier Hermine for his help and mentorship in this project.

Thanks to him, we were able to pursue the work and I was allowed to do my PhD. His humanity and his devoNon are precious for paNents. Merci Olivier pour ces fulgurances et ces échanges plus qu'enrichissants, parfois tardifs pour ne pas dire nocturnes! Je garde en mémoire un départ dans la nuit parisienne en vélo, comme un moment privilégié. I want to thank in my lab: Mathilde Lamarque and her delicious cakes; Hervé Souchet, a sailor climbing partner; Morgane Cheminant, very efficient and a clever mind; Moussab Tagi, a surprising friend. The lab 217 was an amazing atmosphere. Thank you to Francine Côté, a bridge between Montreal and Paris, Joël Babdor, a climbing friend sharing interesNng ideas.

Thanks to all the members of the team Hermine: Flavia Guillem, Geneviève Courtois, Guilleme)e Fouquet, Leila Maouche-ChréNen, Lucile Couronné, Yves LepelleNer… and all the others.

I also need to thank many people from Imagine insNtute, Olivier Pellé and Aude Magerus for their help with the Fortessa, asso YR2I, and colleagues and friends from many labs (Benoît, Antoine, David, Clarisse, Christelle…).

In Lyon, I thank Laurent Jacqueroud, a true friend with whom we have been through the challenging steps of the MSc before we started our PhD. Besides, Pierre Cochat, Charles Dumontet, Caroline Moyret-Lalle, Yves Bertrand and Alexandre Belot were very important for me, especially to build a consistent medical and scienNfic career path.

I also want to thank our collaborators worldwide. Their help is crucial. It makes our science feasible.

Thanks to the ARC FoundaNon for their financial support for the 4 th year of PhD; The Liliane Be)encourt School of Inserm, and the Be)encourt-Schueller FoundaNon for their scholarship during the medical school.

Thanks to Catherine Blish, Alain Fischer, Eric Solary, Felipe Suarez and Thierry Walzer for their role in the evaluaNon of this manuscript. It's a great honor and a privilege for me.

My parents and my brother are the cornerstones of my personality. I owe them everything.

Merci à la famille Nicoler, nous quatre, un solide repère, une direcNon. I started these acknowledgements wriNng it was more than a research work. I'm looking forward to the next stopovers. VSV-G: Vesicular stoma))s virus G-protein WPRE: Woodchuck hepa))s virus poseranscrip)onal regulatory element XHIGM: X-linked immunodeficiency with hyper-IgM XLP: X-linked lymphoprolifera)ve disease X-MAID: X-linked moesin-associated immunodeficiency XMEN: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infec)on, and neoplasia syndrome Table 1: 1.

INTRODUCTION AND PROBLEM STATEMENT

IMMUNE FUNCTION OF NATURAL KILLER CELLS

In 1975, two ar.cles reported the discovery of large granular lymphocytes capable of killing a target cell spontaneously, without requiring neither a priming step nor the expression of major histocompa.bility complex (MHC) molecules on target cells [START_REF] Herberman | Natural cytotoxic reac=vity of mouse lymphoid cells against syngeneic acid allogeneic tumors[END_REF].

Since then, the so-called Natural Killer (NK) cells have been the subject of intensive inves.ga.on, from their ontogeny to their effector func.ons. It is now well-established that these cells contribute to defending the organism against infec.ons and cancer, and also play a role in immune homeostasis through selec.ve elimina.on of cells that were involved in the immune response [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF].

The propor.on of systemic NK cells among peripheral blood mononuclear cells (PBMCs) is generally considered to be between 5-15% [START_REF] Lotze | Natural Killer Cells[END_REF]. NK cells are also found in some organs in which they can represent a more significant part of all lymphocytes, such as secondary lymphoid .ssues, mucosal .ssues, liver (where they can encompass up to 30-50% of all lymphocytes), lungs, skin, uterus, pancreas, joints and central nervous system [START_REF] Björkström | Emerging insights into natural killer cells in human peripheral =ssues[END_REF][START_REF] Shi | Organ-specific features of natural killer cells[END_REF].

Because they exhibit features reminiscent of innate immunity such as the ability to be rapidly ac.vated and respond to mutated or infected cells, and the absence of priming step requirement or of receptor rearrangement from their germline configura.on, NK cells have been considered innate lymphocytes [START_REF] Caligiuri | Human natural killer cells[END_REF][START_REF] Vivier | Natural Killer Cells[END_REF]. However, recent works have challenged this view [START_REF] Cerwenka | Natural killer cell memory in infec=on, inflamma=on and cancer[END_REF][START_REF] Holmes | Natural killer cell memory in context[END_REF][START_REF] Paust | Natural killer cell memory[END_REF][START_REF] Sun | Adap=ve immune features of natural killer cells[END_REF]: adapta.ve or memory NK cells have been iden.fied in humans in response to cytomegalovirus infec.on [START_REF] Schlums | Cytomegalovirus infec=on drives adap=ve epigene=c diversifica=on of NK cells with altered signaling and effector func=on[END_REF] following pioneering work in mice, but also murine hapten-specific liver NK cells [START_REF] Paust | Natural killer cell memory[END_REF], as well as an.gen-specific NK cells in non human primates [START_REF] Reeves | An=gen-specific NK cell memory in rhesus macaques[END_REF][START_REF] Walzer | Memory NK cells discovered in non-human primates[END_REF]. Also, cytokine-induced memory NK cells, that can be generated in vitro, show increased prolifera.on, cell recovery, and effector func.on in vivo [START_REF] Ni | Sustained effector func=on of IL-12/15/18-preac=vated NK cells against established tumors[END_REF][START_REF] Romee | Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia[END_REF]. Furthermore, the expression of the recombina.onac.va.ng gene proteins RAG1 and RAG2 (collec.vely, RAG) and of RAG endonuclease ac.vity during NK cell ontogeny, which play a cri.cal role in T and B cells development as shown in the related severe combined immunodeficiency (SCID) [START_REF] Fischer | Severe combined immunodeficiencies and related disorders[END_REF], appears to impact NK cell phenotype, fitness and func.on both in mice [START_REF] Karo | The RAG Recombinase Dictates Func=onal Heterogeneity and Cellular Fitness in Natural Killer Cells[END_REF] and humans [START_REF] Dobbs | Natural Killer Cells from Pa=ents with Recombinase-Ac=va=ng Gene and Non-Homologous End Joining Gene Defects Comprise a Higher Frequency of CD56(bright) NKG2A(+++) Cells, and Yet Display Increased Degranula=on and Higher Perforin Content[END_REF]. Thus, NK cells blur the well-established func.onal dis.nc.ons between the two arms of the immune system, namely the innate and adap.ve immuni.es (Vivier et al., 2011).

Recently, several groups have redefined the conven.onal immune dichotomy to further dis.nguish adap.ve lymphocytes from innate-like ones, with evidence for overlapping func.ons [START_REF] Bedoui | Parallels and differences between innate and adap=ve lymphocytes[END_REF]. Among the laeer, innate lymphoid cells (ILCs) were conceptually theorized as a large family of cells that mirror the phenotype and ac.vity of differen.ated T cells [START_REF] Eberl | Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology[END_REF][START_REF] Oskarsson | La rechute survient chez environ 20% des pa3ents pédiatriques et 60% des pa3ents adultes (Perova et al[END_REF]. In this model, NK cells are the counterpart of the cytotoxic CD8 + T cells due to their shared ability to kill infected, stressed, or transformed cells (Figure 1).

NK cells are the only cytotoxic ILCs and the major circula.ng subset although all ILC groups can be detected in peripheral blood from children and adults [START_REF] Vély | Evidence of innate lymphoid cell redundancy in humans[END_REF]. As with T- Signals from injured or infected .ssues ac.vate ILCs whose func.on will mirror those of CD8 + and CD4 + T cells. The main difference between both cell types is the kine.cs of the immune response. Adapted from [START_REF] Eberl | Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology[END_REF][START_REF] Oskarsson | La rechute survient chez environ 20% des pa3ents pédiatriques et 60% des pa3ents adultes (Perova et al[END_REF]. [START_REF] Gao | Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells[END_REF].

NK cells (Killer

Overall, many parallels can be drawn between NK cells and CD8 + T cells due to their shared molecular ac.va.on pathways and effector responses, and dis.nct ways of recognizing target cells [START_REF] Narni-Mancinelli | The "T-cell-ness" of NK cells: unexpected similari=es between NK cells and T cells[END_REF][START_REF] Sun | NK cell development, homeostasis and func=on: parallels with CD8⁺ T cells[END_REF].

THE INTERACTION BETWEEN CYTOTOXIC LYMPHOCYTES AND TARGET CELLS

In order to avoid inappropriate immune reac.on promo.ng auto-immunity, i.e. a break of tolerance toward the self, it is of major importance for cytotoxic lymphocytes to efficiently discriminate between infected or transformed cells and normal cells. While CD8 + T cells recogni.on is restricted to an.gens presented by target cells through the major histocompa.bility complex (MHC) class I protein, NK cells immune recogni.on is driven by the "missing self" model, preferen.ally killing cells with low or no MHC class I expression [START_REF] Watzl | Natural killer cell regula=on -beyond the receptors[END_REF]. Such recogni.on allows NK cells to complement T cell-mediated MHC class I-dependent immunosurveillance through so-called natural cytotoxicity [START_REF] Kärre | Selec=ve rejec=on of H-2-deficient lymphoma variants suggests alterna=ve immune defence strategy[END_REF].

This unique feature implies that cells which express MHC-I are protected from NK cellmediated damage by engaging their inhibitory receptors, while cells lacking or with considerably reduced levels of MHC-I are not because they display a "missing-self" phenotype (Figure 2). As a corollary, NK cells also target healthy allogenic cells because they lack self-MHC-I molecules [START_REF] Kärre | Natural killer cell recogni=on of missing self[END_REF].

Nevertheless, the laeer model has been expanded by the recogni.on that both ac.va.ng and inhibitory receptors play a role in NK cell func.on: the expression of "induced self" ligands by target cells can overcome the inhibi.on induced by MHC class I-specific inhibitory receptors (Mar.net and Smyth, 2015). Thus, NK cell ac.va.on is controlled by a wide range of germline-encoded receptors, either inhibitory or ac.va.ng, depending on the nature of the signal they transmit. As a result of the engaged immune synapse, effector cells integrate both types of signals in their "decision to kill" (Figure 2).

Appropriate progression through cri.cal cell biological steps is essen.al for the triggering of the effector phase and subsequent target cell death. These checkpoints include three consecu.ve stages iden.fied respec.vely as recogni.on, effector and termina.on steps [START_REF] Mace | Cell biological steps and checkpoints in accessing NK cell cytotoxicity[END_REF]. Cell adhesion to the target cell is the first and crucial step both to ensure the subsequent stages of the immune synapse and to avoid uncontrolled cytotoxic response.

LFA-1 is a key molecule for NK cell adhesion. Indeed, other NK ac.va.ng receptors induce socalled inside-out signals to dynamically regulate LFA-1 conforma.on and spa.al distribu.on, highligh.ng its key role [START_REF] Bryceson | Minimal requirement for induc=on of natural cytotoxicity and intersec=on of ac=va=on signals by inhibitory receptors[END_REF].

Overall, the interac.on between effector cytoly.c cells and target cells is of major importance as it will determine whether the targets have to be eliminated. For NK cells, both ac.va.ng and inhibi.ng signals are integrated in an equilibrium that controls the triggering of subsequent effector responses.

THE MAIN RECOGNIZED EFFECTOR PATHWAYS

We previously emphasized the similari.es between NK cells and CD8 + T cells which also share complementary immune func.ons [START_REF] Narni-Mancinelli | The "T-cell-ness" of NK cells: unexpected similari=es between NK cells and T cells[END_REF][START_REF] Sun | NK cell development, homeostasis and func=on: parallels with CD8⁺ T cells[END_REF]. The major dis.nc.on between the two is the induced pathways for ac.va.on aoer target recogni.on. As shown by transcriptomic studies in mice [START_REF] Bezman | Molecular defini=on of the iden=ty and ac=va=on of natural killer cells[END_REF] and to some extent in humans [START_REF] Hidalgo | The transcriptome of human cytotoxic T cells: similari=es and dispari=es among allos=mulated CD4(+) CTL, CD8(+) CTL and NK cells[END_REF], many transcripts are shared between both cell types while NK-specific ones were found to be principally related to NK receptors [START_REF] Hidalgo | The transcriptome of human cytotoxic T cells: similari=es and dispari=es among allos=mulated CD4(+) CTL, CD8(+) CTL and NK cells[END_REF] and were some.mes expressed by other cell types of the immune system, apart from T cells [START_REF] Bezman | Molecular defini=on of the iden=ty and ac=va=on of natural killer cells[END_REF].

At the func.onal level, similari.es have been documented as well, especially for granule exocytosis with common molecular requirements, although cytokine produc.on was notably different in its kine.cs and frequency [START_REF] Chiang | Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for ly=c granule exocytosis but differences in cytokine produc=on[END_REF]. Deep immune profiling by mass cytometry also showed that parallels in cytotoxic molecule expression can be drawn between T and NK cells, with granulysin, perforin and various granzymes (A, B, K and M) detected in both cell types [START_REF] Bengsch | Deep immune profiling by mass cytometry links human T and NK cell differen=a=on and cytotoxic molecule expression paqerns[END_REF].

Moreover, these works established dynamic changes linked to the ac.va.on state of the effector cell. Res.ng NK cells are poor effectors at steady state but can acquire their full effector poten.al once the have been primed [START_REF] Narni-Mancinelli | The "T-cell-ness" of NK cells: unexpected similari=es between NK cells and T cells[END_REF], which implies a key role for dendri.c cells in secondary lymphoid organs through membrane-bound IL-15 trans-presenta.on (Hun.ngton et al., 2009;[START_REF] Lotze | Natural Killer Cells[END_REF]. Among CD8 + cells, cytotoxic molecule expression paeerns are also linked to T cell differen.a.on [START_REF] Bengsch | Deep immune profiling by mass cytometry links human T and NK cell differen=a=on and cytotoxic molecule expression paqerns[END_REF].

Focusing on effector func.ons, NK cells do not require prolifera.on for acquisi.on of effector func.ons but rather exhibit a "pre-armed" state with cons.tu.ve expression of perforin, granzyme A, granzyme B and IFN-γ allowing quick response, similarly to memory CD8 + T cells [START_REF] Narni-Mancinelli | The "T-cell-ness" of NK cells: unexpected similari=es between NK cells and T cells[END_REF]. Furthermore, the epigene.c control of NK cell func.ons, that has been linked to some viral infec.ons like the human cytomegalovirus (HCMV) [START_REF] Cichocki | Epigene=c regula=on of NK cell differen=a=on and effector func=ons[END_REF][START_REF] Schlums | Cytomegalovirus infec=on drives adap=ve epigene=c diversifica=on of NK cells with altered signaling and effector func=on[END_REF], introduces a supplemental level of complexity in the ac.ve regula.on of their responses which parallels CD8 + cells.

Apart from their control, effector pathways poten.ally engaged by cytotoxic lymphocytes can be categorized depending on their dependence to cell contact. On one hand, the secre.on of cytokines (such as TNF-α and IFN-γ) is likely to induce target cell changes through the local milieu, while, on the other hand, granule exocytosis as well as death receptors pathways rely on a direct cellular contact between effector and target cells [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]Marsnez-Lostao et al., 2015) (Figure 3).

. 3 . . C O N T A C T -I N D E P E N D E N T P A T H W A Y S

A vast array of cytokines can be produced by NK cells, including IFN-γ, GM-CSF, TNF-α and IL-10, as well as chemokines such as CCL3, CCL4 and CCL5, and also bactericidal pep.des like α-defensins [START_REF] Chalifour | Direct bacterial protein PAMP recogni=on by human NK cells involves TLRs and triggers alpha-defensin produc=on[END_REF][START_REF] Clark | Bacterial Manipula=on of NK Cell Regulatory Ac=vity Increases Suscep=bility to Listeria monocytogenes Infec=on[END_REF][START_REF] Perona-Wright | Systemic but not local infec=ons elicit immunosuppressive IL-10 produc=on by natural killer cells[END_REF][START_REF] Rajasekaran | Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine produc=on in NK cells[END_REF][START_REF] Reefman | Cytokine secre=on is dis=nct from secre=on of cytotoxic granules in NK cells[END_REF]. Although IFN-γ produc.on has been intensively linked to the control of viruses, however it is interes.ng to note that NK cells not only release both pro and an.-inflammatory factors, but also chemoaeractant mediators and molecules with an.infec.ous proper.es, thus providing a link between NK cell effector and regulatory func.ons within the immune system.

Cytokine produc.on is not equally shared among NK cell subsets although all NK cells can produce cytokines. Thus, CD56 bright c e l l s h a v e b e e n c l a s s i c a l l y d e fi n e d a s a n immunoregulatory subpopula.on because of their greater (and faster) ability to produce a wide range of cytokines in response to monokine s.mula.on [START_REF] Cooper | Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset[END_REF]. However, further work on the regula.on of cytokine produc.on upon target cell recogni.on revealed that the CD56 dim subset is a prominent producer in this sepng [START_REF] Fauriat | Regula=on of human NK-cell cytokine and chemokine produc=on by target cell recogni=on[END_REF].

Another key ques.on is whether cytotoxicity and cytokine produc.on are differen.ally controlled by NK cells. Indeed, ac.va.on of NK cells by tumor or infected cells classically leads to both phenomenons, and interac.on with a suscep.ble target cell line can induce a pro-inflammatory profile of chemokine and cytokine secre.on [START_REF] Fauriat | Regula=on of human NK-cell cytokine and chemokine produc=on by target cell recogni=on[END_REF][START_REF] Jenkins | Failed CTL/NK cell killing and cytokine hypersecre=on are directly linked through prolonged synapse =me[END_REF]. Colocaliza.on and trafficking studies of IFN-γ and TNF-α in human NK cells have suggested that compartments and vesicles do not overlap with perforin or other late endosome granule markers [START_REF] Reefman | Cytokine secre=on is dis=nct from secre=on of cytotoxic granules in NK cells[END_REF]. However, the defini.ve answer was provided by a work demonstra.ng that a dedicated signaling pathway consis.ng of the tyrosine kinase Fyn, the adaptor ADAP and the CBM signalosome (which consists of the adaptors Carma1, Bcl-10 and MALT1) was exclusively responsible for the produc.on of inflammatory cytokines but not for cytotoxicity, uncoupling both effector responses [START_REF] Rajasekaran | Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine produc=on in NK cells[END_REF][START_REF] Vivier | ADAPted secre=on of cytokines in NK cells[END_REF].

Importantly, cytokines which are produced by NK cells can impact targets' sensi.vity and the outcome of NK mediated cytotoxicity. For instance, IFN-γ and TNF-α were shown to act synergis.cally to promote NK cell cytotoxicity through NFκB-dependent up-regula.on of ICAM-1 expression in target cells [START_REF] Wang | Natural killer cell-produced IFN-γ and TNF-α induce target cell cytolysis through up-regula=on of ICAM-1[END_REF]. Contradictory reports have also been published regarding the role of IFN-γ either sensi.zing or altering NK cell mediated cytotoxicity among targets through the up-regula.on of various NK ligands such as PD-L1, MHC-I or FAS [START_REF] Aquino-López | Interferon Gamma Induces Changes in Natural Killer (NK) Cell Ligand Expression and Alters NK Cell-Mediated Lysis of Pediatric Cancer Cell Lines[END_REF]. Taken together, this suggests that cytokine effect may vary depending on target cell type.

. 3 . 2 . C O N T A C T -D E P E N D E N T P A T H W

A Y S 1.3.2.1.

GRANULE EXOCYTOSIS

The main effector mechanisms of cytotoxic lymphocytes are strongly associated with their ability to engage an immune synapse through a membrane cellular contact with target cells.

Cytotoxic granule exocytosis is a major contribu.ng pathway to contact-dependent effector func.on. Pore-forming proteins (perforin and granulysin) and proteases (granzymes) are the two types of molecular effectors contained in specialized secretory lysosomes, the so-called cytotoxic granules [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF][START_REF] Voskoboinik | Perforin and granzymes: func=on, dysfunc=on and human pathology[END_REF]. A serglycin matrix holds them in the granule in an acidic environment where they are maintained inac.ve to avoid selfdestruc.on of the killer cells. Death-inducing enzymes are synthesized as proenzymes which are inac.ve precursors, and will be processed in an ac.ve form only once within the cytotoxic granule [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]. Cytosolic serpins (serine protease inhibitors which inac.vate the granzymes) protect the effector cell from any poten.al leak from the granules.

The granule-mediated death is a well-orchestrated process leading, from the ini.al cell adhesion and signal transduc.on from an ac.va.ng immune synapse, to the cytotoxic granule mobiliza.on and exocytosis (Figure 4). There are four main steps to this interac.on [START_REF] De Saint Basile | Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules[END_REF][START_REF] Krzewski | Human NK cell ly=c granules and regula=on of their exocytosis[END_REF][START_REF] Voskoboinik | Perforin and granzymes: func=on, dysfunc=on and human pathology[END_REF]: (1) the ini.al cell contact between the killer cell and its target, (2) the mobiliza.on of a microtubule-organizing centre (MTOC, to which cytotoxic granules are anchored) toward the cell contact area, (3) the cytotoxic granule delivery and release of the molecular death-inducing effectors into the synap.c cleo and (4) the target cell death per se. At the molecular level, the sequen.al stages are the following. First, cytotoxic granules move along the microtubules toward the MTOC and the immune synapse (polariza.on step). Then, they switch to the filamentous ac.n network and navigate toward the cell membrane (docking step), where they undergo a series of changes and become primed (priming step), before eventually fusing with the cell membrane (fusion step) to release their effector molecules. The synapse is .ghtly sealed and different mechanisms have been shown to contribute to effector cell protec.on toward its own death-inducing molecules. Among them, it has been proposed that the lysosomal-associated membrane protein LAMP-1 (CD107a), which is used as a surrogate marker to assess NK cell degranula.on [START_REF] Alter | CD107a as a func=onal marker for the iden=fica=on of natural killer cell ac=vity[END_REF] and plays an important role for perforin trafficking to ly.c granules [START_REF] Krzewski | LAMP1/CD107a is required for efficient perforin delivery to ly=c granules and NK-cell cytotoxicity[END_REF], protects cytotoxic lymphocyte from damage consecu.ve to degranula.on [START_REF] Cohnen | Surface CD107a/LAMP-1 protects natural killer cells from degranula=on-associated damage[END_REF].

Gene.c defects impairing granule exocytosis highlight its importance in host defense toward infec.ons but also for immune homeostasis [START_REF] De Saint Basile | Cytotoxic granule secre=on by lymphocytes and its link to immune homeostasis[END_REF]: hemophagocy.c syndromes (hemophagocy.c lymphohis.ocytosis syndrome or HLH, also named macrophage ac.va.on syndrome) are caused by impaired NK and cytotoxic CD8 + T cell func.on [START_REF] Ramos-Casals | Adult haemophagocy=c syndrome[END_REF]. HLH is a rare disease which can occur at any age and generally has an infec.ve agent as a trigger. Familial HLH (or FHL) are a subgroup of inherited hemophagocy.c syndromes: their study allowed the iden.fica.on of key molecular effector of granule exocytosis, and demonstra.on of the link between cytotoxicity and immune homeostasis (de Saint [START_REF] De Saint Basile | Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules[END_REF][START_REF] Oskarsson | Les données de survie se sont améliorées avec 85 à 90% de survie chez les enfants aKeints mais également des complica3ons sur le long terme liées au traitement par chimiothérapie (Hunger and Mullighan[END_REF][START_REF] Sieni | Familial Hemophagocy=c Lymphohis=ocytosis: When Rare Diseases Shed Light on Immune System Func=oning[END_REF]. Several gene.c disorders have now been associated with occurrence of HLH (Table 1).

PRF1 and UNC13D muta.ons account for ∼70% of FHL cases [START_REF] Sieni | Familial Hemophagocy=c Lymphohis=ocytosis: When Rare Diseases Shed Light on Immune System Func=oning[END_REF]. They illustrate gene.c defects in cytotoxic granule content (perforin deficiency) or granule priming (Munc13-4 deficiency) and show that altera.on of the granule exocytosis pathway at different steps can lead to severe pathology [START_REF] Feldmann | Munc13-4 is essen=al for cytoly=c granules fusion and is mutated in a form of familial hemophagocy=c lymphohis=ocytosis (FHL3)[END_REF][START_REF] Stepp | Perforin Gene Defects in Familial Hemophagocy=c Lymphohis=ocytosis[END_REF].

Of note, the degree of degranula.on defect can change, from total abroga.on to a rela.ve decrease, par.cularly with hypomorphic muta.ons affec.ng one or mul.ple genes involved in the pathway. Interes.ngly, it has been suggested that the importance of the cytotoxic ac.vity defect correlates with clinical HLH severity [START_REF] De Saint Basile | Cytotoxic granule secre=on by lymphocytes and its link to immune homeostasis[END_REF]. This postulate could explain the variable age-onset of the disease, with a higher threshold for triggering the HLH in older people which would be reached through cumula.ve environmental factors [START_REF] De Saint Basile | Cytotoxic granule secre=on by lymphocytes and its link to immune homeostasis[END_REF][START_REF] Ramos-Casals | Adult haemophagocy=c syndrome[END_REF].

Released death-inducing molecules include perforin, granzymes and granulysin. Perforin is a protein that oligomerizes to form membrane-spanning pores at the target cell membrane.

Granzymes are serine proteases and abundant cons.tuents of the cytotoxic granules, released together with perforin (among others). There are five iden.fied granzymes in humans, namely granzymes A, B, H, K and M [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]. However, coexpression paeerns of cytotoxic molecules reveal that each single CD8 + T cell or NK cell does not express all the types of granzymes, nor perforin or granulysin (at least for uns.mulated cells) [START_REF] Bengsch | Deep immune profiling by mass cytometry links human T and NK cell differen=a=on and cytotoxic molecule expression paqerns[END_REF]. Granzymes act synergis.cally with perforin to induce target cell death [START_REF] Voskoboinik | Perforin and granzymes: func=on, dysfunc=on and human pathology[END_REF]. To date, no gene.c disorders affec.ng granzymes have been described in humans [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]. Finally, granulysin is another membrane-perturbing effector whose expression has been reported in many mammals but not in rodents [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF].

How do these molecular actors interact together? Mechanis.cally, at the target cell level, once effector cells have degranulated they release these death-inducing molecules in the synapse. Perforin will then form pores in the target cell membrane: whether granzymes enter directly into the cytosol or through an endocytosis process was the subject of debate [START_REF] Voskoboinik | Perforin and granzymes: func=on, dysfunc=on and human pathology[END_REF]. Perforin, granzyme and granulysin are released by cytotoxic lymphocytes in the synapse. Direct entry of granzyme through cell membrane perforin pores and the "endosomolysis" hypothesis are depicted. Once in the cytosol, granulysin acts through a similar mechanism, inducing pores in intracellular bacteria and parasites which allows granzyme to induce pathogen elimina.on.

Indeed, an "endosomolysis" hypothesis has been suggested proposing a first step where transient pores in the cell membrane trigger the endocytosis of granzyme, perforin and granulysin, and then pore forma.on in endosomes followed by a second step triggering cytosolic release and subsequent cell death [START_REF] Thiery | Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells[END_REF]. However, the role of granulysin is now appreciated for infec.on with intracellular bacteria and parasites with a two-step process to deliver granzyme into the pathogen (Do.wala et al., 2016;[START_REF] Walch | Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes[END_REF] (Figure 5).

Perforin and granulysin respec.ve roles are closely related to their biochemical ac.vity:

while the former is only ac.ve in cholesterol-containing membranes, the laeer preferen.ally disrupts membranes that does not contain cholesterol (which is the case for bacteria, fungi and parasites) [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]. Granulysin induces a pathogen cell death which is largely oxida.ve and based on two complementary processes: superoxide anion produc.on and inac.va.on of the microbial an.oxidant defenses (Do.wala et al., 2016;[START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF][START_REF] Walch | Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes[END_REF]. Once delivered in the cytosol of the target cell, granzyme cleaves BID to trigger the intrinsic apopto.c cascade, or ac.vates directly the terminal caspases 3 and 7

(Figure 5 and §1.3.2.2). Overall, these molecular mechanisms are highly relevant in the sense that they confer mul.ple possible outcomes following the same ini.al process (granule exocytosis) which are appropriate for various pathogens (virus, bacteria, fungi and parasites) as well as transformed or stressed target cells.

DEATH RECEPTORS

Another cytotoxic weapon of the killer cells' arsenal is the expression of death ligands at their cell surface. These death ligands can engage death receptors and induce the so-called death receptor mediated cell death in target cells. Death receptors (DRs) form a subgroup within the TNFR superfamily, characterized by the shared presence among all DRs of an approximately 80 amino acid long cytoplasmic death domain [START_REF] Ashkenazi | Regulated cell death: signaling and mechanisms[END_REF].

To date, six human DRs have been iden.fied: TNF-R1, CD95 (Fas/APO-1), TRAIL-R1 (DR4), TRAIL-R2 (APO-2/TRICK/DR5/KILLER), DR3 (TRAMP/APO-3) and DR6 [START_REF] Walczak | Death receptor-ligand systems in cancer, cell death, and inflamma=on[END_REF]. Their known respec.ve ligands are TNF, CD95L (FasL), TRAIL, TL1A, and APP respec.vely.

FasL, TRAIL and membrane bound TNF expression have been reported for NK cells in conjunc.on with their ac.va.on profile [START_REF] Bengsch | Deep immune profiling by mass cytometry links human T and NK cell differen=a=on and cytotoxic molecule expression paqerns[END_REF][START_REF] Caligiuri | Human natural killer cells[END_REF][START_REF] Caron | Human NK cells cons=tu=vely express membrane TNF-alpha (mTNFalpha) and present mTNFalpha-dependent cytotoxic ac=vity[END_REF][START_REF] Kashii | Cons=tu=ve expression and role of the TNF family ligands in apopto=c killing of tumor cells by human NK cells[END_REF]. FasL exposure at the effector cell surface is linked to the mobiliza.on of lysosome-related vesicles dis.nct from the degranula.on-associated ones [START_REF] He | CTLs contain and use intracellular stores of FasL dis=nct from cytoly=c granules[END_REF]Leeau et al., 2015). TRAIL expression by NK cells plays a role in the control of cancer cells [START_REF] Kashii | Cons=tu=ve expression and role of the TNF family ligands in apopto=c killing of tumor cells by human NK cells[END_REF][START_REF] Takeda | Cri=cal Role for Tumor Necrosis Factor-related Apoptosis-inducing Ligand in Immune Surveillance Against Tumor Development[END_REF] but also in immune homeostasis through the interac.on between NK cells and autologous immature dendri.c cells (DCs) [START_REF] Peppa | Up-regula=on of a death receptor renders an=viral T cells suscep=ble to NK cellmediated dele=on[END_REF] as well as autologous ac.vated CD8 + T cells [START_REF] Melki | Escape of HIV-1-infected dendri=c cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk--a pivotal role of HMGB1[END_REF].

Figure 6. Extrinsic and intrinsic apoptoFc signaling pathways.

Once bound to the appropriate ligand, death receptors signal through adaptor molecules such as FADD (FAS-associated death domain protein) and ac.vate ini.ator caspase-8 and caspase-10. These either directly ac.vate the effector caspase-3 and caspase-7 or will cleave BID, providing a link toward the mitochondria-dependent cell death signaliza.on.

In the laeer, truncated BID ac.vate BAX and BAK which will mediate mitochondrial outer membrane permeabiliza.on (MOMP) and subsequent release of cytochrome c. Through its interac.on with APAF1 (apopto.c protease ac.va.ng factor 1), it leads to the apoptosome assembly and caspase-9 ac.va.on which will ac.vate then the effector caspase-3 and caspase-7 triggering apoptosis.

FADD

DR mediated signaling can induce target cell death through the extrinsic pathway of apoptosis [START_REF] Ashkenazi | Regulated cell death: signaling and mechanisms[END_REF] (Figure 6). Of note, DRs can also mediate nonapoptosis-related func.ons which include regula.on of cell prolifera.on and differen.a.on, chemokine produc.on and inflammatory responses [START_REF] Guicciardi | Life and death by death receptors[END_REF]. Regula.on between these signaling pathways is expected to be linked to the adaptor molecules: thus, the FAS-associated death domain protein (FADD) is more likely to trigger cell death, while signaling with the TNF type 1-associated death domain (TRADD) is more likely to ac.vate cell prolifera.on and inflamma.on [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF].

It is interes.ng to note that granule exocytosis and death receptor mediated pathways both par.ally overlap and diverge in the molecular mechanisms which are implied in target cell death (Figures 5 and6).

EFFECTOR CELLS HETEROGENEITY AND DIVERSITY

How cytotoxic effector cells are dis.nct from each other is an interes.ng ques.on to address. As previously discussed, CD8 + T cells and NK cells have similari.es in their development, homeostasis and immune role [START_REF] Sun | NK cell development, homeostasis and func=on: parallels with CD8⁺ T cells[END_REF] and can be considered as complementary effectors in their mechanism for target cells recogni.on.

As opposed to adap.ve immunity, innate immune response has been considered as a set of non-specific early defenses, regardless of the threat. Nevertheless, this view has now been challenged. Although the T cell receptor (TCR) confers T cell specificity toward the recognized an.gen, not all the NK cells will react to a threat, underlying their heterogeneity and diversity. The laeer can be defined at the phenotypic as well as the behavioral levels, both being intrinsically correlated.

Regarding the phenotypic differences, some have been long known: CD56 dim CD16 + and CD56 bright C D 1 6 -N K c e l l s u b s e t s a r e c l a s s i c a l l y d i s . n g u i s h e d a s c y t o t o x i c a n d immunoregulatory cells respec.vely [START_REF] Caligiuri | Human natural killer cells[END_REF], based on their func.onal ability to engage cytotoxicity or cytokine produc.on more efficiently. It has been proposed that these two subsets correspond to different stages of NK cell differen.a.on, with CD56 bright CD16 - cells being less mature precursors of CD56 dim CD16 + cells, although some groups have found evidence for a different ontogeny between them [START_REF] Wu | Clonal Tracking of Rhesus Macaque Hematopoiesis Highlights a Dis=nct Lineage Origin for Natural Killer Cells[END_REF]. Another dis.nc.on can be made whether circula.ng or .ssue resident NK cells are considered, which deeply impact the rela.ve propor.on of these two subsets [START_REF] Björkström | Emerging insights into natural killer cells in human peripheral =ssues[END_REF]. In that regard, liver NK cells are a good example of organ-adapted effector cell func.on with an important propor.on of immunoregulatory NK cells within this specific site of immunosurveillance.

Although NK cells lack a recombined an.gen-specific receptor, they express a complex array of germline-encoded receptors whose combinatorial expression defines a massive degree of NK cell diversity. Using a mul.parametric study by mass cytometry, it has been es.mated that one could dis.nguish between 6000 and 30,000 phenotypically NK subpopula.ons within an individual and more than 100,000 NK cell subpopula.ons within a cohort of 22 people [START_REF] Horowitz | Gene=c and environmental determinants of human NK cell diversity revealed by mass cytometry[END_REF]. One can consider that this combinatorial diversity and the mul.plicity of ac.va.ng and inhibitory receptors confers a wide range of triggering thresholds for effector responses.

We have previously discussed cytokine produc.on and cytotoxicity, and it has been shown that they can be uncoupled [START_REF] Rajasekaran | Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine produc=on in NK cells[END_REF][START_REF] Vivier | ADAPted secre=on of cytokines in NK cells[END_REF]. From a "behavioral" point of view, the rela.ve contribu.on in the killing of target cells varies between individual NK cells. Indeed, in a degranula.on-dependent model of cytotoxicity, others demonstrated that a subset of "serial killer" NK cells were responsible for an important propor.on of the total number of kills [START_REF] Choi | Imaging burst kine=cs and spa=al coordina=on during serial killing by single natural killer cells[END_REF][START_REF] Vanherberghen | Classifica=on of human natural killer cells based on migra=on behavior and cytotoxic response[END_REF]. Overall, a classifica.on into five categories was proposed based on the contact number and their outcomes: (1) NK cells that don't interact with target cells; (2) NK cells that interact with targets but don't kill them; (3) NK cells that kill all target cells encountered; (4) exhausted NK cells (i.e. that stop killing aoer a certain number of interac.ons); and (5) NK cells that kill stochas.cally (a strong minority of the effector cells).

NK cell subsets also exhibit divergent adapta.on with regard to their local environment. For instance, it has been showed that the CD56 bright CD16 -cells are more resistant to reac.ve oxygen species-induced stress [START_REF] Thoren | The CD16-/CD56bright subset of NK cells is resistant to oxidant-induced cell death[END_REF], which was shown responsible for tumoral immune escape [START_REF] Aurelius | Monocy=c AML cells inac=vate an=leukemic lymphocytes: role of NADPH oxidase/gp91(phox) expression and the PARP-1/PAR pathway of apoptosis[END_REF].

Furthermore, HCMV infec.on drives the expansion of a so-called adap.ve subset with dis.nct func.onal capaci.es which include on one hand reduced effector responses toward autologous ac.vated T cells, and on the other hand enhanced func.on against virus-infected cells [START_REF] Lee | Epigene=c Modifica=on and An=body-Dependent Expansion of Memory-like NK Cells in Human Cytomegalovirus-Infected Individuals[END_REF][START_REF] Schlums | Cytomegalovirus infec=on drives adap=ve epigene=c diversifica=on of NK cells with altered signaling and effector func=on[END_REF].

Among others, Frank Cichocki et al., taking in considera.on these recent discoveries, proposed an updated model of NK cell subsets that dis.nguish four main subgroups with a dichotomic contribu.on to either immunoregula.on or immunosurveillance (Figure 7).

These studies provide further evidence of the complexity of NK cell responses at the cellular level.

PRE-B ACUTE LYMPHOBLASTIC LEUKEMIA AS A TARGET CELL'S MODEL

Among neoplasia, pre-B cell acute lymphoblas.c leukemia (pre-B ALL) is the most common form of childhood cancer. Long-term survival has improved to reach 85-90% in children,

although intensified systemic and central nervous system-directed chemotherapy causes acute and long-term treatment-related complica.ons [START_REF] Hunger | Acute Lymphoblas=c Leukemia in Children[END_REF]Inaba et al., 2013;[START_REF] Oskarsson | Relapsed childhood acute lymphoblas=c leukemia in the Nordic countries: prognos=c factors, treatment and outcome[END_REF]. However, relapse, which is observed in about 20% of pediatric and in more than 60% of adult pa.ents [START_REF] Perova | Therapeu=c Poten=al of Spleen Tyrosine Kinase Inhibi=on for Trea=ng High-Risk Precursor B Cell Acute Lymphoblas=c Leukemia[END_REF], is s.ll a major concern with a poor prognosis that has not significantly improved since the 80s [START_REF] Bhojwani | Relapsed childhood acute lymphoblas=c leukaemia[END_REF]. Thus, relapsed ALL ranks as the fourth most common childhood malignancy and only 30% of the pa.ents survive [START_REF] Mullighan | Genomic analysis of the clonal origins of relapsed acute lymphoblas=c leukemia[END_REF].

Pre-B cell ALL development is a complex mul.step process and the disease e.ology is likely different between young infant and older children or adults, although no single causal mechanism is expected [START_REF] Greaves | Infec=on, immune responses and the ae=ology of childhood leukaemia[END_REF]. Following a classical view of cancer development, a prenatal origin has been proposed for ALL arising in childhood [START_REF] Marshall | The prenatal origins of cancer[END_REF] while the disease in adults could result from acquired muta.ons [START_REF] Hanahan | Hallmarks of cancer: the next genera=on[END_REF].

This view is supported by the observa.on of dis.nct molecular gene.cs between both groups of pa.ents. For example, BCR-ABL1 (or Philadelphia chromosome) is detected in over 25% of adult versus only 3% to 5% of pediatric ALL cases [START_REF] Mullighan | Molecular gene=cs of B-precursor acute lymphoblas=c leukemia[END_REF].

A causal link between childhood pre-B ALL and infec.on has been reported [START_REF] Greaves | Infec=on, immune responses and the ae=ology of childhood leukaemia[END_REF][START_REF] Greaves | Infec=on and the Perils of B-cell Ac=va=on[END_REF]Marsn-Lorenzo et al., 2015;[START_REF] Swaminathan | Mechanisms of clonal evolu=on in childhood acute lymphoblas=c leukemia[END_REF]. At the pre-B cell stage of B lymphopoiesis, cells exhibit an increase suscep.bility to gene.c lesions that can be exacerbated by abnormal repe..ve or chronic infec.ons, through the associated cytokine signaling and inflamma.on. However, there is no precise view of the individual species of viruses, bacteria, or other pathogens involved. An infec.on-triggered selec.on of pre-leukemic clones has been suggested by the occurrence, among 2% of the pediatric pa.ents, of a transient clinical phase of aplasia in the few months preceding ALL. This phase, which is expected to favor the emergence of preleukemic clones [START_REF] Greaves | Infec=on, immune responses and the ae=ology of childhood leukaemia[END_REF] Sorted blast popula.ons defined with the CD34, CD19 and CD20 surface markers shared the ability to recons.tute and reestablish the complete leukemic phenotype in vivo. These results and others establish a clear difference between AML and B-ALL [START_REF] Belderbos | Clonal selec=on and asymmetric distribu=on of human leukemia in murine xenograps revealed by cellular barcoding[END_REF][START_REF] Rehe | Acute B lymphoblas=c leukaemia-propaga=ng cells are present at high frequency in diverse lymphoblast popula=ons[END_REF]: AML arises within the normal HSC compartment and retains a hierarchy similar to normal hematopoiesis while pre-B ALL is highly polyclonal and stochas.c in its stemness (Figure 8). As a corollary, relapsed ALL clonal diversity is comparable to the one observed at the moment of the diagnosis while clonal survival from diagnosis to relapse is not associated with a muta.on burden [START_REF] Ma | Rise and fall of subclones from diagnosis to relapse in pediatric B-acute lymphoblas=c leukaemia[END_REF][START_REF] Mullighan | Genomic analysis of the clonal origins of relapsed acute lymphoblas=c leukemia[END_REF].

AML B-ALL

In this work, we have been interested in studying the immune control of pre-B ALL cells (that we used as targets) by cytotoxic lymphocytes and more specifically NK cells.

PRIMARY IMMUNE DEFICIENCIES TO DECIPHER EFFECTOR LYMPHOCYTES' MOLECULAR PATHWAYS

One par.cular aspect of our approach was the use of effector cells from pa.ents suffering from various primary immune deficiencies (PIDs), as natural human knockout models.

By defini.on, a cytotoxicity between effector NK cells and targets involves two cellular types.

To study the precise molecular mechanisms governing NK mediated killing, many previous works used chemical approaches like the inhibi.on of the granule exocytosis pathway by EGTA/MgCl 2 (EGTA is a chela.ng agent of divalent ca.ons including Ca 2+ to block exocytosis while the presence of Mg 2+ in excess allows to maintain adherence between effector and target cells), or by concanamycin A that degrades perforin by increasing the pH of ly.c granules. However, a major concern with this strategy is the lack of specificity of these methods that affect both the effector and the target cells. In addi.on, the molecular selec.vity of these drugs is ques.onable as many different processes can be impacted. For example, with EGTA/MgCl 2 , Ca 2+ chela.on also inhibits FasL expression on the cell surface of CD8 + c y t o t o x i c T c e l l s [START_REF] He | CTLs contain and use intracellular stores of FasL dis=nct from cytoly=c granules[END_REF], thus rendering any interpreta.on equivocal.

To circumvent these limits, we aimed to separate effector and target cell study. For this purpose, we used NK cells obtained from primary immune deficient pa.ents to study effector cells, and knockouts generated by CRISPR/Cas9 genome edi.ng technology to study the target leukemic cell lines. We used PIDs whose molecular defects could impact: (1) NK cell ac.va.on, (2) the integra.on of the ac.va.ng and inhibitory signals and (3) the effector response per se [START_REF] Long | Controlling natural killer cell responses: integra=on of signals for ac=va=on and inhibi=on[END_REF].

AIMS

Allogeneic HSCT was ini.ally developed for two reasons [START_REF] Copelan | Hematopoie=c stem-cell transplanta=on[END_REF]. First, it allows complete replacement of an abnormal hematopoie.c system by a normal one (which is of par.cular interest for severe primary immune deficiencies as well as hematological malignancies) [START_REF] Gooley | Reduced mortality aper allogeneic hematopoie=c-cell transplanta=on[END_REF]. Second, it allows dose-intensive/myeloabla.ve therapy for pa.ents suffering from cancer, for whom these high dose regimens increase tumoral cell elimina.on but also induce a permanent bone marrow failure that can be rescued by HSCT.

The grao is as an immunocompetent .ssue considering the immune cells it contains which are able to mediate ac.ve immune responses, either deleterious or beneficial, the so-called grao-versus-host disease (GvHD) and grao-versus-tumor (GvT) effect respec.vely. Both T cells and NK cells have been shown to contribute to GvT.

In 2002, Loredana Ruggeri et al. published an important study establishing a role for NK cell alloreac.vity in mismatched hematopoie.c stem cell transplanta.on (HSCT) [START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF].

Their work laid the founda.ons for this phenomenon which was aeributed to a mismatch between killer immunoglobulin-like receptors (KIR) expressed by donor NK cells within the grao, and KIR ligands (KIRL) expressed at the surface of recipient cells, including tumor cells.

Interes.ngly, they provided evidence in AML but not in pre-B ALL that they found to be resistant to NK-mediated killing [START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF][START_REF] Ruggeri | Role of natural killer cell alloreac=vity in HLA-mismatched hematopoie=c stem cell transplanta=on[END_REF]. Since then, different models have been proposed to predict the rela.ve risk of relapse taking into account donor and recipient's phenotype, including the "ligand-ligand" and the "receptor-ligand" models suggested by the Perugia and the Memphis groups respec.vely (Handgre.nger et al., 2016).

If the former considers KIRL expressed both by the donor and the recipient to establish a poten.al mismatch, the laeer considers the KIRs from the donor and the absence of the corresponding KIRLs in the recipient. Of note, alterna.ve models exist such as the "genegene" model which includes KIR genes from both the donor and the recipient in its predic.on (Handgre.nger et al., 2016). Altogether, each model performs beeer in specific condi.ons sugges.ng that there might be no unique rule for NK alloreac.vity but instead several mechanisms which could account differently depending on the disease type and pa.ents (childhood versus adults).

Deciphering the mechanisms governing it is important from both a fundamental point of view (i.e. to understand molecular pathways which contribute to NK cell ac.va.on and/or inhibi.on against this type of target cells as well as control their effector func.ons), and considering poten.al future developments as an immunotherapeu.c tool.

We worked on understanding NK cell alloreacFvity against pre-B ALL and why do these blasts exhibit an increase resistance to effector cell killing?

CHAPTER 2. METHODS

2.

METHODS

PATIENTS RECRUITMENT

Several centers around the world were involved in recrui2ng pa2ents suffering from various primary immunodeficiencies (PID). Because of the scarcity of PIDs that were interes2ng for our study, we obtained samples from pa2ents followed in many ins2tu2ons over the world.

Hence, we could get samples from Hôpital Necker-Enfants Malades (Paris, France), CHU Saint-Jus2ne (Montreal, QC, Canada), Hospices Civils de Lyon (Lyon, France), Hôpital Saint-Louis (Paris, France) and Cincinna2 Children's Hospital Medical Center (Cincinna2, OH, USA).

Healthy control donors were sampled at the same 2me as the pa2ents bearing confirmed muta2ons, and samples were carried together. This study was accepted by the CHU Sainte-Jus2ne IRB (protocole number 3195) and all the involved subjects (pa2ents and controls) signed informed consent forms reviewed and approved by the appropriate ins2tu2onal medical research ethics commiZee (Table 2).

PERIPHERAL BLOOD MONONUCLEAR CELLS ISOLATION

Peripheral blood from pa2ents and controls were collected in heparin tubes and followed the same procedure. Whole blood was diluted 1:1 in Dulbecco's phosphate buffer saline (D-PBS, Gibco TM ) supplemented with 0.6% CPDA (citrate phosphate dextrose adenine solu2on) and carefully layered on top of Ficoll-Paque PLUS (GE Healthcare Life Sciences) in 50 mL tubes at a ra2o of 3 volumes of diluted blood to 1 volume of Ficoll. Arer spinning (400 g for 30 minutes with brakes off), the Ficoll interphase was recovered and washed in 50 mL Falcon tubes with D-PBS/0.6% CPDA (400 g for 10 minutes). Cells were then resuspended in complete medium (RPMI (Gibco TM ) supplemented with 10% heat-inac2vated fetal bovine serum (FBS) and penicillin-streptomycin (100 IU/mL)) before coun2ng on a hemocytometer (Neubauer improved cell coun2ng chamber); dead cells were excluded by the trypan blue viability test. PBMCs in excess were frozen down in FBS with 10% dimethylsulfoxide (DMSO) and stored in liquid nitrogen for long term preserva2on.

CELL CULTURE

. 3 . 1 . C E L L L I N E S

Human cell lines were purchased from the American Type Culture Collec2on (ATCC) and the Deutsche Sammlung von. Mikroorganismen und Zellkulturen GmbH (DSMZ). B cell precursor leukemias REH (DSMZ no. ACC 22), NALM6 (DSMZ no. ACC 128), 697 (DSMZ no. ACC 42), and RS4;11 (DSMZ no. ACC 508); and chronic myelogenous leukemia K562 (ATCC CCL-243) were cultured and maintained in complete RPMI (RPMI 1640 supplemented with 10% FBS and penicillin-streptomycin (100 IU/mL)) according to manufacturer's instruc2ons. K562-mb15-41BBL or K562-tCD19-CD64-CD86-CD137L-mbIL21 (Clone9.mbIL21) feeder cells used for NK cells expansion were kindly provided by Pr Dario Campana (Na2onal University of Singapore, Singapore) and Dr. Dean Lee (Na2onwide Children's Hospital, OH, USA) respec2vely, and cultured and maintained in complete RPMI medium.

. 3 . . IN VIVO EXPANSION OF PRIMARY PRE-B ALL BLASTS AND CELL

CULTURE

Deiden2fied diagnos2c bone marrow samples were obtained from pa2ents arer wriZen informed consent in accordance with the Declara2on of Helsinki. Following bone marrow mononuclear cells (BMMCs) isola2on, cells were either directly injected into NSG (NOD/LtSzscid/IL2Rγ -/-) mice (Jackson Laboratory) or frozen down and stored in liquid nitrogen.

An op2mal blast expansion was achievable by injec2ng intravenously 5.10 6 ALL blasts.

Depending on the cell recovery arer Ficoll, it was common to inject only one NSG mouse with a subop2mal dose of blasts (total unpurified BMMCs are used). For primary expansion, we injected the highest possible cell number to a maximum of 5 x 10 6 cells and three mice.

At the same 2me, we performed a flow cytometry analysis of the recovered BMMCs to evaluate the CD3 + cells propor2on among injected cells. To avoid a xenogeneic grar-versushost disease (GvHD), mice were treated with an2-human CD3 (50 μg/mouse, OKT3)

intraperitoneally on days 1, 3 and 5 arer human cells injec2on. Time to engrarment following xenotransplanta2on was dependent on the injected total BMMCs number as well as on interindividual varia2ons. Development of human ALL in mice was followed by using peripheral blood sampling, and mice were euthanized when they reached endpoints that met accepted animal care guidelines. Expanded primary blasts were recovered from the spleen and used for further in vivo expansion by secondary xenotransplanta2ons or frozen down and stored for long term preserva2on.

For cytotoxicity assay, expanded blasts were thawed and kept in culture a few hours to get rid of the dying cells, in X-VIVO TM 15 (Lonza) medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (100 IU/mL). Although it is known that primary pre-B acute lymphoblas2c cells are difficult to keep in culture, we observed that the remaining cells following this res2ng passage could survive for the 2me of the assay. Cells were then used for a flow cytometry-based assay following a regular protocol ( §2.5).

NK ACTIVATION AND EXPANSION SYSTEMS (NKAES)

Highly ac2vated NK cells were generated either from fresh or frozen PBMCs, using a previously described co-culture system. Briefly, 100 Gy irradiated K562-mb15-41BBL or Clone9.mbIL21 feeder cells (0.5 x 10 5 cells/mL) were used to s2mulate mononuclear cells (0.75 x 10 5 cells/mL) in RPMI medium supplemented with L-glutamine, 10% fetal bovine serum, penicillin-streptomycin (100 IU/mL) and 40 IU/mL recombinant human IL-2 (Proleukin®, Novar2s), in a 20 mL final volume T75 flask. Arer 3 and 5 days, an equivalent volume of fresh complete medium (containing rhIL-2) was added. Cells were eventually harvested arer 7 days and NK cells expansion and purity were assessed by flow cytometry and either directly used for func2onal assays arer CD3-deple2on or put back in culture for a new expansion week.

For subsequent expansion weeks, irradiated feeder cells (0.5 x 10 5 cells/mL) were used to s2mulate NKAES-NK (0.5 x 10 4 cells/mL) at a ra2o of 1 CD56 + CD3 -cell to 10 feeder cells (with a 20 mL star2ng volume in a T75 flask), and rhIL-2 concentra2on was increased to 100 IU/mL.

NKAES-NK cells were harvested arer 14 days and purity was assessed by flow cytometry (Figure 9).

CYTOTOXICITY ASSAY

First introduced in the 60's, the radioac2ve chromium ( 51 Cr)-release has been considered as The final purity of the effector cells (> 80%) is assessed by flow cytometry before using the NK cells for further func2onal assays.

Although a reliable assay, the 51 Cr-release method suffers from a number of limita2ons including: (i) technical disadvantages taking into account the regulatory procedure for radia2on safety training and licensing, the hazardousness to health, the cost, as well as the short half-life of the radioisotope which make it imprac2cal for unplanned experiments; (ii) intrinsic disadvantages depending on target cells like a variability in the ability to uptake a sufficient amount of 51 Cr for a reliable detec2on of lysis or a strong background due to a high spontaneous 51 Cr release which make the assay uninterpretable; (iii) biological concerns regarding the technical readout since not only the results are uniparametric (meaning death

is not quan2fied at a single-cell level) but also some types of cell death might not been detected because they do not involve a rupture in cell membrane integrity (and consequently do not induce 51 Cr release in the supernatant) or require longer coincuba2on 2mes which will be associated with an excessive background.

Taken together, these reasons prompted us to consider a flow cytometry-based cytotoxicity assay as a befer alterna7ve (Figure 10). Briefly: target cells were stained with PKH26, a membrane labeling dye with long alipha2c tails which stably stains cell membrane. Following coincuba2on with effector cells, the absolute count of living targets was calculated thanks to the addi2on of fluorescent microspheres (CountBright TM Absolute Coun2ng Beads, ThermoFisher) as well as the use of a viability dye (7-AAD and fixable viability dyes were used independently depending on the experiments) and a lineage specific target cells marker (CD19 for pre-B ALL).

PKH26 STAINING OF THE TARGET CELLS

The day (or a few hours) before the FCC assay, a maximum of 2 x 10 6 target cells were washed twice in RPMI 1640 or D-PBS and resuspended in 100 μL of diluent C, then 100 μL of PKH26 (8 μM in diluent C) was added and cells were incubated 5 min at room temperature.

The staining was stopped by adding 200 μL of FBS with a supplemental incuba2on for 1 min.

Cells were then washed three 2mes in RPMI 1640 or D-PBS, and resuspended in complete medium for cell culture.

. 5 . . C D 3 P O S I T I V E C E L L S D E P L E T I O N

Our protocol used the Miltenyi magne2c system with LD columns. Expanded NK cells were harvested, washed in complete medium and absolute count determined by flow cytometry.

Magne2c deple2on was performed according to manufacturer's instruc2ons: cells were resuspended in D-PBS supplemented with 0.5% bovine serum albumin (BSA) and 0.6% CPDA (up to 10 x 10 6 cells per 80 μL) and an2-CD3 beads were added to the suspension (20 μL per 10 x 10 6 cells). Cells were incubated 15 min at 4°C in the dark, then washed (1 to 2 mL for 10

x 10 6 cells) and resuspended in 500 μL of buffer. The magne2c column was rinsed with 2 mL of buffer, then cell suspension was applied and two washes (1 mL each) were done. CD3 posi2ve cells were retained on the column (posi2ve frac2on) while NK cells were harvested in the nega2ve frac2on. Deple2on efficiency was assessed by flow cytometry on recovered cells with CD3 + cells < 10% and CD3 -CD56 + > 80% required for further func2onal assays.

FLOW CYTOMETRY-BASED CYTOTOXICITY (FCC) ASSAY

The day of the FCC assay, effector cells were harvested and their absolute count was calculated by flow cytometry. Cells were then resuspended in a cytotoxicity assay medium (RPMI 1640 supplemented with 2% FBS, penicillin-streptomycin (100 IU/mL) and rhIL-2 (20 IU/mL) at a concentra2on of 4 x 10 5 C D 3 -C D 5 6 + per mL and diluted 1:1 to prepare supplemental ra2os (we systema2cally performed the following effector to target ra2os: 4:1, 2:1, 1:1, 1:2). Target cells were harvested, counted on a hemocytometer, and resuspended in the same medium at a concentra2on of 1 x 10 5 per mL.

The assay was performed in 96 wells round boZom plates with 100 μL of the targets suspension (i.e. equivalent to 10 4 cells) and 100 μL of the effector cells suspension for a final volume of 200 μL per well. The plate was centrifuged at 200 g for 1 minute and incubated (37°C and 5% CO 2 ).

Arer various incuba2on 2mes, plates were recovered: cells were centrifuged at 400 g for 5 minute, stained for 25 min at 4°C in the dark with a viability dye and a lineage specific marker (most oren CD19), washed with 175 μL of FACS buffer (D-PBS supplemented with 2% FBS and sodium azide) and finally resuspended in 215 μL of FACS buffer per well. Prior to acquisi2on, 5 μL of CountBright TM beads (equivalent to 5 x 10 3 cells) were added in each well and cells acquisi2on was performed on a BD LSRFortessa TM X-20 cytometer equipped with a high throughput sampler (HTS). Stopping gate was fixed by the acquisi2on of 2500 beads per well which consequently allowed to calculate the absolute count of remaining living targets.

Specific lysis was calculated with the following formula: % Specific lysis = 100-[(absolute count of PKH26 + 7-AAD -CD19 + targets arer incuba2on with effector cells) / (absolute count of PKH26 + 7-AAD -CD19 + targets arer incuba2on alone) X 100].

DEVELOPMENT OF A VERSATILE LENTIVIRAL PLATFORM

Our first len2viral transduc2ons of the pre-B leukemic cell lines faced previously described difficul2es associated with this technology, i.e. variega2on and silencing of the integrated transgene whose expression was ac2vely and dynamically repressed. Therefore, we developed a homemade len2viral pla~orm with the aim to efficiently transduce effector NK cells as well as target cells.

Ubiquitous chroma2n opening elements (UCOEs) have been intensively inves2gated as a solu2on to these obstacles, showing promising results in maintaining high levels of transgene expression in cells over extended periods of 2me, both in vitro and in vivo. Len2viral vectors for transgene overexpression were generated using the promoter (SFFV) based solu2on.

We developed two systems, namely a promoter-based Gateway cloning system with a SFFV expressing Des2na2on vector (pHRSIN UCOE SFFV DEST plasmid) as well as a promoterless one which is based on a Mul2Site Gateway approach and has been developed to allow the cloning of mul2ple DNA fragments. In the promoterless seÄng, it is possible to choose the promoter which will drive the expression of the insert of interest, as well as to use a Des2na2on vector expressing an IRES GFP fragment.

The final len2viral pla~orm is offering several advantages: (i) Entry vectors are small so that the cDNA cloning is easier, (ii) LR cloning efficiency is extremely high (> 90%) with a strong decrease in the genera2on of unwanted cloning ar2facts and (iii) its versa2lity offers the possibility to easily change the promoter, the expression vector type (len2viral or retroviral vectors) or the detec2on of the transgene expression (with the IRES GFP casseZe).

TRANSDUCTION OF PRE-B ALL CELL LINES

In order to transduce pre-B ALL cell lines, we used vesicular stoma22s virus (VSV) G-protein pseudotyped self-inac2va2ng HIV-1-derived len2viral vectors (VSV-G LVs). Transgene expression is under the control of a spleen focus foamy virus (SFFV) promoter and vectors were modified with the UCOE element to maintain long term transgene expression.

VSV-G LVs were generated by transfec2on of 2.4 x 10 6 HEK 293T cells per 10 cm culture plate in DMEM medium (Dulbecco's Modified Eagle's medium, Gibco TM ) supplemented with 10%

FBS and penicillin-streptomycin (100 IU/mL). Co-transfec2on with polyethylenimine (PEI, a stable ca2onic polymer) was performed with the Gag-Pol packaging construct 8.91 (8.6 µg)

and the self-inac2va2ng (SIN) HIV-1-derived vector (pHRSIN; 8.6 µg). For VSV-G-LV produc2on, 5.5 µg of VSV-G encoding plasmid were co-transfected. 24h later, the supernatant was replaced by Op2-MEM or DMEM. Viral supernatant was harvested 48h later (72h post-transfec2on) and filtrated through a 0.45 µm pore-sized membrane. If needed, ultracentrifuga2on (2h, 4°C, 25000 rpm) was performed to concentrate vectors 300-fold. We also directly used filtrated culture supernatant to perform cell lines transduc2on. In any case, concentrated vectors were aliquoted and stored at -80°C.

Cell lines transduc2on was performed by pla2ng 200 x 10 3 blasts per well in a 48 wells plate and adding 1 mL of non-concentrated viral supernatant with protamine sulfate (8 µg/mL).

The plate was then centrifuged (700 g, 45 min) and the supernatant is replaced 24h later by fresh culture medium. Depending on the len2viral vector, cells were either FACS-sorted or selected with the appropriate an2bio2c selec2on star2ng 72h post transduc2on.

TRANSDUCTION OF PRIMARY ACTIVATED NK CELLS

It is notoriously difficult to efficiently transduce NK cells. However, we developed a protocol showing strong and reproducible transduc2on rates in NKAES-NK cells with the use of len2viral vectors pseudotyped with a baboon retroviral envelope glycoprotein, kindly provided by Dr. Els Verhoeyen (ENS Lyon, France), and further displayed BaEVRless-LVs (BaEVRless-gp was constructed by dele2on of the R pep2de sequence from BaEVwt-gp). For len2viral produc2on, 7 μg of plasmid coding for this glycoprotein was co-transfected (the procedure was otherwise exactly the same than with VSV-G LVs).

NK cells transduc2ons were performed in a 12 wells non-treated 2ssue-culture plate previously coated for 2h at room temperature with retronec2n solu2on (1 µg/µL) followed by coa2ng with 2% BSA solu2on for 30 min. Viral par2cles were plated onto the retronec2ncoated wells for 4h at 37°C. 250 x 10 3 NKAES-NK cells were added per well with protamine sulfate (8 µg/mL), rhIL-2 (100 IU/mL) and IL-15 superagonist/IL-15RαSushi-Fc fusion complex (10 ng/mL, IL-15SA/IL-15RαSu-Fc; ALT-803, Altor BioScience Corpora2on, USA). The plate was then centrifuged (1000 g, 1h) before an overnight incuba2on; 24h later, fresh complete RPMI 1640 medium was added and 72h post-transduc2on, len2viral transduc2on efficiency was assessed by FACS.

CRISPR/CAS9-BASED GENOME ENGINEERING

Two complementary strategies, adapted from the dual vector system developed by Dr. Feng

Zhang lab (MIT, MA, USA) [START_REF] Shalem | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells[END_REF] based on len2viral delivery of the CRISPR/ Cas9 components in the pre-B ALL cell lines, were used depending on the experiments and the number of delivered single guide RNAs (sgRNAs): an uniplex (Figure 12) and a mul2plex genome engineering methods (Figure 13). Len2viral expression vectors for Cas9 and sgRNA mammalian delivery using a dual vector system.

UNIPLEX GENOME ENGINEERING

For individual gene knockout (KO), cell lines were first transduced with the len2Cas9-Blast plasmid (Addgene #52962, Appendix 2 and Figure 12) to derive stably expressing Cas9 cell lines arer blas2cidin selec2on (5 µg/mL). The appropriate sgRNA was cloned into len2Guide-Puro (Addgene #52963, Appendix 3) and the final expression vector was used to transduce Cas9-expressing cell lines. Arer puromycin selec2on (5 µg/mL), single clones were isolated by FACS sor2ng into 96 wells plates.

MULTIPLEX GENOME ENGINEERING

For mul2ple gene knockouts (double KO or DKO in this study), we combined two previously published strategies (Figure 13) [START_REF] Shalem | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells[END_REF][START_REF] Kabadi | Mul=plex CRISPR/Cas9-based genome engineering from a single len=viral vector[END_REF].

CTRL Mul2plex ph7SK-sgRNA CTRL_sgE0_Rv5 AAACCATGTCTCGCCCCGATATGGC CASP3 Mul2plex phU6-sgRNA CASP3_sgE4_Fw4_p1 CACCGGGAAGCGAATCAATGGACTC CASP3 Mul2plex phU6-sgRNA CASP3_sgE4_Rv4_p1 AAACGAGTCCATTGATTCGCTTCCC CASP3 Mul2plex ph7SK-sgRNA CASP3_sgE4_Fw5_p1 CCTCGATTATACATAAACCCATCTC CASP3 Mul2plex ph7SK-sgRNA CASP3_sgE4_Rv5_p1 AAACGAGATGGGTTTATGTATAATC CASP7 Mul2plex pmU6-sgRNA CASP7_sgE5_Fw6_p1 TTGTTTGACTTTGATAAAGTGACAGGT CASP7 Mul2plex pmU6-sgRNA CASP7_sgE5_Rv6_p1 AAACACCTGTCACTTTATCAAAGTCAA CASP7 Mul2plex phH1-sgRNA CASP7_sgE6_Fw5_p1 TCCCAGAAGCACTTGAAGAGCGCCT CASP7 Mul2plex phH1-sgRNA CASP7_sgE6_Rv5_p1 AAACAGGCGCTCTTCAAGTGCTTCT CASP8 Mul2plex pmU6-sgRNA CASP8_sgE5_Fw2_p1 TTGTTTGATGGGTTCTTGCTTCCTTTG CASP8 Mul2plex pmU6-sgRNA CASP8_sgE5_Rv2_p1 AAACCAAAGGAAGCAAGAACCCATCAA CASP8 Mul2plex phU6-sgRNA CASP8_sgE5_Fw2_p2 CACCGGATGTTATTCCAGAGACTCC CASP8 Mul2plex phU6-sgRNA CASP8_sgE5_Rv2_p2 AAACGGAGTCTCTGGAATAACATCC CASP10 Mul2plex phH1-sgRNA CASP10_sgE2_Fw3_p1 TCCCAGGACTTCTCCAGCTTCTTGT CASP10 Mul2plex phH1-sgRNA CASP10_sgE2_Rv3_p1 AAACACAAGAAGCTGGAGAAGTCCT CASP10 Mul2plex ph7SK-sgRNA CASP10_sgE2_Fw3_p2 CCTCGTTTTGAACATCTCTTGGCAG CASP10 Mul2plex ph7SK-sgRNA CASP10_sgE2_Rv3_p2 AAACCTGCCAAGAGATGTTCAAAAC BAK1 Mul2plex pmU6-sgRNA BAK1_sgE3_Fw1_p1 TTGTTTGCACTCCTGCCTGGGAGGACC BAK1 Mul2plex pmU6-sgRNA BAK1_sgE3_Rv1_p1 AAACGGTCCTCCCAGGCAGGAGTGCAA BAK1 Mul2plex phU6-sgRNA BAK1_sgE3_Fw1_p2 CACCGCTGCCCTCTGCTTCTGGTAA BAK1 Mul2plex phU6-sgRNA BAK1_sgE3_Rv1_p2 AAACTTACCAGAAGCAGAGGGCAGC BAX Mul2plex phH1-sgRNA BAX_sgE4_Fw1_p1 TCCCAAGTAGAAAAGGGCGACAACC BAX Mul2plex phH1-sgRNA BAX_sgE4_Rv1_p1 AAACGGTTGTCGCCCTTTTCTACTT BAX Mul2plex ph7SK-sgRNA BAX_sgE4_Fw1_p2 CCTCGTGCTCAAGGTGGGCAGCTGC BAX Mul2plex ph7SK-sgRNA BAX_sgE4_Rv1_p2 AAACGCAGCTGCCCACCTTGAGCAC
Control sgRNAs are depicted in blue.

Regions of the sgRNA complementary to the protospacer are shown in red.

2.6.3.4.

IMMUNOBLOT SCREENING OF CANDIDATE CLONES

For immunoblots, total protein was extracted with 1× RIPA lysis buffer (Millipore) with 1×

protease inhibitor (Roche) and protein concentra2on was determined using the BCA assay (Thermo/Pierce). Cell lysates were resolved on 12% SDS-PAGE gels (Bio-Rad), transferred to PVDF membranes (Millipore) which were blocked overnight at 4°C, and incubated 1h30 at room temperature with the appropriate primary an2bodies: an2-Caspase-3 (clone 3G2; Cell Signaling 9668), an2-Caspase-7 (clone 4G2; MBL Interna2onal M053-3), an2-Caspase-8

(clone 5F7; MBL Interna2onal M032-3), an2-Caspase-9 (clone 5B4; MBL Interna2onal M054-3), an2-Caspase-10 (clone 4C1; MBL Interna2onal M059-3), an2-BAX (clone 3/Bax; BD Biosciences), an2-BAK (clone G317-2; BD Biosciences) and an2-β-ac2n (clone AC-15, Novus Biologicals). Signals were detected using a HRP-conjugated secondary an2body (sc-2005; Santa Cruz Biotechnology) and SuperSignal West Pico Chemiluminescent Substrate (Thermo/ Pierce). Images were captured using G:BOX Chemi WRQ imaging system (Syngene).

CELL CONJUGATION ASSAY

NKAES-NK cells were labeled with PKH26 as previously described ( §2.5.1) and target cells were stained with 5-6 carboxyfluorescein diacetate succinimidyl ester (CFSE; BD Biosciences).

Briefly: targets were washed in D-PBS and then resuspended in a solu2on of CFSE diluted in D-PBS at 0,625 nmol/mL for 7 min at room temperature in the dark, vortexing cells every 1-2 min. The staining was stopped by adding 1 mL of cold FBS for 1 min on ice. Targets were washed twice with culture medium.

For the assay, both effector and target cells are resuspended at 2 x 10 6 cells/mL and mixed with a 1:1 ra2o using 100 μL of each suspension (equivalent to 2 x 10 5 cells). Cell mix was spun at 1000 rpm for 5 min and incubated for 1, 2, 5, 10, or 20 min at 37°C. Arer gentle resuspension, cells were analyzed by flow cytometry.

In this assay, we studied as targets K562 with (nega2ve control) or without (posi2ve control)

EDTA 1 mM, as well as NALM6 and REH cell lines.

DEGRANULATION ASSAY

Evalua2on of NK cells degranula2on was performed via the detec2on of CD107a and CD107b on the cell surface arer s2mula2on with various target cells. Unstained effector and target cells were cul2vated at an E:T ra2o of 1:1 in a final volume of 200 μL following the same cell culture condi2ons than a conven2onal cytotoxicity assay ( §2.5). A mix of an2-CD107a (clone H4A3; BD Biosciences) and an2-CD107b (clone H4B4; BD Biosciences) an2bodies were added from the beginning of the assay. Arer a 1h incuba2on, 13.3 µL of a diluted 1:100 monensin solu2on (GolgiStop, BD Biosciences) were added per well and incuba2on was pursued 3 h. At the end of the assay, cells were washed in D-PBS and a cell surface staining was performed with an2-CD3 (clone UCHT1; BD Biosciences) and an2-CD56 (clone B159; BD Biosciences)

an2bodies before acquisi2on at the FACS. The percentage of NK degranula2on was compared between effector cells s2mulated with various condi2ons against targets or medium alone.

BLOCKING EXPERIMENTS

In these experiments, NK cells FcR blockade was performed with FcR blocking reagent from Miltenyi Biotec according to manufacturer's instruc2ons to avoid induced an2bodydependent cell-mediated cytotoxicity (ADCC). Up to 10 x 10 6 cells were resuspended in 90 µL of a D-PBS/0.5% BSA/0.6% CPDA buffer and 20 µL of FcR blocking reagent, then incubated for 15 min at 4°C in the dark and washed with buffer.

To study their rela2ve contribu2on to the cytotoxicity effect, unconjugated an2bodies directed against NKG2D (clone M585; Amgen) and DNAM-1 (clone DX11; BD Biosciences) on NK cells, or against DR4 (TRAIL-R1, clone HS101; Enzo Life Sciences) and DR5 (TRAIL-R2, clone HS201; Enzo Life Sciences) on target cells were preincubated with the appropriate cells at a final concentra2on of 20 μg/mL for 30 min at room temperature. An irrelevant isotype an2body (MOPC21; BD Biosciences) was used as control.

In some experiments, NKp30, NKp44, NKp46 and NKp80 surface markers were blocked with Fc chimera proteins (Figure 14) to prevent these NCRs engagements. Targets were pre-incubated with a satura2ng Ig Fc concentra2on (5 μg/mL per chimera) for 30 min at room temperature before pursuing a conven2onal cytotoxicity assay.

FLOW CYTOMETRY STAININGS

Regular cell surface staining was performed by incuba2ng cells for 25 min at 4°C with an2bodies and a viability dye in D-PBS/2% FBS/sodium azide (0.2 mg/mL) in a final volume of 50 μL. Cells are then washed with this buffer.

For intracellular staining (BD Cytofix/Cytoperm; BD Biosciences), cells were fixed and permeabilized with 100 µL of Cytofix/Cytoperm buffer for 20 min at 4°C, then washed twice in Perm/Wash buffer. Conjugated an2bodies were then added in 50 µL of Perm/Wash buffer for 20 min at 4°C. Cells were finally washed twice with Perm/Wash buffer and resuspended in regular FACS buffer before acquisi2on. The following PE-conjugated an2bodies were purchased from Santa Cruz Biotechnology: an2-p22-phox (clone 44.1), an2-p47-phox (clone D-10), an2-p67-phox (clone D-6) and irrelevant isotype control an2bodies IgG1 and IgG2a.

An2-flavocytochrome b558 (clone 7D5; MBL Interna2onal D162-5) was used both for intracellular and cell surface stainings.

ANNEXIN V STAINING

Cells were harvested, washed in D-PBS and resuspended in Binding Buffer (BD Biosciences).

Staining was performed (15 min at room temperature in the dark) adding 2 µL of annexin-V and others cell surface an2bodies. Arer washing, cells were resuspended in Binding Buffer with 7-AAD and acquisi2on to the flow cytometer was performed within 1h (Figure 15).

MITOCHONDRIAL DEPOLARIZATION ASSAY

We used the ra2ometric fluorescent dye JC-1 to assess mitochondrial poten2al, a key readout of mitochondrial func2on. Briefly: we prepared a JC-1 stock solu2on by dilu2ng a vial of dye in 230 µL of DMSO (then kept at 4°C

MEASUREMENT OF INTRACELLULAR CASPASES ACTIVATION

To determine intracellular ac2va2on of specific caspases, fluorescent labelled inhibitors of caspases (FLICA) probe assays (ImmunoChemistry Technologies) were performed. Each FLICA probe contains a 3 or 4 amino acid sequence targeted by a specific ac2vated caspase (there is no interference from pro-caspases or the inac2ve form of the enzymes). FLICA probes are cell-permeable and covalently bind to the ac2ve forms of specific caspases. Arer washing, FLICA fluorescent signal is specifically retained within cells containing the appropriate ac2ve form of the caspase while the reagent is washed away in cells lacking the appropriate ac2ve caspase.

Briefly: we prepared FLICA stock solu2ons by dilu2ng vial of probes in 50 µL of DMSO (then kept at -20°C). Staining was performed by adding 2 µL of a working solu2on (stock solu2on diluted 1:5 in D-PBS) into a final volume of 200 µL per well. Cells were incubated 15 min at 37°C, then washed twice in Apoptosis Wash Buffer from the kit and finally resuspended in the same buffer before acquisi2on. If required, subsequent cell surface as well as annexin-V stainings were performed in the same buffer.

CHARGE-BASED CAPILLARY NANO-IMMUNOASSAY (NANOPRO ASSAY)

The NanoPro 1000 system (ProteinSimple) is built on an automated, capillary-based immunoassay pla~orm and enables a rapid and quan2ta2ve analysis of specific proteins.

RNA-SEQUENCING

EXPERIMENTAL DESIGN

Blood mononuclear cells from three pa2ents suffering from X-linked chronic granulomatous disease (X-CGD) with complete CYBB gene dele2on (depicted P12, P17 and P19 in Table 2) , as well as those from four normal donors, were isolated by Ficoll-Paque centrifuga2on ( §2.2).

Res2ng NK cells were purified from PBMCs cells by nega2ve selec2on with magne2c

MicroBeads (Miltenyi Biotech) using LS columns. The purity of the isolated cells was verified by flow cytometry and used as a correc2on factor in downstream bioinforma2cs analysis (Figure 17).

CD3 depleted NKAES-NK cells, either arer a 2 weeks expansion or following respec2ve exposure to a sensi2ve (REH) or a resistant (RS4;11) pre-B ALL cell line, were isolated by CD56 posi2ve selec2on. Magne2c MicroBeads (Miltenyi Biotech) using LD (for CD3 + deple2on) and MS (for CD56 + selec2on) columns were used. The purity of the isolated cells was verified by flow cytometry and used as a correc2on factor in downstream bioinforma2cs

analysis (see below). Cells (approximately 1 x 10 6 per condi2on) were immediately lysed in 600 µL of QIAzol and frozen at -80°C.

RNA EXTRACTION, LIBRARY PREPARATION AND SEQUENCING

Once recruitment was completed, samples were all shipped to Montreal. Total RNA was extracted using the miRNeasy kit (QIAGEN) and RNA quan2ty was evaluated spectrophotometrically. A total of seven donors (3 X-CGD and 4 normal donors) were recruited which represented 24 samples for further sequencing and analy2c steps. libraries per pool) in equimolar amounts and were sequenced with single-end 100bp reads on an Illumina HiSeq2500. We thank Calcul Québec and Compute Canada for providing access to the supercomputer Briaree from the University of Montreal.

DATA PROCESSING

Samples were used to define eight different groups (Figure 18).

The sequences were treated following the following pipeline. The reads within the samples were trimmed using TrimGalore! (v0.2.7, using Cutadapt v.1.11) to remove any adapter sequence or bad quality (BQ>20) ler within the sequencing read. The sequences were then aligned to the reference transcriptome on the hg19 release and using gene defini2on file based on ENSEMBL v75 using kallisto (v. 0.43.0). The tximport (v. 1.0.3) R package was used to reconstruct from the isoforms-levels the gene-level counts.

We used mixed models (package EMMREML), to fit two models:

• Model 1: NKAES-model: all groups but 1 and 2 (to deal with NKAES behavior pre and post exposure to leukemia in healthy donors versus pa2ents);

• Model 2: res2ng NK vs NKAES (no exposure to leukemia) to explore effects of NK cells ac2va2on on gene expression, and how it varies between controls and pa2ents.

In each model, the gene-set to analyze was selected independently, by filtering out lowexpressed genes. To do that, in each of them, we get the within-group median gene expression, per each gene and group, for all groups present in the model, and selected only genes with a median>2 (log2 cpm) in at least one of the groups present in the model Normal donors and X-CGD pa2ents defined respec2vely groups 1, 3, 5, 6, and 2, 4, 7, 8.

In both cases, using EMMREML, the models have the following structure:

Fixed effects nested design for deconvolve samples content: Model 1

We assumed that each sample had two components (a frac2on of pure NKAES-NK cells and a frac2on of impuri2es, whatever those are made of), each of which could be modeled independently through two auxiliar models M_nk and M_imp:

• M_nk: to model the frac2on of interest (NK cells), as follows: E~cohort+Condi2on*cohort

• M_imp: to model the complementary frac2on (impuri2es before exposure and/or cancer cells arer). Modeled as E~Cytotoxic_target Once defined M_nk and M_imp, we defined the complete model as follows:

Which could be declared like this: (Purity_comp=1-p, Purity_raw=p).

We defined eleven contrasts following Model 1 (Table 4). • M_nk: to model the frac2on of interest (NK cells), as follows: E~cohort+Condi2on*cohort

• M_imp: to model the complementary frac2on (impuri2es before exposure to leukemia).

Modeled as E~Intercept

We defined four contrasts following Model 2 (Table 5).

55 ! "# : %~' ( )1: +,@./0 ' ( + ' 23456 )3: +,8%9@./0 ' ( + ' : )2:+,@<8/=%+/ ' ( + ' : + ' 23456 + ' > @ 23456 )4: +,8%9@<8/=%+/

! "#$ : &~( ) *)#$ +1:-.@012 ( ) *)#$ +3: -.4&5@012 ( ) *)#$ +2:-.@7418&-1 ( ) *)#$ +4: -.4&5@7418&-1 ! " = $! %& + 1 -$ ! *+, :
.~$ 0 1 + (1 -$)0 1 41+, 51:67@9:; $(0 1 + 0 <=>?@ ) + (1 -$)0 1 41+, 53:67B.C@9:; $(0 1 + 0 , ) + (1 -$)0 1 41+, 52: 67@EB:F.6: $(0 1 + 0 , + 0 <=>?@ + 0 G I <=>?@ ) + (1 -$)0 1 41+, 54: 67B.C@EB:F.6: Contrast 1 differen2al expression between pa2ents and controls for res2ng NK cells: (G2-G1)

Contrast 5 ac2va2on effect on controls: (G3-G1)

Contrast 6 ac2va2on effect on pa2ents: (G4-G2) Contrast 7 differences in ac2va2on effects between pa2ents and controls: (G4-G2)-(G3-G1)

Groups used for contrasts' defini2on are depicted in Figure 18.

STATISTICAL ANALYSIS

Sta2s2cal analyses were performed with Prism v6.0h for MacOS (GraphPad Sorware Inc.), and the open-source sta2s2cal package R (hZp://www.r-project.org).

CHAPTER 3. RESULTS AND DISCUSSION

3.

RESULTS AND DISCUSSION

PRE-B ALL CELLS ARE SENSITIVE TO NK CELL KILLING IN A LONG-TERM CYTOTOXICITY ASSAY

Pre-B ALL cells rela-ve resistance to NK cell mediated cytotoxicity has been well documented [START_REF] Imai | Gene=c modifica=on of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells[END_REF][START_REF] Kübler | Both mature KIR+ and immature KIR-NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice[END_REF][START_REF] Romanski | Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblas=c leukemia[END_REF][START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF][START_REF] Ruggeri | Role of natural killer cell alloreac=vity in HLA-mismatched hematopoie=c stem cell transplanta=on[END_REF]. We studied NK cell cytotoxicity against pre-B ALL cell lines as well as primary blasts amplified in vivo (Figure 19) by using a flow cytometry-based assay (FCC). In agreement with previous studies, we observed markedly impaired killing of pre-B ALL following a 4h incuba-on with allogeneic NK cells in comparison to the cytotoxicity measured against the HLA-I nega-ve, posi-ve control K562 cell line, a condi-on which is similar to a conven-onal short term cytotoxicity assay. However, we observed a strong increase of ALL target cells' death when the assay was pursued up to 25h (Figures 19A and 19B). Of note, we confirmed the specificity of this delayed killing because one cell line (RS4;11) was constantly resistant and blasts were not affected by a 25h coculture with allogeneic CD4 + cells (data not shown). The late increase of NK cytotoxicity was not restricted to NK cells ac-vated in our IL-15-based system [START_REF] Imai | Gene=c modifica=on of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells[END_REF] since the same result was observed when using res-ng NK cells from peripheral blood as well as NK cells ac-vated in an IL-21-based system [START_REF] Denman | Membrane-Bound IL-21 Promotes Sustained Ex Vivo Prolifera=on of Human Natural Killer Cells[END_REF] (Figures S1A andS1B).

(legend on next page)

We then addressed the important ques-on of the relevance of this observa-on by using pa-ents' primary leukemic blasts. Since pre-B ALL blasts are known to be difficult to keep in culture in vitro, we used as target cells in a FCC assay primary cells from pediatric pre-B ALL pa-ents which had been previously amplified in Nod/SCID/IL2Rγ -/-(NSG) mice. Primary blasts could be separated in two groups respec-vely iden-fied as sensi-ve and resistant based on NK mediated cytotoxicity (Figures 19C and19D), with cells ini-ally resistant being killed in a late -me point while others remained insensi-ve aber long exposure to NK cells.

This data shows that pre-B ALL cells from pa-ents are sensi-ve to NK cells cytotoxicity in a prolonged assay.

CHARACTERIZATION OF THE EFFECTOR PATHWAY

NK cells can kill target cells through several effector pathways shared with effector CD8 T cells [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF]Marenez-Lostao et al., 2015;[START_REF] Russell | Lymphocyte-mediated cytotoxicity[END_REF]Sun and Lanier, 59 2011). Schema-cally, three main pathways are known to be engaged by cytotoxic cells: 1granule exocytosis of ly-c molecules (perforin, granzymes and granulysin); 2-death receptors mediated killing; and 3-secre-on of soluble factors like cytokines that may exert direct as well as indirect (sensi-za-on of target cells) effects (Figure 20). We aimed to decipher the rela-ve contribu-on to each separate pathway to the late killing of pre-B ALL cells by NK cells by a methodical, systema-cal and dual approach exploring both molecular requirements of NK cells ac-va-on and effector response, as well as those of engaged cell death among blasts. To do so, we studied this cytotoxic interac-on assessing each steps of a simplified model (Figure 20).

A CONTACT-DEPENDENT MECHANISM

We first assessed the requirement of a contact between NK cells and blasts. It has been previously postulated that the rela-ve resistance to pre-B ALL cells was due to a defec-ve adhesion step [START_REF] Ruggeri | Role of natural killer cell alloreac=vity in HLA-mismatched hematopoie=c stem cell transplanta=on[END_REF], although further works tended to exclude this hypothesis as a major systema-cal mechanism of resistance [START_REF] Mengarelli | Adhesion molecule expression, clinical features and therapy outcome in childhood acute lymphoblas=c leukemia[END_REF][START_REF] Romanski | Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblas=c leukemia[END_REF]. Albeit lower with pre-B ALL cells than with K562, early forma-on of conjugates by NK cells was s-ll detectable (Figure 21A) and higher than in a condi-on preven-ng cell adhesion (Figure 21B). Exploring the hypothesis of a poten-al requirement for an ini-al NK cells priming, we also measured early conjugates forma-on with NK cells In this model, one can dis-nguish the following sequence of events: adhesion between NK and target cells (1), signals integra-on leading to either an ac-va-on or an inhibitory synapse (2) and effector phase per se, i.e. mechanisms responsible for targets' death (3).

F

We then performed a transwell experiment choosing the 697 leukemic cell line which had shown the lower propor-on of NK and target cells conjugates (Figures 21C-21E). When cell contact was prevented, blast killing was fully abrogated as shown by the absolute count of targets in the transwell upper compartment which remained unchanged in the presence of an increasing quan-ty of NK cells in the lower compartment, at 4h as well as 25h incuba-on -mes (Figure 21F). One assump-on could have been that NK cells ac-va-on step was contact-dependent while the effector phase would have relied on a produced soluble factor such as cytokines. Therefore, we compared the absolute counts of targets in the upper compartment when the lower one was either seeded with NK cells only or with NK and target cells (i.e. an ongoing cytotoxicity). In both condi-ons, absolute counts were comparable at 4h and 25h incuba-on -mes (Figures 21F and21H), sugges-ng that no soluble factor could explain the late killing effect.

The precise cytotoxicity kine-cs was explored to assess whether the killing increased at 25h was sudden or progressive over the course of the experiment. We designed a FCC assay with mul-ple -me point readouts together with a quan-fica-on of apoptosis at 4h, 12h and 25h (Figure 22A).

Addi-onally, we used XVA143, an allosteric LFA-1 inhibitor [START_REF] Shimaoka | Small Molecule Integrin Antagonists that Bind to the β 2 Subunit I-like Domain and Ac=vate Signals in One Direc=on and Block Them in the Other[END_REF], to study the temporal impact of NK and target cells interac-on. XVA143, which has been shown to be a potent modulator of NK cell func-on, is not only able to prevent cell aggrega-on but also to disaggregate exis-ng clusters as well as to interfere with NK cell killing and ac-va-on (Weitz- [START_REF] Weitz-Schmidt | Allosteric LFA-1 Inhibitors Modulate Natural Killer Cell Func=on[END_REF]. We took advantage of these proper-es to interrupt effector and target cells interac-on aber various incuba-on -mes while assessing the delayed cytotoxicity (Figure 22A). When XVA143 was added to the culture following 3h incuba-on, killing was measured at the usual 4h and 25h -me points corresponding to serngs in which cells were allowed to interact 3h and then leb together without further cell contact for 1h and 22h respec-vely. Proceeding this way, we addressed whether the late killing that we measured at 25h was resul-ng from an early signal received by NK cells during the co-culture or was requiring a permanent cell contact.

Real--me cytotoxicity assessment revealed a con-nuous, progressive and cumula-ve killing effect with a final plateau corresponding to a maximal effect (Figure 22B), together with a constant decrease in the target absolute counts over the 25h incuba-on (Figure 22C). Blasts were undergoing apoptosis with Annexin-V + 7-AAD -cells (Figure 22D), sugges-ng that 4h classical cytotoxicity assay underes-mated cells engaged in a cell-death process. Cell adhesion inhibi-on with XVA143 fully abrogated short term as well as long term NK killing when the allosteric inhibitor was added at the beginning of the co-culture (Figure 22E and 22F), confirming cell contact requirement. Interes-ngly, we observed that further adhesion inhibi-on following an ini-al cell contact was stopping the killing at its real--me value (once included the part of apopto-c cells engaged in the cell death process), sugges-ng that cell adhesion was required throughout the co-culture (Figure 22B-22F).

In summary, we established permanent cell contact requirement between NK and target cells for a progressive and cumula-ve killing.

A DEGRANULATION-INDEPENDENT MECHANISM

We then searched for evidence regarding the involvement of the degranula-on pathway which is the main cytotoxic arm used by CD8 T cells and NK cells [START_REF] Lieberman | Cytotoxic Lymphocytes[END_REF][START_REF] Sun | NK cell development, homeostasis and func=on: parallels with CD8⁺ T cells[END_REF]. We observed that NK cells had a strongly reduced CD107a/b expression when co-cul-vated with pre-B ALL in a 4h conven-onal degranula-on assay: less than 10% cells were posi-ve while more than 40% against K562, posi-ve control (Figures 23A and23B).

Previous reports demonstrated that the NK killing poten-al is not equally shared among effector cells with a minority of so-called serial killer cells responsible for a majority of the measured cytotoxicity [START_REF] Choi | Imaging burst kine=cs and spa=al coordina=on during serial killing by single natural killer cells[END_REF][START_REF] Vanherberghen | Classifica=on of human natural killer cells based on migra=on behavior and cytotoxic response[END_REF]. This led us to hypothesize that the small propor-on of degranula-ng NK cells could s-ll be responsible for the 25h killing, with the idea that a smaller frac-on of effector cells could require more -me to achieve the same effect. To confirm or rule out this hypothesis, we interfered with the degranula-on pathway through different approaches: 1-cell surface blockade of known key receptors of this pathway; 2-target cells gene-c modifica-on to increase their resistance; and 3-use of effector cells from pa-ents with selected immune deficiencies. (legend on next page)

Among surface receptors, NKG2D and DNAM-1 have been shown to be crucial to trigger NK cells degranula-on through the signaling-competent adaptor DNAX-ac-va-ng protein 10 (DAP10) [START_REF] Billadeau | NKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway[END_REF] although signals from co-ac-va-ng receptors are necessary too [START_REF] Bryceson | Minimal requirement for induc=on of natural cytotoxicity and intersec=on of ac=va=on signals by inhibitory receptors[END_REF][START_REF] Lanier | NK cell recogni=on[END_REF][START_REF] Long | Controlling natural killer cell responses: integra=on of signals for ac=va=on and inhibi=on[END_REF]. Thus, as a prototypic model, deficiency in the MAGT1 gene causing XMEN disease with altered cell surface NKG2D expression among pa-ent T and NK cells is associated with a defec-ve degranula-on [START_REF] Chaigne-Delalande | Mg2+ regulates cytotoxic func=ons of NK and CD8 T cells in chronic EBV infec=on through NKG2D[END_REF]. We assessed this triggering pathway by interfering with NKG2D and NCRs (NKp30, NKp44, NKp46 and NKp80) engagement using blocking an-body or Fc chimera proteins respec-vely (Figures 23C and23D). Interes-ngly, we observed a biphasic response with a strong inhibi-on (∼70%) of the 4h killing when NKG2D receptor was blocked,

and almost no inhibi-on of the 25h killing. This observa-on was further confirmed by NKG2D

and DNAM-1 co-blockade (Figures 23E and23F), sugges-ng that at least two mechanisms could have been engaged, the early one relying more on degranula-on than the later one.

Cell surface expression of CD107a (LAMP1) by NK cells has been shown to protect effector cells from self-killing [START_REF] Cohnen | Surface CD107a/LAMP-1 protects natural killer cells from degranula=on-associated damage[END_REF]. Therefore, we transduced ALL target cells with the same sCD107a (the cytoplasmic domain was truncated by removing the cytoplasmic mo-f implicated in lysosomal targe-ng of newly synthesized molecules; the construc-on was kindly provided by Pr. Carsten Watzl (IfADo, Leibniz, Germany)). When used as targets in a FCC assay, sCD107a expressing blasts were killed as efficiently as untransduced cells (Figures 23G and23H, see NALM6 cell line), or if an early inhibi-on was detected following a 4h incuba-on, it didn't prevent the 25h increase in killing (Figure 23I, see REH cell line). In line with the CD107a/CD107b assay and blocking experiments, these observa-ons suggest that degranula-on is not the main cytotoxic pathway involved in the observed 25h killing. [START_REF] Feldmann | Munc13-4 is essen=al for cytoly=c granules fusion and is mutated in a form of familial hemophagocy=c lymphohis=ocytosis (FHL3)[END_REF][START_REF] Stepp | Perforin Gene Defects in Familial Hemophagocy=c Lymphohis=ocytosis[END_REF] and life-threatening HLH.

We amplified NK cells from PRF1 and UNC13D deficient pa-ents and used them in a FCC assay against K562, NALM6, REH and 697 cell lines with 4h and 25h co-incuba-ons. As expected, FHL pa-ents exhibited a severe defect in K562 killing in a conven-onal 4h assay, compared to a normal donor (Figures 23J and23N). Defect was also no-ced against pre-B ALL blasts although more importantly with FHL3 NK cells (Figures 23K-23M and 23O-23Q).

However, all FHL pa-ent NK cells showed a strong increase against all tested targets aber 25h incuba-on (Figures 23J-23Q), ruling out the possibility that the delayed detected cytotoxic effect was dependent on granule exocytosis.

Thus, taking together, our results demonstrate that NK cells can induce target cells to die through a late mechanism which is independent of the degranula-on pathway.

APOPTOSIS-LIKE CELL DEATH OF THE TARGET CELLS

To gain further insight into the mechanism used by NK cells to kill targets over a prolonged incuba-on, we performed an annexin-V staining aber 4h, 10h and 25h incuba-on with effector cells, postula-ng that dis-nc-on between apopto-c and necro-c like cell deaths would orientate our working hypothesis.

Results showed an apopto-c-like induced cell death among pre-B ALL targets with annexin-V + 7-AAD -single posi-ve cells (early apopto-c-like) preceding annexin-V + 7-AAD + double posi-ve cells (late apopto-c-like) (Figure 24). Apoptosis is a form of programmed cell suicide linked to the ac-va-on of the cysteine-dependent aspartate-driven proteases (caspases), which cleave key intracellular substrates to promote cell death [START_REF] Green | Apoptosis: physiology and pathology[END_REF][START_REF] Taylor | Apoptosis: controlled demoli=on at the cellular level[END_REF].

Apoptosis is controlled in mammals through two major cellular signaling pathways, namely the intrinsic and the extrinsic pathways. The former relies on mitochondria while the later is associated to the transmission of signals from extracellular death ligands. However, both pathways oben act directly and/or indirectly to reinforce one another [START_REF] Ashkenazi | Regulated cell death: signaling and mechanisms[END_REF]. Caspase proteins are highly regulated from the moment of their synthesis.

They are produced in zymogen form lacking protease ac-vity [START_REF] Parrish | Cellular Mechanisms Controlling Caspase Ac=va=on and Func=on[END_REF]. Intrinsic and extrinsic apopto-c pathways relie on dis-nct ini-ator caspases whose ac-va-on will then trigger effector caspases through a cascade-like proteoly-c s-mula-on.

We found evidence for pan-caspase ac-va-on among pre-B ALL cells which were harboring apopto-c-like features (Figures 25A and25B). A detailed study of ini-ator (caspases 8, and 10) and execu-oner (caspases 3 and 7) caspases revealed a global ac-va-on profile (Figure 25C) as well as mitochondrial depolariza-on assessed by a JC-1 staining (Figure 25D), compa-ble with concomitant ac-va-on of both apopto-c cellular signaling pathways. Ac-ve caspases 8 and 10 are reminiscent of the involvement of the extrinsic apopto-c pathway but can also be a late consequence in the hierarchical ordering of caspases ac-va-on following engagement of the intrinsic pathway [START_REF] Inoue | Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway[END_REF][START_REF] Slee | Ordering the Cytochrome c-ini=ated Caspase Cascade: Hierarchical Ac=va=on of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9-dependent Manner[END_REF]. Because the study of caspases ac-vity was compa-ble with ac-va-on of both apopto-c cellular signaling pathways (Figure 25C), we performed a caspase 8 vs caspases 3&7 co-staining by flow cytometry to further characterize the ordering in their ac-va-on. We didn't highlight a clear ac-ve caspase 8, unac-ve caspases 3&7 popula-on (Figure 26) but our results could suggest ac-va-on of the extrinsic pathway considering that the profile was similar to the one induced by the SuperKillerTRAIL posi-ve control (data not shown).

Thus, these results suggest that NK cells trigger an apopto-c-like cell death among pre-B ALL targets with poly-caspase ac-va-on and mitochondrial depolariza-on. Representa-ve dot plots show target cells ac-ve caspase 8 / caspases 3&7 co-staining (FLICA) following incuba-on with NK cells (4:1 effector to target ra-o). Blue and orange histograms depict living and early apopto-c cells respec-vely (Figure 25A). NALM6, REH and 697 cell lines were used as targets. Observa-ons were the same following 4h, 10h and 25h incuba-on.

A DEATH RECEPTOR PATHWAY-INDEPENDENT MECHANISM

At this point, we established that our killing mechanism was contact-dependent, did not rely on granule exocytosis and induced an apopto-c-like cell death with caspases and mitochondria altera-ons. Consequently, we next aimed to study whether death receptor pathway could be involved.

CD8 + T cells and NK cells are known to express several death ligands including FASL and TRAIL which can trigger target cells apoptosis through death receptors engagement [START_REF] Ashkenazi | Regulated cell death: signaling and mechanisms[END_REF][START_REF] Guicciardi | Life and death by death receptors[END_REF]Marenez-Lostao et al., 2015;[START_REF] Russell | Lymphocyte-mediated cytotoxicity[END_REF]. Besides, induced cell death through this pathway can take from 12h to 24h [START_REF] Jedema | New CFSE-based assay to determine suscep=bility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell popula=on[END_REF], substan-ally longer than perforin and granzymes induced cell death which typically occurs within minutes following contact between effector and target cells [START_REF] Jedema | New CFSE-based assay to determine suscep=bility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell popula=on[END_REF][START_REF] Lopez | Rapid and Unidirec=onal Perforin Pore Delivery at the Cytotoxic Immune Synapse[END_REF][START_REF] Voskoboinik | Perforin and granzymes: func=on, dysfunc=on and human pathology[END_REF][START_REF] Vrazo | Rapid detec=on of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease[END_REF].

71

(legend on next page)

REH cell line

We first evaluated the expression of FAS, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) death receptors on pre-B ALL cell lines by flow cytometry (Figures S2A-S2C). We observed that each one showed a weak expression of FAS and a variable expression of DR4 and/or DR5, promp-ng us to test cell lines sensi-vity to FASL and TRAIL mediated apoptosis. APO-1 and SuperKillerTRAIL treatments respec-vely revealed a resistance to FASL and a strong sensi-vity to TRAIL mediated apoptosis among the three pre-B cell lines (Figures S2D-S2G).

Based on these observa-ons, we tried to block DR4 and DR5 on ALL cell lines targets. Indeed, these two receptors mediate TRAIL induced death signals while the other TRAIL receptors are considered as nondeath ones [START_REF] Guicciardi | Life and death by death receptors[END_REF][START_REF] Walczak | Death receptor-ligand systems in cancer, cell death, and inflamma=on[END_REF]. We observed no inhibi-on of the NK killing when blocking an-bodies against DR4 and DR5 were added to the assay (Figure 27A) while these blocking an-bodies could significantly inhibit TRAILinduced ALL cell death. Since TNFα can also induce leukemic cells apoptosis [START_REF] Schimmer | Receptor-and mitochondrial-mediated apoptosis in acute leukemia: a transla=onal view[END_REF], we decided to block membrane bound TNFα, which is expressed and used by NK cells to induce target cells death [START_REF] Caron | Human NK cells cons=tu=vely express membrane TNF-alpha (mTNFalpha) and present mTNFalpha-dependent cytotoxic ac=vity[END_REF]. Contrary to infliximab, etanercept does not induce reverse signaling and apoptosis of effector cells [START_REF] Horiuchi | Transmembrane TNFalpha: structure, func=on and interac=on with an=-TNF agents[END_REF][START_REF] Mitoma | Mechanisms for cytotoxic effects of an=-tumor necrosis factor agents on transmembrane tumor necrosis factor α-expressing cells: Comparison among infliximab, etanercept, and adalimumab[END_REF]. Therefore, we decided to use etanercept to block membrane bound TNFα. Although we could not set up a control condi-on demonstra-ng the efficacy of our blocking experiment, no inhibi-on of the NK killing was no-ced when etanercept was added during the experiment (Figure 27B). Although our pre-B cell lines were not sensi-ve in vitro to APO-1 mediated apoptosis (Figures S2D andS2E), we decided to use a broader and more reliable strategy to inhibit death receptor mediated death. (G-J) Graphs showing early and late leukemic killing of K562, NALM6, REH and 697 cell lines (control or hFADD-DN transduced) using NK cells from a perforin deficient pa-ent. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. (A-J) Shown are mean ± SD.

To do so, we gene-cally modified target cells to make them resistant to death receptor apopto-c signaling. FADD and TRADD are two keys death receptor signal transducers, the former being associated with cell death signalling whereas the later is linked to cell prolifera-on and survival [START_REF] Strasser | Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases[END_REF][START_REF] Wilson | Death receptor signal transducers: nodes of coordina=on in immune signaling networks[END_REF]. Also, when TRADD is involved in death signaling, it has been described to involve FADD [START_REF] Strasser | Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases[END_REF][START_REF] Wilson | Death receptor signal transducers: nodes of coordina=on in immune signaling networks[END_REF]. We transduced pre-B ALL cells with a dominant-nega-ve FADD to induce stable resistance [START_REF] Wajant | Dominant-nega=ve FADD inhibits TNFR60-, Fas/Apo1-and TRAIL-R/Apo2-mediated cell death but not gene induc=on[END_REF] (Appendix 12 modified from LMBP #4627 plasmid, BCCM, and Figure S2H). As expected and as a control of our strategy, FADD-DN expressing target cells became resistant to TRAIL mediated apoptosis (Figure S2H). However, they remained efficiently killed by NK cells in a long-term FCC assay (Figures 27C-27F), sugges-ng that cytotoxicity was independent of the death receptor pathway.

To definitely prove that our mechanism was dis-nct from the two main described contactdependent killing pathways and was not par-ally caused by each of them, we amplified NK cells from a PRF1 deficient pa-ent and used them in a 4h and 25h FCC assay against K562, NALM6, REH and 697 cell lines transduced with a control GFP or hFADD-DN IRES GFP vector (Appendices 11 and 12). As previously observed (Figures 23J-23N), perforin-deficient NK cells exhibited a severe killing defect in a conven-onal 4h assay against K562, compared to a normal donor (Figures 27G-27J). However, perforin-deficient NK cells showed an increased cytotoxicity against all tested ALL targets aber 25h incuba-on, which was comparable to the increased cytotoxicity observed with normal donor cells (Figures 27G-27J). Thus, we demonstrated in a same assay that NK cells which couldn't efficiently engage the granule exocytosis cytotoxic pathway were able to kill target cells rendered resistant to death receptor induced cell death, ruling out the possibility that our mechanism was dependent, even par-ally, on any of them.

MINIMAL MOLECULAR REQUIREMENTS AMONG EFFECTOR CELLS

Our results tend to demonstrate that NK cells can kill ALL target cells through an unconven-onal contact-dependent pathway. To gain insight into both molecular control of this pathway by NK cells as well as the sequence of events inducing target cell death, we used a complementary approach studying either NK cells from pa-ents suffering from molecularly characterized primary immunodeficiencies which could affect effector cells func-on, or gene-cally modified target cells whose we altered key molecular nodes. Of note, isolated NK cells deficiencies have not been reported so far [START_REF] Fischer | Primary immunodeficiencies suggest redundancy within the human immune system[END_REF] meaning that other immune cell subsets are also affected among these pa-ents. (legend on next page)

A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
Using PIDs as a screening pla}orm, we decided to select molecular defects which were described to impact NK cell cytotoxicity (Table 6). Some NK deficiencies were described to be (some-mes par-ally) rescued by IL-2: because we were using an ac-va-on and expansion system with exogenous rhIL-2, these ones were excluded.

Our previous results showed that the three used pre-B ALL cell lines were not equally sensi-ve to NK mediated killing, with 697 being the more resistant (with inter-individual variability) while NALM6 and REH are more constantly killed (Figure 19B). Besides, rela-ve contribu-on to the various cytotoxic pathways (granule exocytosis, death receptors and our unconven-onal pathway, although minor in the late killing for the first two (Figures 23 and27)) differed. Depending on the samples, we some-mes had limited number of NK cells so that we decided, when limited, to privilege K562 (posi-ve control) and REH cell lines before NALM6 and then 697 in FCC assays. Based on these observa-ons, REH was a good candidate to further characterize our pathway.

UNC13D

Type 3 familial hemophagocy-c lymphohis-ocytosis (FHL3) [START_REF] Bryceson | A prospec=ve evalua=on of degranula=on assays in the rapid diagnosis of familial hemophagocy=c syndromes[END_REF][START_REF] De Saint Basile | Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules[END_REF][START_REF] Feldmann | Munc13-4 is essen=al for cytoly=c granules fusion and is mutated in a form of familial hemophagocy=c lymphohis=ocytosis (FHL3)[END_REF][START_REF] Marcenaro | Analysis of natural killer-cell func=on in familial hemophagocy=c lymphohis=ocytosis (FHL): defec=ve CD107a surface expression heralds Munc13-4 defect and discriminates between gene=c subtypes of the disease[END_REF]

PRF1

Type 2 familial hemophagocy-c lymphohis-ocytosis (FHL2) Some of the gene-c defects were also of interest because previous works suggested altered NK cell response to B-EBV lymphoblastoid cell lines (Table 6). We postulated these cells to be a relevant model with shared ontogeny with pre-B cells and because we also observed an increase in B-EBV LCL killing by NK cells following a 25h incuba-on (data not shown).

None of the tested molecular defect exhibited a constant inhibi-on in the late killing of pre-B ALL cell lines (Figure 28), with the notable excep-on of moesin (MSN) deficient NK cells.

Moesin is a member of the ezrin-radixin-moesin (ERM) family of proteins that are important for organizing membrane domains and receptor signaling. For example, CD43 removal at the T cell immune synapse has been shown to be regulated by moesin [START_REF] Delon | Exclusion of CD43 from the immunological synapse is mediated by phosphoryla=on-regulated reloca=on of the cytoskeletal adaptor moesin[END_REF], while CD43 could be an ac-va-ng receptor in NK cells [START_REF] Mccann | The Size of the Synap=c Clep and Dis=nct Distribu=ons of Filamentous Ac=n, Ezrin, CD43, and CD45 at Ac=va=ng and Inhibitory Human NK Cell Immune Synapses[END_REF][START_REF] Nieto | Signaling through CD43 induces natural killer cell ac=va=on, chemokine release, and PYK-2 ac=va=on[END_REF].

Moesin is expressed in res-ng and ac-vated NK cells [START_REF] Ramoni | Differen=al expression and distribu=on of ezrin, radixin and moesin in human natural killer cells[END_REF][START_REF] Schmidt | 2-D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of func=onally relevant proteins in leukemic and ac=vated human NK cells[END_REF]. MSN deficient pa-ents have low circula-ng NK count [START_REF] Lagresle-Peyrou | X-linked primary immunodeficiency associated with hemizygous muta=ons in the moesin (MSN) gene[END_REF], which made our expansion system crucial to assess their cytotoxic func-on. NK cells from a MSN deficient showed impaired killing of the REH ALL cell line compared to a normal donor (Figure 28Z), which could suggest an ac-n-dependent receptor clustering [START_REF] Orange | Forma=on and func=on of the ly=c NK-cell immunological synapse[END_REF] at an ac-va-ng immune synapse [START_REF] Mccann | The Size of the Synap=c Clep and Dis=nct Distribu=ons of Filamentous Ac=n, Ezrin, CD43, and CD45 at Ac=va=ng and Inhibitory Human NK Cell Immune Synapses[END_REF] although more pa-ents should be tested.

In addi-on to the exclusion of the granule exocytosis contribu-on, we concluded from these experiments that NK ac-va-on and effector response to leukemic blasts was independent of the SLAM family receptors, CD40L, NFκB, STAT3, BTK or PKCδ mediated signaling, nor required GATA2 protein.

MINIMAL MOLECULAR REQUIREMENTS AMONG TARGET CELLS

We then focused on trying to characterize the induced cell death mechanism among ALL target cells. A beter understanding of the molecular events leading to pre-B ALL death would also help elucida-ng NK cell effector mechanism.

We previously discussed that NK cells trigger an apopto-c-like cell death among pre-B ALL targets with poly-caspase ac-va-on and mitochondrial depolariza-on (Figures 25C and25D).

Using CRISPR/Cas9 generated KO, we assessed the pre-B ALL target cell killing by NK cells by knocking out key selected genes: caspase 9 KO to interfere with the intrinsic apopto-c pathway; BAX/BAK double KO (DKO) to block mitochondrial depolariza-on; caspases 8 and 10 DKO to inhibit the extrinsic apopto-c pathway; and caspases 3 and 7 DKO to block effector caspase ac-va-on. Again, we ini-ally focused on REH cell line.

A B

Interes-ngly, pre-B ALL cells were differently sensi-ve to hydrogen peroxyde and we observed that it was mimicking their rela-ve resistance to NK cell, with RS4;11 (the only tested pre-B cell line which was constantly resistant to NK cells) being the less sensi-ve to H 2 O 2 induced stress (Figures 31A and31B). These preliminary results prompted us to modify targets to make them beter to manage oxida-ve stress, and then assess their sensi-vity or resistance to NK cell killing. We adapted a series of previously reported gene-c tools [START_REF] Walter | Exit from dormancy provokes DNA-damage-induced aqri=on in haematopoie=c stem cells[END_REF] We couldn't detect any difference among living targets (Annexin-V -7-AAD -) which were in contact with effector cells, neither exploring glutathione redox poten-al nor hydrogen peroxide level (data not shown). However, apopto-c Annexin-V + 7-AAD -cells displayed altered phenotype for both readouts but this observa-on appears to be a general property of apopto-c cells since we observed it regardless of the inducing s-mulus (targets treated with SuperKillerTRAIL gave the same results, data not shown).

To our surprise, combined overexpression of the SOD2 and the catalase didn't confer protec-on from NK killing (Figure 32).

NALM6 GFP

These results, together with the experiment using NAC, raised the hypothesis that ROS produc-on could be important in NK cells and not in pre-B ALL targets.

EVIDENCE FOR A NADPH COMPLEX IN NK CELLS

All experiments exploring biological interference through chemical inhibi-on were of limited interest in our project because of effector and target cells were both impacted. To address the role of ROS in NK cell func-on, we looked for primary cells which could present a gene-c defect in their produc-on.

Pa-ents with chronic granulomatous disease (CGD) have a well-described hereditary defect in reac-ve oxygen intermediates produc-on by neutrophils and monocytes [START_REF] Kuhns | Residual NADPH oxidase and survival in chronic granulomatous disease[END_REF]: NADPH oxidase ac-vity is dras-cally impaired due to muta-ons in any one of five subunits of phagocyte-derived NADPH oxidase, including gp91 phox (CYBB, ∼65% of pa-ents), p22 phox (CYBA, <5%), p47 phox (NCF1, ∼30%), p67 phox (NCF2, <5%), and p40 phox (NCF4, one case iden-fied). We decided to assess whether NK cells from CGD pa-ents displayed normal killing ac-vity in our FCC assay against pre-B ALL cells Surprisingly, we observed a significa-ve defect in NK cell mediated late killing of pre-B ALL cells using effector cells from a X-CGD pa-ent (Figure 33A). To further characterize our mechanism, we decided to increase pa-ent recruitment: we tested ten different X-CGD pa-ents and observed a significant decrease in cytotoxic abili-es against NALM6 and REH cell lines, more specifically following 25h incuba-on (Figures 33D and33E). Of note, we no-ced that NK cells from X-CGD pa-ents proliferated less than cells from controls or other PIDs (data not shown). Then, we tested cells from CGD pa-ents harboring muta-ons affec-ng another protein of the phagocy-c NADPH oxidase complex, to see whether altered cytotoxicity was a shared characteris-c among CGD pa-ents. We included two pa-ents with NCF1 muta-ons (AR47-CGD) and one with CYBA muta-ons (AR22-CGD). Not only all of them were prolifera-ng very well in our system (data not shown), but they displayed strong cytotoxicity against pre-B ALL blasts (Figures 33B and33C).

These results suggest that CYBB could play a role in NK cell func-on. However, to our knowledge, neither biological role nor NADPH expression have been reported in NK cells. We first assessed CYBB mRNA expression in PBMCs, res-ng and amplified NK cells, as well as those of the main components of the NADPH oxidase and NOX1 and NOX4, two NOX2 (CYBB) homologues which can interact with p22 phox too (Figures 34A-34C) [START_REF] Nguyen | NADPH oxidases, Nox: new isoenzymes family][END_REF][START_REF] Lambeth | Regula=on of Nox and Duox enzyma=c ac=vity and expression[END_REF].

(legend conJnued on next page)

Apart from NOX1 and NOX4 whose expression was reported only in non-immune cells, res-ng NK cells expressed all the genes at the transcriptomic level, but ac-vated NK cells displayed a selec-ve loss of CYBB mRNA expression (Figures 34B and34C).

A previous study from Dr. Dario Campana lab explored gene-c features of NKAES-NK cells, comparing the global gene expression profiles of these cells with those of uns-mulated NK cells [START_REF] Fujisaki | Expansion of Highly Cytotoxic Human Natural Killer Cells for Cancer Cell Therapy[END_REF]. Supplemental data from their work reported a 55 fold repression of CYBB in NKAES-NK compared to res-ng cells. Besides, we confirmed this observa-on by performing a PrimeFlow RNA assay on PBMCs with a CYBB targe-ng probe. We observed that res-ng NK cells (both CD56 bright and CD56 dim ) expressed CYBB mRNA at a similar level as monocytes, and stronger than CD19 + B cells which are known to express a phagocyte-type NADPH oxidase complex [START_REF] Cotugno | Defec=ve B-cell prolifera=on and maintenance of long-term memory in pa=ents with chronic granulomatous disease[END_REF] (Figures 34D and34E).

gp91 phox and p22 phox are the two subunits of flavocytochrome b558, a heterodimeric integral membrane protein which is the key component of the phagocyte NADPH oxidase system [START_REF] Jesai=s | Structure of human phagocyte cytochrome b and its rela=onship to microbicidal superoxide produc=on[END_REF]. Its expression has been reported both at the cell surface of neutrophils, monocytes and B cells, as well as intracellularly in those same cell types. We evaluated flavocytochrome b558 expression using the 7D5 monoclonal an-body which recognizes an epitope on gp91 phox when its conforma-on is within the gp91 phox / p22 phox heterodimer (Burrit et al., 2001;[START_REF] Campion | New insights into the membrane topology of the phagocyte NADPH oxidase: characteriza=on of an an=-gp91-phox conforma=onal monoclonal an=body[END_REF]. To ensure that the an-body was specific, we tested as a nega-ve control a CYBA deficient pa-ent (AR22-CGD) together with a normal donor.

Figure 34. NK cells express gp91 phox and p22 phox but do not display a phagocyte-type NADPH oxidase complex. (A-C) End-point PCR was performed on PBMCs (A, posi-ve control), sorted res-ng NK cells (B) and sorted NKAES-NK cells (C) to check mRNA expression of the following genes related to NADPH oxidase: NRROS, NOX1, NOX4, CYBA (p22 phox ), NCF4 (p40 phox ), NCF1 (p47 phox ), NCF2 (p67 phox ), CYBB (gp91 phox ) and the B2M housekeeping gene. Representa-ve gel (at least three different donors were tested per condi-on). (D and E) PrimeFlow RNA assay was performed on total PBMCs (D, ga-ng strategy). RLP13a mRNA was used as a posi-ve control among PBMCs subpopula-ons and CYBB mRNA expression was studied (E). Representa-ve histogram of one of three different donors showing similar results. (F-H) Cell surface (F) and intracellular (G) flavocytochrome b558 expression (clone 7D5; MBL Interna-onal) was assessed by flow cytometry on PBMCs, as well as intracellular p22 phox expression (H) (clone 44.1; Santa Cruz Biotechnology). Normal donor and AR22-CGD pa-ent are depicted as blue and red histograms respec-vely. PBMCs from four normal donors were tested with the same results. (I) Nanofluidic proteomic immunoassay against CYBB (clone 54.1; Santa Cruz Biotechnology) was performed using res-ng NK cells (isolated by nega-ve selec-on) from a normal donor (in green) and a X-CGD pa-ent (in blue) harboring a complete CYBB gene dele-on.

We detected flavocytochrome b558 on monocytes and B cells, both intracellularly and at the cell surface, and confirmed an-body specificity since no expression was detected for the AR22-CGD pa-ent (Figures 34F and34G). We didn't detect any flavocytochrome b558 expression in res-ng NK cells from PBMCs, in agreement with another work [START_REF] Vrazo | Rapid detec=on of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease[END_REF] (Figures 34F and34G). Although a team previously reported an expression among CD3 + T cells [START_REF] Jackson | T cells express a phagocyte-type NADPH oxidase that is ac=vated aper T cell receptor s=mula=on[END_REF], we did not detect any flavocytochrome expression b558 in T cells.

Then we postulated that gp91 phox and p22 phox are expressed in cytotoxic lymphocytes in a complex which differ from the phagocyte-type flavocytochrome b558. We looked for evidence of a p22 phox proteic expression by flow cytometry with an an-body which recognizes an intracellular epitope of p22 phox . Again, an-body specificity was confirmed using cells from our AR22-CGD pa-ent. Interes-ngly, we showed that res-ng NK cells (both CD56 bright and CD56 dim ), as well as monocytes, B cells and with a lower intensity T cells, did express p22 phox (Figure 34H).

Since we could not find an an-body suitable for flow cytometry which could specifically recognize gp91 phox in a conforma-on independent of the flavocytochrome b558, we took advantage of a nanofluidic proteomic immunoassay to evaluate gp91 phox proteic expression with small amounts of proteins. Cells from a X-CGD pa-ent with complete CYBB dele-on were used to validate our approach and our an-body. We found res-ng NK cells to express gp91 phox at the proteomic level (Figure 34I). Thus, our results showed that NK cells from PBMCs expressed all the components of the NADPH oxidase complex at the transcriptomic level as well as NRROS which has been described to regulate flavocytochrome b558 expression in phagocytes by regula-ng gp91 phox degrada-on [START_REF] Noubade | NRROS nega=vely regulates reac=ve oxygen species during host defence and autoimmunity[END_REF]. They also expressed gp91 phox and p22 phox , the two subunits of the flavocytochrome b558 which is a key component of the NADPH oxidase complex, at the proteic level but did not display a phagocyte-type flavocytochrome b558, sugges-ng that these proteins could be involved in an alterna-ve complex.

We found surprising that CYBB mRNA was virtually completely absent in ac-vated NK cells: then, how could we link the func-onal defect we observed in X-CGD pa-ents considering that cells from normal donors didn't express the gene?

We decided to test CYBB mRNA expression in NKAES-NK cells from normal donors aber they encountered targets (K562 or pre-B ALL cells).

Our results showed that CYBB was re-expressed when NKAES-NK cells were incubated for 25h with pre-B ALL cells but not with K562 targets (Figure 35). These results demonstrated that CYBB expression is ac-vely and dynamically regulated by prolifera-ng NK cells and, together with our results regarding late killing of X-CGD pa-ent NK cells against pre-B ALL blasts (Figures 33D and33E), suggested that its expression could be important for the unconven-onal cytotoxic pathway that we described.

To gain insight into the molecular defects among X-CGD pa-ents, we performed RNAseq on res-ng NK cells and ac-vated NKAES-NK cells either unexposed to targets or following 24h incuba-on with a NK-sensi-ve (REH) or resistant (RS4;11) pre-B ALL cell line. We tested four controls and three X-CGD pa-ents with a complete CYBB gene dele-on; because we had a limited amount of ac-vated cells for pa-ents, only one of them could be also exposed to targets.

We performed unsupervised clustering using PCA and observed a perfect separa-on of samples per condi-on and donor (Figure 36A). Responses to REH or RS4;11 cells are, in general, larger in effect size in the pa-ent's sample than in the controls (data not shown) and similar among controls regardless of the cell line (Figure 36B).

Differen-ally expressed genes were ploted per contrast of interest (Figures 36C-36G and

Table S1) and a gene ontology enrichment analysis was performed into Cytoscape using the ClueGO plugin. End-point PCR was performed on PBMCs (posi-ve control), sorted NKAES-NK cells before and aber incuba-on with targets (K562 or REH), to check mRNA expression of the following genes related to NADPH oxidase: NRROS, CYBA (p22 phox ), NCF4 (p40 phox ), NCF1 ( p 4 7 phox ), NCF2 (p67 phox ), CYBB (gp91 phox ) and the B2M housekeeping gene. Representa-ve gel of one among several donors. NALM6 and 697 cell lines were tested with results similar to REH.

For this analysis, we had contrasts with more differen-ally expressed genes than for others.

However, the enrichment analyses work differently depending on the number of genes in the target gene sets. In our experience using this program, the most informa-ve results were usually obtained when target gene sets are around ~500-1000 genes, so that we changed the fdr threshold between contrasts to retrieve target gene sets of sizes as comparable as possible (Tables S2 andS3). Label -controls:

Label -patients: Regarding the comparison of res-ng NK cells from X-CGD pa-ents to normal donors, results

• • • • • • • • • • • • • • • • • -2 -1 0
showed that func-ons (strongly) enriched among genes more expressed in pa-ent cells were related to phagocytosis (which is directly evoca-ve of the pathophysiology of the CGD), endocytosis, receptor-mediated endocytosis and Fc receptor signaling (Figure 37A). Func-ons (slightly) enriched among genes less expressed in NK from pa-ents were associated to protein localizaJon to membrane (Figure 37B).

Responses to REH and RS4;11 were highly similar in intensity and the category of the func-ons enriched, the main difference being the increased number of terms in response to REH, both up and down-regulated. Shared func-ons enriched among up-regulated genes in response to either REH or RS4;11 included regulaJon of apoptosis-related terms, T cell acJvaJon, JAK-STAT cascade, leukocyte homeostasis and response to DNA damage (Figures 37C and37E). Shared func-ons enriched among down-regulated genes in response to either REH or RS4;11 were related to carbohydrates metabolism, degranulaJon (of different cell types, though), type I IFN response and response to virus (Figures 37D and37F). When looking at the differen-al expression between NKAES-NK cells exposed to RS4;11 vs REH among controls, an interes-ng patern appeared: func-ons enriched among genes more highly when s-mulated with RS4;11 were mainly metabolic (Figure 37G) while those enriched with REH as a s-mulus included sodium ion transport, regulaJon of metal ion transport, water homeostasis, and other terms closely related to ion pumping processes (Figure 37H). For cellular components terms, the enrichment was weaker; however there was a huge enrichment of immunoglobulin complex genes in the target gene set comparing res-ng NK cells from X-CGD pa-ents to normal donors (Figure 37I).

Thus, our RNAseq study highlighted strong differences between X-CGD pa-ents and controls.

It also showed that NK cell responses either to a NK-sensi-ve (REH) or resistant (RS4;11) pre-B ALL cell line were similar in effect size, which could be indica-ve that their differen-al behavior in sensi-vity to NK cells was intrinsic to targets. Besides, our results suggested a role of metabolic processes in NK cells responses. Biological process (A-H) and cellular component (I-P) related terms were studied. We dis-nguished up-regulated and down-regulated genes for the analysis.

In the graphs, size is propor-onal to the fdr of the result (following a scale roughly logarithmic), but the significance scales vary from some runs to others.

METABOLIC SUPPORT OF NK CELLS ACTIVITY

In our transcriptomic study of NK cells, most of the hits were related to metabolism. We aimed to evaluate metabolic changes in NK cells that were engaged in a killing through our unconven-onal pathway. (A-F) OCR was measured using NK cells from a normal donor (in blue) and a AR47-CGD (NCF1 deficient; in red) either exposed or not to various target cell lines. (G-L) OCR was measured using NK cells from a normal donor (in blue) and a X-CGD (CYBB deficient; in green) either exposed or not to various target cell lines.

A B C D E F G H I J K L
We posi-vely purified by magne-c separa-on NK cells (from normal donors or an AR47-CGD and a X-CGD pa-ents) which had been s-mulated for ∼20h with various target cell lines, and then evaluated OXPHOS and glycolysis (Figures 38 and39). (A-F) ECAR was measured using NK cells from a normal donor (in blue) and a AR47-CGD (NCF1 deficient; in red) either exposed or not to various target cell lines. (G-L) ECAR was measured using NK cells from a normal donor (in blue) and a X-CGD (CYBB deficient; in green) either exposed or not to various target cell lines.

We observed that NK cells which had been exposed to K562 cell line decreased their OXPHOS use and (Figures 38B and38H) but not their glycoly-c ac-vity (Figures 39B and39H).

When s-mulated with pre-B ALL cell lines, NK cells didn't strongly modify their OXPHOS ac-vity (Figures 38C-38F and 38I-38L). However, facing 697 and RS4;11 cell lines (which are the more resistant to our late NK killing), we observed an increase in NK cells glycoly-c ac-vity (Figures 39K and39L).

Interes-ngly, we didn't observe any difference in OXPHOS and glycolysis use by NCF1

deficient or control NK cells, which both killed efficiently pre-B ALL cells. On the other hand, CYBB deficient NK cells which were s-mulated with 697 and RS4;11 targets, didn't increase their glycoly-c ac-vity, in contrast to control cells.

Our results revealed opposite metabolic profile when NK cells were engaged in K562 killing or pre-B ALL cell lines. They also suggested that a hyperglycoly-c step could be required for efficient engagement of our unconven-onal cytotoxicity pathway.

CYBB deficient NK cells didn't exhibit hyperglycolysis, which is evoca-ve of a work showing that NADPH oxidase has a func-on in suppor-ng hyperglycolysis in s-mulated neutrophils [START_REF] Baillet | Unexpected func=on of the phagocyte NADPH oxidase in suppor=ng hyperglycolysis in s=mulated neutrophils: key role of 6-phosphofructo-2-kinase[END_REF].

The NK cell ac-va-on and expansion system we used relies on K562-mb15-41BBL feeder cells and therefore on IL-15 signaling. Previous works showed mTOR dependent metabolic changes induced by IL-15 signaling in murine and human NK cells [START_REF] Gardiner | What Fuels Natural Killers? Metabolism and NK Cell Responses[END_REF][START_REF] Kea=ng | Metabolic Reprogramming Supports IFN-γ Produc=on by CD56bright NK Cells[END_REF][START_REF] Marçais | The metabolic checkpoint kinase mTOR is essen=al for IL-15 signaling during the development and ac=va=on of NK cells[END_REF].

We checked CD98 (Figures 40A-40C) and CD71 (Figures 40D-40F) expression as well as glucose uptake (Figures 40G and40H) in NK cells following incuba-on with K562 or pre-B ALL cell lines.

We found opposite results when effector cells were engaged in a conven-onal degranula-on-dependent cytotoxicity against K562, with increased CD71 expression and no difference for CD98, compared to uns-mulated NK cells. However, pre-B ALL cell lines induced an early CD98 up-regula-on (delayed with K562) and no CD71 modula-on compared to control. Glucose uptake was increased at 4h with K562 only, while we could observe a later increase (at 25h) with pre-B ALL cell lines, sugges-ng that both metabolic requirement and its kine-cs were dis-nct depending on the killing pathway that NK cells were engaging.

Finally, we also assessed the basal metabolism in target ALL cells. Compared to K562 cell line, pre-B ALL cells had a lower oxida-ve phosphoryla-on (OXPHOS) with glycolysis being substan-ally the same (Figures 41A and41B), and overall, equally relied on OXPHOS and glycolysis (Figure 41C). The NK-resistant cell line RS4;11 showed a very low OXPHOS and we generally observed that pre-B ALL cell lines which were relying mainly on glycolysis because of this low OXPHOS were also the more resistant to NK killing (Figure 41C).

These preliminary results in ALL cell lines showed that spare respiratory capacity could poten-ally be correlated to their sensi-vity to ac-vated NK cells and suggest that target cell metabolism should be further inves-gated to understand NK cell induced death process.

CONCLUDING REMARKS AND PERSPECTIVES

CONCLUDING REMARKS

The work reported in this manuscript was aimed at understanding the NK-mediated killing of pre-B ALL. Previous publica@ons indicated that these targets were resistant, but the mechanisms responsible for this resistance remain largely unknown [START_REF] Imai | Gene=c modifica=on of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells[END_REF][START_REF] Kübler | Both mature KIR+ and immature KIR-NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice[END_REF][START_REF] Romanski | Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblas=c leukemia[END_REF][START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF][START_REF] Ruggeri | Role of natural killer cell alloreac=vity in HLA-mismatched hematopoie=c stem cell transplanta=on[END_REF]. Our results conclusively establish that pre-B ALL are not intrinsically resistant to NK cell cytotoxicity. In our experiments, despite these blasts were poorly killed aQer a 4h incuba@on period, we found a striking increase in this killing when the incuba@on was extended up to 25h (Figure 19). Is this experimental setup biologically relevant?

We believe the answer is yes, for at least two reasons: first, we observed resistant targets (both the RS4;11 cell line and some primary leukemic samples), meaning that not all long-term co-cultures with NK cells induce target death; second, when CD4 + T cells were used as a nega@ve control for effector cells, no effector-induced cell death was measured, proving that the effect was linked to the cytotoxic poten@al of NK cells and not simply to the prolonged allogeneic co-culture (data not shown).

In our assay, we used NK cells amplified and ac@vated with feeder cells in a mbIL-15dependent system; we decided to use such a protocol because of the limited number of NK cells present in the blood, par@cularly with pa@ent samples. Importantly, we showed that NK cells ac@vated in a dis@nct system (a mbIL-21-dependent, feeder cell-based system), as well as res@ng NK cells displayed the same ability to kill the pre-B ALL cells in a delayed manner (Figure S1). We also demonstrated that primary pediatric pre-B ALL blasts behaved similarly to cell lines and dis@nguished NK-sensi@ve and resistant primary blasts. In sum, we used NKAES-NK cytotoxicity against pediatric pre-B ALL cell lines as a model that includes a number of intrinsic limita@ons but allows flexible work on both effector and target cells.

Having established this cytotoxicity, we aimed to understand the precise molecular mechanisms that governing it. We found that both killing ini@a@on and the effector phase were contact-dependent (Figures 21 and22). In par@cular, we researched the rela@ve contribu@on of the two recognized contact-dependent cytotoxicity pathways, namely the granule exocytosis and the death receptor pathway. To assess the role of these pathways, we used NK cells from PIDs with a complete absence of the granule exocytosis to demonstrate that the late killing by NK cells was s@ll preserved (Figure 23). We also generated target cells resistant to death receptor mediated cell death and again, we found no inhibi@on in the NK mediated killing (Figure 27). In conclusion, our results strongly suggest that the delayed killing of ALL cells by NK cells expanded in the NKAES system relies on a novel unconven@onal cytotoxicity pathway. This novel pathway is independent of both granule exocytosis and death receptor pathway.

In parallel, we became interested in the minimal molecular requirements for target cell death, postula@ng that understanding the engaged pathway was complementary to the study of the effector cells. We found evidence for an apopto@c-like cell death among pre-B ALL (Figure 24) with pan-caspase ac@va@on and mitochondrial depolariza@on (Figures 25 and26). From these empirical observa@ons, we wanted to establish their absolute requirement.

We generated knockout cell lines using the CRISPR/Cas9 technology and, to our surprise, found that caspases 3 and 7 DKO were s@ll efficiently killed (Figure 29), which demonstrates that the ac@va@on of effector caspases is not required for NK-induced cell death. Caspases 8 and 10 DKO targets were also s@ll killed, sugges@ng that these ini@ator caspases are not required either. Finally, disrup@on of the mitochondrial depolariza@on by double knockout of BAX and BAK did not prevent cell death, nor caspase 9 KO, although there was a tendency sugges@ng that mitochondrial signaling amplifies the molecular signaling for cell death commitment.

We have yet to characterize the type of engaged cell death since the pre-B ALL cells consistently died regardless of the canonical apopto@c gene(s) we disrupted. Giving an appropriate name to our cell death is important but following the recommenda@ons of the Nomenclature Commihee on Cell Death (NCCD) [START_REF] Kroemer | Classifica=on of cell death: recommenda=ons of the Nomenclature Commiqee on Cell Death 2009[END_REF], it does not correspond to any category [START_REF] Galluzzi | Molecular defini=ons of cell death subrou=nes: recommenda=ons of the Nomenclature Commiqee on Cell Death 2012[END_REF]. In par@cular, we expect caspases 3 and 7

DKO cells to be resistant to both intrinsic and extrinsic apoptosis [START_REF] Lakhani | Caspases 3 and 7: key mediators of mitochondrial events of apoptosis[END_REF] leading us to limit our descrip@on to an "apopto@c-like cell death" of the pre-B ALL cells.

Importantly, the observa@on that the killing of the BAX BAK DKO targets is preserved (Figure 29) excludes both the possibility of a truncated BID (tBID) mediated cell death [START_REF] Wei | Proapopto=c BAX and BAK: a requisite gateway to mitochondrial dysfunc=on and death[END_REF] as well as the one of the unfolded protein response (UPR) consecu@ve to endoplasmic re@culum (ER) stress [START_REF] Hetz | Proapopto=c BAX and BAK modulate the unfolded protein response by a direct interac=on with IRE1alpha[END_REF], or lysosomal membrane permeabiliza@on induced cell death [START_REF] Boya | Lysosomal membrane permeabiliza=on induces cell death in a mitochondriondependent fashion[END_REF].

In the mean@me, we demonstrated that interfering with ROS with N-acetylcysteine, a ROS scavenger, par@ally inhibited pre-B ALL killing (Figure 30). However, oxida@ve metabolism did not play a significant role in target cell death (Figure 32). Because the NADPH oxidase complex is associated with oxygen-derived species in neutrophils, monocytes/macrophages and B-cells, we studied NK cells from pa@ents suffering from chronic granulomatous disease (CGD) whose ability to produce such deriva@ves is severely compromised in phagocytes [START_REF] Roos | Chronic granulomatous disease[END_REF]. Although no NK cell deficiency has been reported to date among these pa@ents, X-linked CGD (CYBB deficient) ac@vated NK cells had a defect in the late killing of pre-B ALL (Figure 33), but not AR22 (CYBA deficient) or AR47 (NCF1 deficient)-CGD. Of note, to date, no report described a phagocyte-type NADPH complex in NK cells. We demonstrated the expression of the NADPH oxidase components in res@ng NK cells, in par@cular gp91 phox (CYBB) and p22 phox (CYBA). These two proteins heterodimerize to form the flavocytochrome b558 in phagocytes. We showed that these two proteins do not interact in res@ng NK cells in the same conforma@on than in phagocytes and B-cells (Figure 34), and concluded that they had to be part of a different, yet s@ll uncharacterized, molecular complex.

Transcriptomic analysis comparing res@ng NK cells and NKAES-NK either unexposed to targets or following 24h incuba@on with a NK-sensi@ve (REH) or resistant (RS4;11) pre-B ALL cell line revealed interes@ng paherns which suggest deep metabolic changes in effector cells (Figure 37). However, this transcriptomic analysis also showed marked differences in CYBB deficient NK cells, with a strong enrichment for phagocytosis, endocytosis, receptor-mediated endocytosis and Fc receptor signaling related gene ontology terms (Figure 37). The former suggest a compensatory mechanism for NK cells in the semng of a neutrophil defect. To some extent, this observa@on is reminiscent of previous works establishing that NK cells express signaling proteins which are molecular signatures of other immune lineages, including myeloid cell-related proteins (e.g. FcεRγ, SYK, and EAT-2) [START_REF] Schlums | Cytomegalovirus infec=on drives adap=ve epigene=c diversifica=on of NK cells with altered signaling and effector func=on[END_REF].

Func@onal studies addressing cellular fitness in NK cells that are engaged in a killing interac@on, established dis@nct metabolic programs depending on target cell type and, as a corollary, on the effector pathway. We found a striking difference between the metabolic profile of NKAES-NK co-cultured with K562 (which engage granule exocytosis) versus pre-B ALL cell lines (which engage our new cytotoxicity pathway). When exposed to pre-B ALL, NKAES-NK showed a higher spare respiratory capacity (the maximal oxygen consump@on reachable by mitochondrial oxida@ve phosphoryla@on ac@vity) and a higher glycoly@c poten@al, indica@ng that the engagement of different cytotoxic pathways were accompanied by different energe@c requirements and, as a consequence, by different metabolic profiles (Figures 38 and39). Interes@ngly, CYBB deficient NKAES-NK cells (which display a restricted ability to kill pre-B ALL cells, Figure 33) showed a remarkably altered metabolic profile, with a decrease of both spare respiratory capacity and glycoly@c poten@al when exposed to ALL targets as compared to NKAES expanded from a normal donor (Figures 38 and39). These observa@ons suggest a role for gp91 phox in the achievement of a metabolic fitness that correlates with killing capacity and are consistent with a recent work showing that the phagocyte-type NADPH oxidase supports hyperglycolysis in s@mulated neutrophils [START_REF] Baillet | Unexpected func=on of the phagocyte NADPH oxidase in suppor=ng hyperglycolysis in s=mulated neutrophils: key role of 6-phosphofructo-2-kinase[END_REF].

The work involved in this manuscript entails major paradigm shiQs. First, we have conclusively demonstrated the existence of an unconven@onal contact-dependent cytotoxicity pathway, dis@nct from the granule exocytosis and death receptor pathway, which has not been described to-date. In this process, metabolic versa@lity appears crucial.

Second, mirroring the fact that we are facing a novel effector mechanism in NK cells, our results strongly suggest that we have iden@fied a new death mechanism in pre-B ALL target cells, far more complex than a regular apopto@c process in the sense that neither effector caspases 3 and 7, nor mitochondrial signaling appear to be required for the process to reach comple@on.

Finally, we challenge the view of the CGD pathogenesis by establishing (i) that key molecular components of the NADPH oxidase are expressed in NK cells but not in a phagocyte-type complex and (ii) that the components of the complex may have a dis@nct func@on, due to their contribu@on to several different complexes. We found that CYBB is dynamically and specifically regulated during NK cell ac@va@on and prolifera@on (Figure 35). CYBB deficient NK cells were impacted both in their transcrip@onal profile and cytotoxic func@on (impac@ng our new cytotoxicity pathway) while CYBA and NCF1 deficient NK cells were not, at least for their killing behavior. Some teams reported a NADPH oxidase among CD8 + T cells [START_REF] Bai | NADH oxidase-dependent CD39 expression by CD8(+) T cells modulates interferon gamma responses via genera=on of adenosine[END_REF][START_REF] Wen | NADPH oxidase deficiency underlies dysfunc=on of aged CD8+ Tregs[END_REF] and at least one publica@on showed T cell blast prepara@ons expression of a phagocyte-type NADPH oxidase recognized by the 7D5 monoclonal an@body [START_REF] Jackson | T cells express a phagocyte-type NADPH oxidase that is ac=vated aper T cell receptor s=mula=on[END_REF], an observa@on that we and others [START_REF] Vrazo | Rapid detec=on of intracellular p47phox and p67phox by flow cytometry; useful screening tests for chronic granulomatous disease[END_REF] did not reproduce on res@ng CD3 + T cells (Figure 34). We believe the molecular role of CYBB in nonphagocyte cell lineages deserves further explora@on and characteriza@on. Notably, we tested NK cells in a flow cytometric dihydrorhodamine 123 (DHR) assay (data not shown). In this test, oxida@on of DHR by ROS to a fluorescent compound, rhodamine 123, can be assessed.

NK cells were nega@ve in this assay, meaning that they did not produce detectable amounts of ROS. However, although this test is important for CGD diagnosis in clinical labs, complete myeloperoxidase (MPO) deficiency can cause a false-posi@ve result for CGD in the DHR flow cytometric assay (i.e. phagocytes do not oxidize DHR) [START_REF] Mauch | Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in rou=ne tes=ng for CGD[END_REF]. Thus, the DHR test is not always correlated to the clinical severity of the disease since MPO deficient pa@ents usually do not display severe symptoms [START_REF] Lekstrom-Himes | Immunodeficiency diseases caused by defects in phagocytes[END_REF] while their cells can be DHR nega@ve similarly to CGD pa@ents cells. In other words, some aspects could contribute to CGD physiopathology independently of their inability to produce oxygen deriva@ves.

What could be the relevance of our findings? Although experimental limita@ons should be emphasized (most of our work was performed in vitro and used amplified and ac@vated effector cells), we point out what we believe are interes@ng considera@ons:

(i) previous teams reported poten@ated killing abili@es among cytotoxic lymphocytes (CD8 + and CD4 + cytotoxic T cells as well as NK cells) in long-term assays, usually with incuba@on periods up to 24h [START_REF] Baetz | Loss of cytotoxic T lymphocyte func=on in Chediak-Higashi syndrome arises from a secretory defect that prevents ly=c granule exocytosis[END_REF][START_REF] Konomi | Cytotoxic ac=vity of CD4+ T cells against autologous tumor cells[END_REF][START_REF] Lee | Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-ac=vated killer cells[END_REF][START_REF] Reeves | An=gen-specific NK cell memory in rhesus macaques[END_REF]. The interac@on between ac@vated NK cells and autologous immature dendri@c cells was also intensively inves@gated in the early 2000s and shown to require prolonged incuba@on @mes too [START_REF] Piccioli | Contact-dependent s=mula=on and inhibi=on of dendri=c cells by natural killer cells[END_REF][START_REF] Reeves | An=gen-specific NK cell memory in rhesus macaques[END_REF][START_REF] Walzer | Natural-killer cells and dendri=c cells: "l'union fait la force[END_REF]. The precise engaged cytotoxic mechanism has not been specifically addressed but it is interes@ng to note that CTL from Chediak-Higashi pa@ents exhibited a delayed killing [START_REF] Baetz | Loss of cytotoxic T lymphocyte func=on in Chediak-Higashi syndrome arises from a secretory defect that prevents ly=c granule exocytosis[END_REF] despite a strong defect in granule exocytosis among these pa@ents [START_REF] Bryceson | A prospec=ve evalua=on of degranula=on assays in the rapid diagnosis of familial hemophagocy=c syndromes[END_REF][START_REF] Jessen | Subtle differences in CTL cytotoxicity determine suscep=bility to hemophagocy=c lymphohis=ocytosis in mice and humans with Chediak-Higashi syndrome[END_REF], which suggests the engagement of alterna@ve cytotoxic pathway(s) in this late effect.

(ii) many paradigms in NK cell func@on have been established in short term co-cultures between effector and target cells. Of note, biological relevance of limi@ng func@onal studies to short incuba@ons because of technical constraints can be (and has been)

challenged. For instance, the classical dichotomy between the two main subsets, i.e.

CD56 bright and CD56 dim NK cells respec@vely considered as immunoregulatory (in link to cytokine produc@on) and cytotoxic cell subpopula@ons, was queried by inves@ga@ng the kine@cs of their effector func@ons. Indeed, it was shown that the CD56 dim subset actually produces cytokines such as IFN-γ within a very short @me span (2-4h) concomitant to their cytotoxic ac@vity [START_REF] De Maria | Revisi=ng human natural killer cell subset func=on revealed cytoly=c CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on ac=va=on[END_REF]. The authors pointed out that cytokine produc@on by NK cells had been rou@nely analyzed at late intervals (≥ 16 h aQer cell s@mula@on) and that early cytokine produc@on had not been appropriately explored before. By analogy, our results suggest that cytotoxicity could be assessed at late intervals and not only within 4h, to unravel unappreciated killing pathways.

(iii) func@onal explora@on of NK cells usually relies on degranula@on assay and, if applicable, intracellular cytokine staining to evaluate cytokine produc@on following appropriate s@mula@ons [START_REF] Bryceson | Func=onal analysis of human NK cells by flow cytometry[END_REF]. The former, based on CD107a up-regula@on at the cell surface, are widely and efficiently used for the rapid diagnosis of familial hemophagocy@c syndromes [START_REF] Alter | CD107a as a func=onal marker for the iden=fica=on of natural killer cell ac=vity[END_REF][START_REF] Bryceson | A prospec=ve evalua=on of degranula=on assays in the rapid diagnosis of familial hemophagocy=c syndromes[END_REF]. Our results demonstrate that these tests do not allow a full explora@on of NK cell func@ons, but rather skew func@onal studies toward preconceived no@ons.

(iv) as a corollary, we suggest that the elucida@on of our novel cytotoxicity pathway could find at least two further paths for development. First, it would open new fields of inves@ga@on in NK cell contribu@on to innate immunity and pathology beyond an@viral and an@tumoral cytotoxicity; second, it would establish the need for new func@onal assay(s) to improve complete NK cell explora@on. Nevertheless, these future developments would make sense on the condi@on that this novel cytotoxicity pathway could be shown to contribute significantly to clinically relevant disorders.

(v) finally, we believe that this novel effector mechanism is also engaged by CD8 + T cells.

These cytotoxic lymphocytes share molecular ac@va@on pathways and effector responses with NK cells [START_REF] Narni-Mancinelli | The "T-cell-ness" of NK cells: unexpected similari=es between NK cells and T cells[END_REF][START_REF] Sun | NK cell development, homeostasis and func=on: parallels with CD8⁺ T cells[END_REF] and no effector func@on was reported to be strictly specific of either one. However, their respec@ve effector behavior are not necessarily iden@cal. Thus, a study exploring effector lymphocytes from Chediak-Higashi pa@ents showed that CD8 + CTL, but not NK cells, could exhibit a normal cytotoxic func@on further correla@ng with late-onset or absent HLH [START_REF] Jessen | Subtle differences in CTL cytotoxicity determine suscep=bility to hemophagocy=c lymphohis=ocytosis in mice and humans with Chediak-Higashi syndrome[END_REF]. Nevertheless, this observa@on could be explained by dis@nct ac@va@on rules and/or threshold between both effector cell types. Preliminary data in our labs showed that cytokine-induced killer cells (CIK), which are generated in vitro from CD3 + T cells [START_REF] Do=wala | Implica=on of different effector mechanisms by cord blood-derived and peripheral blood-derived cytokine-induced killer cells to kill precursor B acute lymphoblas=c leukemia cell lines[END_REF], also have an increased cytotoxicity against pre-B ALL targets when co-incubated for 24h (Figure S3). Interes@ngly, this cytotoxicity did not involve granule exocytosis since CIK cells did not significantly degranulate (data not shown).

In conclusion, we demonstrate that ac@vated NK cells can engage a previously unappreciated contact-dependent cytotoxicity pathway which is dis@nct from granule exocytosis and death receptors. Furthermore, the corresponding apopto@c-like engaged cell-death appears to rely on unconven@onal molecular requirements. Eventually, we establish a dynamic metabolic fitness in NK cells depending on the target cells they encounter and possibly the cytotoxicity pathway they engage. Dealing with pre-B ALL target cells, gp91 phox expression is important for the NK cell cytotoxicity. We further demonstrate that gp91 phox is expressed and dynamically regulated among NK cells and suggest a poten@al role for this protein in the metabolic control of effector cell func@on.

These discoveries deserve further explora@on and characteriza@on to establish their contribu@on to the immune func@on.

. 2 . P E R S P E C T I V E S

A basic model for further inves@ga@on is presented in Figure 42. It depicts the NK killing process as a sequence of defined events providing key nodes to decipher.

We ini@ally made the choice not to work with a mouse model but instead to favor primary human NK cells that have been ac@vated and amplified in vitro to generate a sufficient amount of cells [START_REF] Imai | Gene=c modifica=on of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells[END_REF]. Murine NK cells display common features with their human counterparts but they also display noteworthy dis@nc@ons that would have limited the scope of our study. For instance, granulysin, which is an important molecule for the elimina@on of intracellular bacteria and parasites by cytotoxic lymphocytes in humans, has no known murine homolog (Do@wala et al., 2016;[START_REF] Walch | Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes[END_REF]. Similarly for target cell death, caspase 10, which is a close homolog of caspase 8 in humans, is absent in rodents [START_REF] Reed | Compara=ve analysis of apoptosis and inflamma=on genes of mice and humans[END_REF]. Caspase 10 is considered to (1) play a role as an ini@ator of the extrinsic apopto@c pathway mediated by TRAIL and FASL [START_REF] Sprick | Caspase-10 is recruited to and ac=vated at the na=ve TRAIL and CD95 death-inducing signalling complexes in a FADDdependent manner but can not func=onally subs=tute caspase-8[END_REF] with some similari@es to caspase 8, and also to (2) par@cipate to proper signaling involved in apoptosis and autophagy [START_REF] Lamy | Control of autophagic cell death by caspase-10 in mul=ple myeloma[END_REF][START_REF] Lee | AK2 ac=vates a novel apopto=c pathway through forma=on of a complex with FADD and caspase-10[END_REF]. More recently, it has been suggested that, far from having redundant func@ons, caspase 10 nega@vely regulates DISC-mediated caspase 8 ac@va@on and contributes to cell commitment between survival and cell death [START_REF] Horn | Caspase-10 Nega=vely Regulates Caspase-8-Mediated Cell Death, Switching the Response to CD95L in Favor of NF-κB Ac=va=on and Cell Survival[END_REF]. Also, we postulate that in vivo studies will be of significant relevance. Indeed, a recent study has demonstrated the ability of ac@vated NK cells to control pediatric pre-B ALL in NSG Effector cells recognize target cells by ac@va@ng and inhibi@ng membrane receptors which engage various signaling pathways, all integrated in the "decision to kill" (inputs). The effector response (outputs), possibly via a metabolic control, is in the form of cytokine produc@on, granule exocytosis, death receptor engagement and our described unconven@onal cytotoxicity pathway.

Cytokine production

Granule exocytosis

Death receptor engagement

Unconventional cytotoxicity pathway

Target recognition

Signal transduction

Signal integration

Effector response Metabolic control?

Inputs Outputs

No effector response mice [START_REF] Kübler | Both mature KIR+ and immature KIR-NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice[END_REF]. We developed exper@se in humanized mice models as well as in pa@ent-derived xenograQs (PDX) and plan to validate our findings using these preclinical mouse models.

Based on our work, subsequent experiments should be performed to beher assess the role of gp91 phox func@on in the above-men@oned model (Figure 42):

A -Biochemical characterizaLon of gp91 phox in NK cells

We have demonstrated that both gp91 phox and p22 phox proteins can be detected in res@ng NK cells, but not within a phagocyte-type NADPH complex.

On one hand, we will establish protein subcellular localiza@on: cellular frac@ona@on will be performed by centrifuga@on on an Op@Prep gradient as described [START_REF] Baginska | Granzyme B degrada=on by autophagy decreases tumor cell suscep=bility to natural killer-mediated lysis under hypoxia[END_REF][START_REF] Noubade | NRROS nega=vely regulates reac=ve oxygen species during host defence and autoimmunity[END_REF] and frac@ons will be tested by nanofluidic proteomic immunoassay.

On the other hand, to iden@fy physiologically-relevant protein-protein interac@ons, we propose to perform co-immunoprecipita@on coupled to mass spectrometry. This approach has already been performed with monoclonal an@bodies directed against gp91 phox (clone 54.1, that we also used for nanofluidic proteomic immunoassays) or p22 phox (clone 44.1) [START_REF] Noubade | NRROS nega=vely regulates reac=ve oxygen species during host defence and autoimmunity[END_REF][START_REF] Raad | Regula=on of the phagocyte NADPH oxidase ac=vity: phosphoryla=on of gp91phox/NOX2 by protein kinase C enhances its diaphorase ac=vity and binding to Rac2, p67phox, and p47phox[END_REF][START_REF] Zhu | Dele=on mutagenesis of p22phox subunit of flavocytochrome b558: iden=fica=on of regions cri=cal for gp91phox matura=on and NADPH oxidase ac=vity[END_REF]. We will apply this protocol to both res@ng and ac@vated NK cells and will use CGD cells as nega@ve controls to determine irrelevant an@bodies' binding.

This combined strategy should allow us to characterize gp91 phox cell localiza@on and its molecular partners.

B -InvesLgaLon of NK cells anLfungal acLvity

CGD pathophysiology is tradi@onally linked to phagocytes (neutrophils, monocytes and macrophages) dysfunc@on due to defec@ve NADPH oxidase ac@vity [START_REF] Kuhns | Residual NADPH oxidase and survival in chronic granulomatous disease[END_REF].

Clinically, it is characterized by severe recurrent bacterial and fungal infec@ons with a limited spectrum of pathogenic species as well as dysregulated inflammatory response resul@ng in granuloma forma@on and inflammatory disorders such as coli@s [START_REF] Marciano | Common severe infec=ons in chronic granulomatous disease[END_REF][START_REF] Song | Chronic granulomatous disease: a review of the infec=ous and inflammatory complica=ons[END_REF]. The most frequently involved extracellular but also intracellular pathogens include Staphylococcus, Serra8a, Nocardia, Burkholderia and Aspergillus species [START_REF] Marciano | Common severe infec=ons in chronic granulomatous disease[END_REF]. Fungal infec@ons by Aspergillus species remain the leading cause of mortality [START_REF] Leiding | Chronic granulomatous disease[END_REF]. Of note, although phagocytes of all CGD pa@ents (regardless of the causa@ve mutated gene) display defec@ve NADPH oxidase func@on with abnormal respiratory burst [START_REF] Kuhns | Residual NADPH oxidase and survival in chronic granulomatous disease[END_REF], there are remarkable clinical differences between gp91 phox -deficient (X-linked) and other CGD pa@ents. The former are more prone to

Aspergillus infec@on [START_REF] Marciano | Common severe infec=ons in chronic granulomatous disease[END_REF] and display a lower survival rate [START_REF] Kuhns | Residual NADPH oxidase and survival in chronic granulomatous disease[END_REF]. Although this observa@on could be explained by a generally lower residual ROS produc@on among X-CGD pa@ents [START_REF] Kuhns | Residual NADPH oxidase and survival in chronic granulomatous disease[END_REF][START_REF] Marciano | Common severe infec=ons in chronic granulomatous disease[END_REF], it does not rule out a possible contribu@on of other immune cell popula@ons. For instance, Kostmann's syndrome, which is caused by muta@ons in the HAX1 gene and oQen considered as the paradigm of congenital neutropenia, usually presents with bacterial but no fungal infec@ons except as a complica@on of prolonged an@bio@c therapy [START_REF] Antachopoulos | Invasive fungal infec=ons in congenital immunodeficiencies[END_REF][START_REF] Donadieu | Congenital neutropenia: diagnosis, molecular bases and pa=ent management[END_REF][START_REF] Skokowa | Severe congenital neutropenias[END_REF].

We hypothesize that NK cells could be involved in the control of Aspergillus infec@on and that this role could depend on gp91 phox . NK cells cytotoxicity (either directly or through their crosstalk with neutrophils) against yeast fungus such as Cryptococcus and Candida species has been well documented [START_REF] Bär | IL-17 regulates systemic fungal immunity by controlling the func=onal competence of NK cells[END_REF][START_REF] Li | The NK receptor NKp30 mediates direct fungal recogni=on and killing and is diminished in NK cells from HIV-infected pa=ents[END_REF][START_REF] Ma | NK cells use perforin rather than granulysin for an=cryptococcal ac=vity[END_REF]. A few works have also addressed their func@on against filamentous fungus such as Aspergillus species and suggested a link between NK-cell counts aQer HSCT and invasive aspergillosis [START_REF] Stuehler | Immune Recons=tu=on Aper Allogeneic Hematopoie=c Stem Cell Transplanta=on and Associa=on With Occurrence and Outcome of Invasive Aspergillosis[END_REF] or even showed a direct NK cell an@fungal ac@vity against Aspergillus hyphae but not conidia [START_REF] Schmidt | Human natural killer cells exhibit direct ac=vity against Aspergillus fumigatus hyphae, but not against res=ng conidia[END_REF]2013). More recently, CD56 has been demonstrated to play a role in the Aspergillus mediated NK cell ac@va@on and was proposed as a pathogen recogni@on receptor [START_REF] Ziegler | CD56 Is a Pathogen Recogni=on Receptor on Human Natural Killer Cells[END_REF]. Besides, it has been widely documented that there is crosstalk between neutrophils and NK cells serving to reciprocally modulate their responses [START_REF] Scapini | Social networking of human neutrophils within the immune system[END_REF].

As our data suggests that metabolism is a key feature of gp91 phox -dependent NK cell func@on, we propose to study two readouts using primary cells from normal donors or CGD pa@ents:

(i) NKAES-NK cells cytotoxicity against Aspergillus fumigatus and (ii) fungus-induced metabolic changes when res@ng or ac@vated NK cells will be facing Aspergillus hyphae or conidia.

Furthermore, we believe that studying inflammasome-derived cytokines induced changes (such as IL-18 and IL-1β (de Zoete et al., 2014;[START_REF] Guo | Inflammasomes: mechanism of ac=on, role in disease, and therapeu=cs[END_REF] on NK cells metabolism would be interes@ng since inflammasome-derived IL-18 has been showed to prime NK cell cytotoxicity to kill infected hepatocytes in mice infected with Chromobacterium violaceum, an intracellular bacteria [START_REF] Maltez | Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infec=on by a Ubiquitous Environmental Bacterium[END_REF] which is an opportunis@c pathogen for CGD pa@ents [START_REF] Song | Chronic granulomatous disease: a review of the infec=ous and inflammatory complica=ons[END_REF].

C -NK cells heterogeneity: idenLficaLon of the novel effector mechanism

We previously discussed phenotypic as well as behavioral diversity among cytotoxic lymphocytes ( §1.4). As shown by others [START_REF] Choi | Imaging burst kine=cs and spa=al coordina=on during serial killing by single natural killer cells[END_REF][START_REF] Vanherberghen | Classifica=on of human natural killer cells based on migra=on behavior and cytotoxic response[END_REF][START_REF] Vasconcelos | Individual Human Cytotoxic T Lymphocytes Exhibit Intraclonal Heterogeneity during Sustained Killing[END_REF], cytotoxicity is not a rapid and homogeneous response to target cells. In vitro, some effector clones are endowed with a serial killing ac@vity when facing target cells. However, in vivo studies tend to support a lower rate of killing at the single effector cell level and a sustained role for dynamic interac@ons and coopera@on among cytotoxic cells [START_REF] Halle | In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interac=ons and T Cell Coopera=vity[END_REF]. There is currently no consensus on the order of magnitude of in vivo killing rates in either experimental models or human diseases. In vivo compara@ve studies also showed some differences between NK cells and CD8 + T cells [START_REF] Deguine | Dynamics of NK cell interac=ons in vivo[END_REF][START_REF] Deguine | Intravital imaging reveals dis=nct dynamics for natural killer and CD8(+) T cells during tumor regression[END_REF]. In comparison to the laher, the former establish contacts which have been described as mainly dynamic.

Our RNAseq experiment showed that the responses to REH or RS4;11 cells are, in general, larger in effect size in X-linked CGD pa@ent's samples than in the controls (Figure 43).

Because we had only one X-CGD pa@ent for which NK cell expansion generated a sufficient amount of cells to be tested against pre-B ALL cell lines, we s@ll consider these results as very preliminary (even though we observed for other samples a perfect separa@on per condi@on and donor with unsupervised clustering using PCA (Figure 36A)).

However, we observed that the specific loss of the CYBB gene led to a drama@c change in the effect sizes for response to sensi@ve or resistant pre-B ALL cell lines (Figure 43). In other words, this implies that among ac@vated NK cells exposed to targets, CYBB deficiency induces many other genes to vary in terms of expression level, which could suggest a role for gp91 phox as a node in a gene regulatory network.

Among NK cell effector func@ons, cytokine produc@on and cytotoxicity have been uncoupled [START_REF] Rajasekaran | Signaling by Fyn-ADAP via the Carma1-Bcl-10-MAP3K7 signalosome exclusively regulates inflammatory cytokine produc=on in NK cells[END_REF][START_REF] Vivier | ADAPted secre=on of cytokines in NK cells[END_REF]. However, at the single cell level, it has not been studied whether single effector func@ons are characteris@c of specific commihed subsets or can be engaged by a same unique NK cell (Figure 44).

Why don't NK cells degranulate when they face pre-B ALL cells? Rouce et al. suggested that TGF-β is a major mediator of immune evasion in childhood pre-B ALL [START_REF] Rouce | The TGF-β/SMAD pathway is an important mechanism for NK cell immune evasion in childhood Bacute lymphoblas=c leukemia[END_REF].

Deep modula@on of NK cell effector func@ons by TGF-β has been documented and this modula@on was linked to effector cell metabolism [START_REF] Gao | Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells[END_REF][START_REF] Viel | TGF-β inhibits the ac=va=on and func=ons of NK cells by repressing the mTOR pathway[END_REF]. It is possible that leukemia-derived TGF-β is a triggering signal for a metabolic reprogramming and effector switch in NK cell func@ons.

Our results show different metabolic profiles depending on the target cell type co-cultured with NKAES-NK cells. They also demonstrate that gp91 phox /CYBB is expressed and dynamically regulated among NK cells and suggest that NKAES-NK cells from X-CGD pa@ents have a defect in our novel cytotoxic pathway.

As a working hypothesis, we propose that a global and versa@le metabolic playorm could control NK cell effector func@on. In this model, gp91 phox could be involved in the control of our novel cytotoxicity pathway.

To gain further insight, we propose to study the respec@ve importance of oxida@ve phosphoryla@on and glycolysis as well as to dissect the metabolic pathways through integra@ve metabolomics and proteomics profiling of NKAES-NK cells facing pre-B ALL targets [START_REF] Geiger | L-Arginine Modulates T Cell Metabolism and Enhances Survival and An=-tumor Ac=vity[END_REF][START_REF] Hukelmann | The cytotoxic T cell proteome and its shaping by the kinase mTOR[END_REF][START_REF] Ross | Phosphoproteomic Analyses of Interleukin 2 Signaling Reveal Integrated JAK Kinase-Dependent and -Independent Networks in CD8(+) T Cells[END_REF][START_REF] Tan | Integra=ve Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenerge=cs Pathways Underlying T Cell Ac=va=on[END_REF]. To precisely map each pathway, effector cells from pa@ents with defec@ve granule exocytosis (such as Cytotoxic cell output can engage an effector response (Figure 42) in the form of cytokine produc@on, granule exocytosis, death receptor engagement and our unconven@onal cytotoxicity pathway. At the single cell level, one may propose two alterna@ve models: a plas@c and permissive model (A) which postulates possible switches between effector responses, and a commihed and restric@ve model (B) which proposes that dis@nct NK clones engage each type of response. Whatever the model, we hypothesize the metabolic state to be a central checkpoint, possibly through a crosstalk with epigene@c regula@on. PRF1 or UNC13D deficient ones) and the use of death receptor resistant target cells will be of significant interest.

To some extent, this view (Figure 44) is reminiscent of the epigene@c landscape depicted by Conrad Waddington [START_REF] Waddington | The strategy of the genes. A discussion of some aspects of theore=cal biology. With an appendix by H. Kacser. The Strategy of the Genes a Discussion of Some[END_REF]. Interes@ngly, epigene@c modifica@ons have been linked to memory NK cell development [START_REF] Lee | Epigene=c Modifica=on and An=body-Dependent Expansion of Memory-like NK Cells in Human Cytomegalovirus-Infected Individuals[END_REF][START_REF] Schlums | Cytomegalovirus infec=on drives adap=ve epigene=c diversifica=on of NK cells with altered signaling and effector func=on[END_REF] and to the regula@on of NK cell effector func@ons [START_REF] Cichocki | Epigene=c regula=on of NK cell differen=a=on and effector func=ons[END_REF]. Cell fate and func@on integrate an interplay between metabolism, epigene@cs and transcrip@on [START_REF] Etchegaray | Interplay between Metabolism and Epigene=cs: A Nuclear Adapta=on to Environmental Changes[END_REF][START_REF] Kaelin | Influence of metabolism on epigene=cs and disease[END_REF][START_REF] Lu | Metabolic regula=on of epigene=cs[END_REF], which has also shown to be relevant for the immune system func@on [START_REF] Xu | Metabolic control of TH17 and induced Treg cell balance by an epigene=c mechanism[END_REF]. Thus, we think that studying epigene@c and metabolic control of NK cell effector func@ons could be an interes@ng and complementary approach. Eventually, understanding the precise metabolic requirements could open new perspec@ves to restore an effector func@on [START_REF] Fisicaro | Targe=ng mitochondrial dysfunc=on can restore an=viral ac=vity of exhausted HBV-specific CD8 T cells in chronic hepa==s B[END_REF][START_REF] Kawalekar | Dis=nct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells[END_REF][START_REF] Schurich | Dis=nct Metabolic Requirements of Exhausted and Func=onal Virus-Specific CD8 T Cells in the Same Host[END_REF] or favor the switch between effector func@ons by cellular reprogramming.

D -Target cell sensiLvity or resistance: idenLficaLon of the death process in pre-B ALL

Our results support a death mechanism which has never been reported. In the mean@me, understanding the mechanism of pre-B ALL cells death is crucial to unravel the new cytotoxic pathway. When we looked at the metabolic profile of pre-B ALL and K562 cell lines, we observed that although glycolysis was similar, pre-B ALL cells had a much lower oxida@ve phosphoryla@on than K562 cells, with the lowest OXPHOS status being observed in the resistant RS4;11 cell line (Figure 41). Besides, we demonstrated that primary pre-B ALL blasts could be separated between sensi@ve and resistant cells (Figures 19C and 19D). We think that a deeper inves@ga@on of pre-B ALL cells' metabolism is necessary: in par@cular, we propose to test whether basal metabolism could predict the sensi@vity of blasts to NK cell killing.

To discover the main gene@c components of our novel cell death pathway in pre-B ALL cells, we propose to perform a genome-scale CRISPR/Cas9-based screening (Figure 45).

Following this strategy, we expect that for sensi@ve pre-B ALL cell lines, the cell viability will be nega@vely impacted with the excep@on of the subset of cells that contain dele@ons in genes essen@al for cell death associated with our new pathway. This will enrich cells that contain sgRNAs that have produced the deficiency in expression of essen@al cell death genes (posi@ve selec@on). On the contrary, for the resistant RS4;11 cell line, which normally survives well facing the cytotoxic insult of NKAES-NK, we expect a deple@on of the cells that contain the sgRNAs that produced the dele@on of genes (nega@ve selec@on) that counteract our new pathway.

Such an approach has been recently successfully used for the iden@fica@on of genes which are essen@al for CTL effector func@on [START_REF] Patel | Iden=fica=on of essen=al genes for cancer immunotherapy[END_REF]. However, it has never been employed for NK cell mediated cytotoxicity or to decipher essen@al genes specific of each of the engaged effector pathways.

Finally, although this screening would allow key molecular pathways to be pinpointed, we think that our results strongly support the hypothesis of a non-gene@c origin of target cell resistance to NK cell mediated cytotoxicity (Figure 46). Through the CRISPR/Cas9-based strategy, we generated knockout clonal target cell lines.

Interes@ngly, when clonal pre-B cell lines have been used, we measured a similar NK killing rate compared to parental bulk cell lines (for the sensi@ve cell lines NALM6, REH and 697). This observa@on suggests that resis@ng pre-B target cells (among an otherwise sensi@ve cell line) do not display a heritable phenotype but rather a non-gene@c origin of cellular variability to NK cell induced-apoptosis, which calls to mind previous works in the field of Sensi@ve and resistant pre-B ALL cell lines are infected at low mul@plicity of infec@on (MOI) to ensure that blasts receive only a single viral par@cle (here, a strategy using a two-plasmid system is depicted; [START_REF] Shalem | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells[END_REF]. For each cell line, a first group is exposed to NKAES-NK cells. The second group of cells, ac@ng as the nega@ve control group cultured under the same condi@on, is not exposed to effector cells. Following incuba@on, the genomic DNA is extracted and sgRNA amplified with specific primers.

cellular drug resistance [START_REF] Flusberg | Surviving apoptosis: life-death signaling in single cells[END_REF][START_REF] Flusberg | Cells surviving frac=onal killing by TRAIL exhibit transient but sustainable resistance and inflammatory phenotypes[END_REF][START_REF] Shaffer | Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance[END_REF][START_REF] Spencer | Non-gene=c origins of cell-to-cell variability in TRAIL-induced apoptosis[END_REF]. Analysis of these complex phenotypes could be conducted combining genome-scale CRISPR/Cas9-based screening and single cell RNAseq [START_REF] Dixit | Perturb-Seq: Dissec=ng Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Gene=c Screens[END_REF], a technology that we are currently using for ongoing experiments [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF].

To conclude, our findings challenge the dogma of pre-B ALL resistance to NK cell cytotoxicity;

and of the absence of gp91 phox expression and role in NK cells. Further works should provide a more complete understanding of this novel cytotoxicity pathway and induced target cell death.

Figure 46. AlternaLve models for resistance to NK cell mediated cytotoxicity.

(A) Resistant cells are gene@cally dis@nct from their sensi@ve counterparts. In that model, when a satura@ng amount of effector cells is used only resistant clones are recovered. (B) Resistant cells represent a transient state. Here, when a satura@ng amount of effector cells is used, both resistant and sensi@ve clones will be recovered following NK cell cytotoxicity.

A

Sensitive cell

Genetic mutation model (heritable phenotype)

Resistant cell For each contrast, + or -stand for up-regulated or down-regulated genes respec,vely.

Co-culture with NK cells

Appendix 1. pHRSIN UCOE0.7 SFFV DEST (plasmid map).

Appendix 2. lenHCas9-Blast (plasmid map).

de déficits immunitaires primi3fs ainsi que dans celui des hémopathies malignes) [START_REF] Gooley | Reduced mortality aper allogeneic hematopoie=c-cell transplanta=on[END_REF] ; ensuite, elle autorise le recours à un traitement myéloabla3f "à haute dose" pour les pa3ents aKeints de cancer, pour lesquels cela permet d'accentuer l'élimina3on des cellules tumorales mais conduit aussi à une perte de fonc3on médullaire permanente nécessitant donc le secours d'une allogreffe.

Dans ce contexte, l'immunobiologie de l'allogreffe de cellules souches hématopoïé3ques le cadre de l'allogreffe de cellules souches hématopoïé3ques [START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF]. Leurs travaux ont établi que les leucémies aiguës myéloïdes (ou LAM) du receveur peuvent être éliminées par les cellules NK du donneur présentes dans le greffon. Cependant, les LAL pré-B ont été décrites résistantes à cet effet cytotoxique [START_REF] Imai | Gene=c modifica=on of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells[END_REF][START_REF] Kübler | Both mature KIR+ and immature KIR-NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice[END_REF][START_REF] Romanski | Mechanisms of resistance to natural killer cell-mediated cytotoxicity in acute lymphoblas=c leukemia[END_REF][START_REF] Ruggeri | Effec=veness of Donor Natural Killer Cell Alloreac=vity in Mismatched Hematopoie=c Transplants[END_REF][START_REF] Ruggeri | Role of natural killer cell alloreac=vity in HLA-mismatched hematopoie=c stem cell transplanta=on[END_REF]. Cette cytotoxicité est contact-dépendante mais ni la voie de l'exocytose des granules cytotoxiques ni celle des récepteurs de mort n'y contribuent. La mort cellulaire des cibles est de profil apoptotique mais indépendante des caspases ; la signalisation mitochondriale l'amplifie partiellement. Interférer avec les dérivés de l'oxygène par un antioxydant diminue la cytotoxicité. Nous montrons que les cellules NK de patients atteints de granulomatose septique chronique liée à l'X présentent un défaut de cette nouvelle cytotoxicité. Nous démontrons l'expression par les NK des composants clefs d'une NADPH oxydase distincte du complexe utilisé par les phagocytes. Nos travaux établissent l'existence d'une voie de cytotoxicité non conventionnelle et en définissent les principaux prérequis moléculaires. Title: Natural Killer cells and pre-B acute lymphoblastic leukemia: evidence for an unconventional cytotoxicity pathway Keywords: Natural Killer cells; acute lymphoblastic leukemia; cytotoxicity; cell death; chronic granulomatous disease; immune deficiencies Abstract: Natural Killer (NK) cells are innate lymphoid cells with anti-infectious and antitumoral activities. Among neoplasia, pre-B acute lymphoblastic leukemias (pre-B ALL) represent the most common form of cancer in childhood and were shown to be resistant to NK cell mediated cytotoxicity although the mechanisms explaining this phenomenon are incompletely understood. In the present work, we investigated the relative immune resistance of pediatric pre-B ALL targets to activated NK cells. We developed a flow cytometry based cytotoxicity assay to assess the NK activity and the involvement of long term cytotoxic pathways. Although pre-B ALL blasts were strongly resistant at 4h, we found a considerable delayed NK killing at 25h. Further investigations revealed that cell contact was mandatory for efficient killing but also that neither the granule exocytosis nor the death receptor pathway were involved. Target cell death was caspase independent but mitochondria signaling amplified it. We then showed that NK cells from patients with Xlinked chronic granulomatous disease could not kill efficiently ALL blasts and that NK cells expressed key components of a NADPH oxidase complex that was distinct from the phagocyte type. Our work reveals an uncharacterized effector pathway among cytotoxic lymphocytes and establishes key molecular requirements for this unconventional pathway.

  MOLECULAR REQUIREMENTS AMONG EFFECTOR CELLS .......................................... p 3.2.6. MINIMAL MOLECULAR REQUIREMENTS AMONG TARGET CELLS .......................................... p 3.2.7. ROLE OF REACTIVE OXYGEN SPECIES (ROS) ............... p 3.2.8. EVIDENCE FOR A NADPH COMPLEX IN NK CELLS .........Innate lymphoid cells IL-X : Interleukin-X (X depicts a number) IRES : Internal ribosome entry site IU: Interna)onal unit KIR(L): Killer immunoglobulin-like receptors (ligand) KO: Knockout LAMP-1: Lysosomal-associated membrane protein 1 LFA-1: Lymphocyte func)on-associated an)gen 1 MFI : Mean fluorescence intensity MHC: Major histocompa)bility complex MOMP: Mitochondrial outer membrane permeabiliza)on MPO: Myeloperoxidase MTOC: Microtubule-organizing centre mTOR: mammalian Target of rapamycin NAC: N-acetylcystein NADPH oxidase: nico)namide adenine dinucleo)de phosphate-oxidase NCRs: Natural cytotoxicity receptors NEMO: NFκB essen)al modulator NFκB: Nuclear factor-kappa B NK: Natural Killer cells NKAES : Natural Killer ac)va)on and expansion system NSG: Nod/SCID/IL2Rγ -/-mice OCR : Oxygen consump)on rate OXPHOS : Oxida)ve phosphoryla)on PBMCs: Peripheral blood mononuclear cells PCA: Principal component analysis PCR: Polymerase chain reac)on PD-(L)1: Programmed cell death-(ligand) 1 PDX: Pa)ent-derived xenograR PE : Phycoerythrin PIDs: Primary immune deficiencies RAG: Recombina)on-ac)va)ng gene RNAseq: RNA sequencing ROS: Reac)ve oxygen species SCID: Severe combined immunodeficiency sgRNA: single guide RNA SLAM: Signaling lymphocyte ac)va)on molecule TCR: T cell receptor TGF-β: Transforming growth factor-beta (mb)TNF-α: (membrane-bound) Tumor necrosis factor-alpha TNFR superfamily: Tumor necrosis factor-receptors superfamily TRADD: Tumor necrosis factor receptor type 1-associated death domain protein TRAIL: Tumor-necrosis-factor related apoptosis inducing ligand UCOEs: Ubiquitous chroma)n opening elements UPR: Unfolded protein response

Figure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :Figure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 26 :Figure 27 :Figure 28 :Figure 29 :Figure 30 :Figure 31 :Figure 32 :Figure 33 :Figure 34 :Figure 35 :Figure 36 :Figure 37 :Figure 38 :Figure 39 :Figure 40 :Figure 41 :Figure 42 :LISTFigure 43 :Figure 44 :Figure 45 :Figure 46 :Figure S1 :Figure S3 :

 12345678910111213141516171819202122232425262728293031323334353637383940414243444546S1S3 Figure 1: Innate lymphoid cells and their adap2ve lymphocytes counterpart: a mirrored model ......................................... p

Figure 1 .

 1 Figure 1. Innate lymphoid cells and their adapFve lymphocytes counterpart: a mirrored model.Signals from injured or infected .ssues ac.vate ILCs whose func.on will mirror those of CD8 + and CD4 + T cells. The main difference between both cell types is the kine.cs of the immune response. Adapted from[START_REF] Eberl | Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology[END_REF][START_REF] Oskarsson | La rechute survient chez environ 20% des pa3ents pédiatriques et 60% des pa3ents adultes (Perova et al[END_REF]

Figure 2 .

 2 Figure 2. A model of NK cells acFvaFon: signal integraFon and outcome depending on target cell type. Both ac.va.ng and inhibi.ng signals are integrated following the adhesion of NK cells to target cells. Self MHC-I molecule provides strong inhibi.on through the engagement of inhibitory receptors. In an allogeneic sepng or in case of infec.on or tumoral transforma.on, ac.va.ng signals become predominant and trigger NK cell effector response (mainly granule exocytosis). Of note, cytokine produc.on is regulated in the same way.

Figure 3 .

 3 Figure 3. Different effector pathways can be engaged by cytotoxic lymphocytes. (A) Granule exocytosis mainly involves perforin, granzymes and granulysin secre.on. (B) Death receptors engagement are part of contact-dependent pathways. (C) Cytokine secre.on (TNF-α and IFN-γ) can sensi.ze target cells to cytotoxic lymphocytes.

Figure 4 .

 4 Figure 4. Cytotoxic killing of a cognate target cell through the granule exocytosis.

Figure 5 .

 5 Figure 5. Induced target cell death through the granule exocytosis pathway.Perforin, granzyme and granulysin are released by cytotoxic lymphocytes in the synapse. Direct entry of granzyme through cell membrane perforin pores and the "endosomolysis" hypothesis are depicted. Once in the cytosol, granulysin acts through a similar mechanism, inducing pores in intracellular bacteria and parasites which allows granzyme to induce pathogen elimina.on.

Figure 8 .

 8 Figure 8. AML and B-ALL cancer stem cell divergent models.

  Figure 9. NK cells ac7va7on and expansion system (NKAES) from PBMCs. The final purity of the effector cells (> 80%) is assessed by flow cytometry before using the NK cells for further func2onal assays.

Figure 10 .

 10 Figure 10. Flow cytometry-based cytotoxicity (FCC) assay (representa7ve dot plots with REH cell line as target and an E:T ra7o = 4:1).NKAES-NK cells were purified from the cell culture by CD3 posi2ve cells deple2on while target cells were stained with PKH26. Cytotoxicity assays included effector:target ra2os from 4:1 to 1:2 and at least two 2me points (4h and 25h) to assess short and long term cytotoxicity. Specific lysis was calculated by determining the absolute count of targets (PKH26 + 7-AAD -CD19 + ) in presence or in absence of effector cells.

Len2viralFigure 11 .

 11 Figure 11. Homemade Gateway based len7viral plakorm. Len2viral vectors for transgene overexpression were generated using the promoter (SFFV) based solu2on.

Figure 12 .

 12 Figure 12. Uniplex genome engineering strategy.Len2viral expression vectors for Cas9 and sgRNA mammalian delivery using a dual vector system.

Figure 13 .

 13 Figure 13. Mul7plex genome engineering strategy.Len2viral expression vectors for Cas9n (D10A mutated nickase) (A) and mul2ple sgRNAs mammalian delivery using a dual vector system (B).

  first transduced with the len2Cas9n-Blast plasmid (Addgene #63593,

Figure 14 .

 14 Figure 14. NCRs blockade using Fc chimeras. Natural Cytotoxicity Receptors (NCRs) engagement was blocked by satura2on of the corresponding ligands with a large excess of NCRs chimera proteins.

Figure 15 .

 15 Figure 15. Parameters for the annexin-V staining.

Figure 17 .

 17 Figure 17. Experimental design for sample processing in the RNA-sequencing experiment.A total of seven donors (3 X-CGD and 4 normal donors) were recruited which represented 24 samples for further sequencing and analy2c steps.

Figure 18 .

 18 Figure18. Group design in the RNA-sequencing experiment analysis. Normal donors and X-CGD pa2ents defined respec2vely groups 1,3, 5, 6, and 2, 4, 7, 8.

  j) equal to 1 if sample I corresponds to donor j and 0 otherwise. -u: individual-wise random effect, follow: !~#$% 0, ( ) * + Fixed effects nested design for deconvolve samples content: Model 2 Again, we have two auxiliar models M_nk and M_imp:

Table 4 .

 4 Differen7al expression contrasts retrieved from Model 1. Contrast 2 disease effect on NKAES cells: (G4-G3) Contrast 3 disease effect on NKAES cells exposed to REH: (G7-G5) Contrast 4 disease effect on NKAES cells exposed to RS4;11: (G8-G6) Contrast 8 REH effect in controls' NKAES's: (G5-G3) Contrast 9 REH effect in pa2ent' NKAES's: (G7-G4) Contrast 10 Difference between REH effect in pa2ent minus controls' NKAES's: (G7-G4)-(G5-G3) Contrast 11 RS4;11 effect in controls' NKAES's: (G6-G3) Contrast 12 RS4;11 effect in controls' NKAES's: (G8-G4) Contrast 13 Difference between RS4;11 effect in pa2ent minus controls' NKAES's: (G8-G4)-(G6-G3) Contrast 14 RS4;11 minus REH effect in controls' NKAES's: (G6-G5) Contrast 15 RS4;11 minus REH effect in pa2ent' NKAES's: (G8-G7) Groups used for contrasts' defini2on are depicted in Figure 18.

Figure 19 .

 19 Figure 19. Pre-B ALL are not intrinsically resistant to NK cell mediated cytotoxicity. (A) Representa-ve graphs showing early and late leukemic killing by NKAES-NK cells. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. Shown are mean ± SD. (B) Results from twenty-one (NALM6 cell line), twenty-three (REH cell line) and eleven (697 cell line) normal donors respec-vely. (C) Leukemic blasts from four different pa-ents (amplified in NSG mice) were assessed using NKAES-NK from three different normal donors. Each primary blasts were tested against two different NK donors. (D) Leukemic blasts from three different pa-ents (amplified in NSG mice) were assessed using NKAES-NK from three different normal donors. Each primary blasts were tested against two or three different NK donors. Sta-s-cs: Wilcoxon matched-pairs signed rank test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001).

Figure 20 .

 20 Figure 20. A simplified three-step model of the NK killing process.In this model, one can dis-nguish the following sequence of events: adhesion between NK and target cells (1), signals integra-on leading to either an ac-va-on or an inhibitory synapse (2) and effector phase per se, i.e. mechanisms responsible for targets' death (3).

  25h with the same target cells but didn't detect any difference (data not shown), i.e. the pre-incuba-on step did not increase the forma-on of conjugates.

Figure 21 .

 21 Figure 21. Killing iniVaVon and effector phase are contact-dependent. (A and B) Conjugate forma-on was measured within minutes following NK and target cells co-incuba-on. K562 (with -nega-ve control -or without -posi-ve control -EDTA 1mM), NALM6, REH and 697 cell lines were studied. NK and target cells were stained with PKH26 and CFSE respec-vely, and double posi-ve conjugates were quan-fied by flow cytometry. (C-E) Experimental condi-ons used for the transwell experiment. The lower compartment contained 30 x 10 3 NK cells (blue cells on the scheme) and blasts (green cells, C and E) according to the E:T ra-o (15 x 10 3 and 120 x 10 3 targets for 1:2 and 4:1 ra-os). The upper compartment contained a fixed number of 20 x 10 3 VPD450 + blasts (yellow cells, D and E). 697 cell line was used as target. (F -H) Cell contact between target and NK cells was either allowed (G, control) or prevented by a transwell membrane and the lower compartment seeded with NK cells alone or co-incubated with targets (F and H), corresponding to C, D and E respec-vely. Shown are percentages of cytotoxicity (G) or VPD450 + target cells absolute counts in the upper compartment (F and H), rela-ve to cultures with no effector cells in co-cultures las-ng 4 and 25h. Representa-ves results shown are mean ± SD.

Figure 22 .

 22 Figure 22. NK cell killing is cumulaVve and requires permanent cell adhesion. (A) Experimental design. Red lines represent -mes that XVA143 was added to co-cultures. Blue and green arrows depict the two referen-al 4h and 25h incuba-ons. (B) Real--me killing measurements (gray bars; 4h and 25h incuba-on values are shown by red dashed lines as references). Blue and green dashed curves depict cytotoxicity values measured aber 4h and 25h respec-vely (A) for each -me point XVA143 was added. (C) Evolu-on of target cell (PKH26 + CD19 + ) absolute counts (gray dashed line: control, i.e. targets alone; red line: targets with NK cells). (D) Annexin-V staining assay was performed at 4h, 12h and 25h to precise cell death process. (E and F) Histograms show the killing inhibi-on of the 4h and 25h cytotoxicity (respec-vely E and F) achieved by XVA143 addi-on at different -me points. Representa-ve results of one experiment shown are mean ± SD against REH cell line and 4:1 effector to target ra-o.

Figure 23 .

 23 Figure 23. NK cell killing is independent of the perforin and degranulaVon pathway. (A) Dot plots show CD107a/CD107b surface expression on NK cells leb alone or exposed for 4h to K562 (posi-ve control), NALM6, REH and 697 cell lines (A). (B) The graph depicts frequencies of NK cell subsets posi-ve for CD107a/CD107b (related to A).(C and D) Effect of the NCRs (NKp30, NKp44, NKp46 and NKp80) or NKG2D blockade was evaluated on the cytotoxicity against REH cell line, at 4h and 25h (C). The corresponding percentage of inhibi-on was calculated (D). (E and F) Effect of the blockade of NKG2D and DNAM-1 alone or in combina-on was evaluated on the cytotoxicity against 697 cell line, at 4h and 25h (E). The corresponding percentage of inhibi-on was calculated (F). (G-I) Graphs showing early and late leukemic killing of K562, NALM6 and REH cell lines either unmodified or transduced with sCD107a. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. Overlays showing sCD107a expression on transduced targets are also shown. (J-M) Graphs showing early and late leukemic killing of K562, NALM6, REH and 697 cell lines using NK cells from a normal donor or a PRF1 deficient pa-ent. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. Two pa-ents were tested with the same results. (N-Q) Graphs showing early and late leukemic killing of K562, NALM6, REH and 697 cell lines using NK cells from a normal donor or a UNC13D deficient pa-ent. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. Two pa-ents were tested with the same results. (A-Q) Shown are mean ± SD.

Figure 24 .

 24 Figure 24. ApoptoVc-like flow cytometry profile of pre-B ALL cells. Representa-ve dot plots show target cells annexin-V / 7-ADD co-staining following 4h, 10h and 25h incuba-on with NK cells (4:1 effector to target ra-o). Soluble human recombinant SuperKillerTRAIL (250 ng/mL; Enzo Life Sciences) was used as posi-ve control.

Figure 25 .

 25 Figure 25. ApoptoVc pre-B ALL cells acVvate both intrinsic and extrinsic pathways. (A) Representa-ve dot plots show target cells annexin-V / 7-ADD co-staining among pre-B ALL targets (PKH26 + CD19 + ). Blue and orange rectangles depict living and early apopto-c cells respec-vely: these two colors are used to define these two same popula-ons in the other panels (B-D) and Figure 26. (B) Representa-ve overlay histogram of poly-caspase ac-vity (FLICA). Gray dashed line histogram show the target cells alone control condi-on. (C) Representa-ve overlay histograms of caspases 3&7, 8, 9 and 10 ac-vi-es (FLICA). Gray dashed line histogram show the target cells alone control condi-on. Gray dashed line histogram show the target cells alone control condi-on.(D) The ra-ometric ΔΨm indicator JC-1 was used to evaluate mitochondrial outer membrane permeabiliza-on (MOMP). CCCP treatment was used as posi-ve control. Representa-ve results against NALM6, REH and 697 cell lines and 4:1 effector to target ra-o are shown. Observa-ons were the same following 4h, 10h and 25h incuba-on.

Figure 26 .

 26 Figure 26. Hierarchical ordering of the acVvaVon of the apoptoVc extrinsic pathway.Representa-ve dot plots show target cells ac-ve caspase 8 / caspases 3&7 co-staining (FLICA) following incuba-on with NK cells (4:1 effector to target ra-o). Blue and orange histograms depict living and early apopto-c cells respec-vely (Figure25A). NALM6, REH and 697 cell lines were used as targets. Observa-ons were the same following 4h, 10h and 25h incuba-on.

Figure 27 .

 27 Figure 27. Pre-B ALL cell killing is independent of the death receptor pathway. (A) Effect of the TRAIL receptors (DR4 and DR5) blockade was evaluated on the cytotoxicity against 697 cell line following 4h and 25h incuba-on. K562 cell line was used as a posi-ve control. (B) Effect of etanercept pre-treatment was evaluated on the cytotoxicity against REH cell line following 4h and 25h incuba-on. (C-F) Graphs showing early and late leukemic killing of K562, NALM6, REH and 697 cell lines (control or hFADD-DN transduced) using NK cells from a normal donor. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. Results are representa-ve of three different donors. Overlays showing GFP expression on sorted targets transduced with the hFADD-DN IRES GFP transgene are also shown (unstransduced controls are depicted in gray).(G-J) Graphs showing early and late leukemic killing of K562, NALM6, REH and 697 cell lines (control or hFADD-DN transduced) using NK cells from a perforin deficient pa-ent. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h. (A-J) Shown are mean ± SD.

Figure 28 .

 28 Figure 28. NK cell mediated cytotoxicity towards pre-B ALL targets: PIDs' screening.Representa-ve graphs showing early and late leukemic killing by NKAES-NK cells. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h (mean ± SD). NK cells were amplified from CD40L (A-D), SH2D1A (E and F), NFκB (G and H), PKCδ (I-L), GATA2 (M-P), BTK (Q-T), STAT3 (U-W) and MSN (X-Z) deficient pa-ents and tested against K562, NALM6, REH and/or 697 cell lines. Control normal donors and pa-ents are respec-vely displayed in blue and in red.

Figure 29 .Figure 30 .Figure 31 .

 293031 Figure 29. AcVve caspases and mitochondrial depolarizaVon in pre-B ALL cell death are not mandatory for NK cell induced killing. Representa-ve graphs showing early and late leukemic killing by NKAES-NK cells. Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h (mean ± SD). Caspases 8 and 10 DKO (A), caspase 9 KO (B), caspases 3 and 7 DKO (C-E, 3 tested donors) and BAX BAK DKO (F-H) NALM6, REH and/or 697 cell lines (or control sgRNA cell lines) were used as targets.(legend conJnued on next page)

  : pre-B ALL cell lines were transduced to stably express a CAT-2A-SOD IRES GFP transgene allowing combined overexpression of the ROS-detoxifying enzymes manganese superoxide dismutase (SOD2) and catalase (Appendix 13, provided by Dr. Michael Milsom (HI-STEM, Germany); a roGFP-based sensor of glutathione redox poten-al either expressed in the cytosol (Grx1-roGFP2, Appendix 14) or in the mitochondria (Mito-Grx1-roGFP2, Appendix 15); and a roGFP-based sensor to measure hydrogen peroxide in the cytosol (roGFP2-ORP1, Appendix 16) or in the mitochondria (Mito-roGFP2-ORP1, Appendix 17). These four biosensors are based on gene-cally encoded redox-sensi-ve green fluorescent proteins (roGFPs), which allow sensi-ve and compartment-specific insights into redox homeostasis, and were provided by Pr. Tobias Dick (DKFZ, Germany).

Figure 32 .

 32 Figure 32. Catalase and SOD2 over-expressing targets do not show increased resistant to NK killing. Early and late leukemic killing by NKAES-NK cells against NALM6 cell line (control or CAT-2A-SOD transduced). Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h (mean ± SD). Overlays showing GFP expression on sorted targets transduced with the CAT-2A-SOD IRES GFP transgene are also shown (unstransduced control is depicted in gray).

Figure 33 .

 33 Figure 33. CYBB deficient NK cells display significantly decreased cytotoxicity against pre-B ALL cells. (A-C) Representa-ve graphs showing early and late leukemic killing by NKAES-NK cells from a CYBB deficient pa-ent (X-CGD) (A), a NCF1 deficient pa-ent (AR47-CGD) (B) and a CYBA deficient pa-ent (AR22-CGD) (C). Shown are percentages of cytotoxicity (rela-ve to cultures with no effector cells) in cocultures las-ng 4 and 25h (mean ± SD). (D) Results from nineteen normal donors and ten X-CGD pa-ents against NALM6 cell line. (E) Results from twenty normal donors and ten X-CGD pa-ents against REH cell line. Sta-s-cs: Mann-Whitney test (* = p < 0.05, ** = p < 0.01).

  Figure 35. CYBB mRNA is acVvely regulated by NK cells.End-point PCR was performed on PBMCs (posi-ve control), sorted NKAES-NK cells before and aber incuba-on with targets (K562 or REH), to check mRNA expression of the following genes related to NADPH oxidase: NRROS, CYBA (p22 phox ), NCF4 (p40 phox ), NCF1 ( p 4 7 phox ), NCF2 (p67 phox ), CYBB (gp91 phox ) and the B2M housekeeping gene. Representa-ve gel of one among several donors. NALM6 and 697 cell lines were tested with results similar to REH.

Figure 36 .

 36 Figure 36. NK cells from X-CGD paVents and normal donors have different transcriptomic expression profile. (A) Principal component analysis was performed on tested samples. (B) Comparison of effect sizes for response to REH (and RS4;11, and their difference) for NKAES-NK cells from controls (n = 4 per condi-on). (C-G) Volcano plots showing differen-ally expressed genes (FDR < 0.05) between res-ng NK cells (C) or NKAES-NK cells (D) from X-CGD pa-ents vs controls (C); NKAES-NK cells from controls exposed to REH (E) or RS4;11 (F) vs uns-mulated NKAES-NK cells; and NKAES-NK cells s-mulated with REH vs RS4;11 (G). The number of hits is also given.

Figure 37 .

 37 Figure 37. Gene ontology funcVonal network of biological process and cellular components terms.ClueGO analysis was performed for res-ng NK cells from X-CGD pa-ents vs controls (A and B; I and J) and NKAES-NK cells (C and D; K and L) from X-CGD pa-ents vs controls as well as NKAES-NK from controls exposed to REH (E and F; M and N) or RS4;11 (G and H; O and P) vs uns-mulated NKAES-NK. Biological process (A-H) and cellular component (I-P) related terms were studied. We dis-nguished up-regulated and down-regulated genes for the analysis. In the graphs, size is propor-onal to the fdr of the result (following a scale roughly logarithmic), but the significance scales vary from some runs to others.

  Figure 38. Oxygen ConsumpVon Rate (OCR) among NK cells from AR47-CGD and X-CGD paVents (compared to controls).(A-F) OCR was measured using NK cells from a normal donor (in blue) and a AR47-CGD (NCF1 deficient; in red) either exposed or not to various target cell lines. (G-L) OCR was measured using NK cells from a normal donor (in blue) and a X-CGD (CYBB deficient; in green) either exposed or not to various target cell lines.

  Figure 39. Extracellular AcidificaVon Rate (ECAR) among NK cells from AR47-CGD and X-CGD paVents (compared to controls).(A-F) ECAR was measured using NK cells from a normal donor (in blue) and a AR47-CGD (NCF1 deficient; in red) either exposed or not to various target cell lines. (G-L) ECAR was measured using NK cells from a normal donor (in blue) and a X-CGD (CYBB deficient; in green) either exposed or not to various target cell lines.

Figure 40 .

 40 Figure 40. NK cell metabolic profile discriminates sVmulaVon with K562 or pre-B ALL cell lines. (A-C) Expression of CD98 on NK cells following 4h (A) or 25h (B) incuba-on with targets (rela-ve to uns-mulated effector cells) was assessed by flow cytometry. Absolute MFI values are also displayed (C). (D-F) Expression of CD71 on NK cells following 4h (D) or 25h (E) incuba-on with targets (rela-ve to uns-mulated effector cells) was assessed by flow cytometry. Absolute MFI values are also displayed (F). (G and H) NK cells incorpora-on of 2-NBDG following 4h (G) or 25h (H) incuba-on with targets (rela-ve to uns-mulated effector cells) was assessed by flow cytometry.

Figure 41 .

 41 Figure 41. Metabolic profile of target cell lines. (A) Oxygen Consump-on Rate (OCR) at baseline among K562, NALM6, REH and RS4;11 cell lines. (B) Extracellular Acidifica-on Rate (ECAR) at baseline among K562, NALM6, REH and RS4;11 cell lines. (C) OCR/ECAR ra-o at baseline was used as a parameter to evaluate the rela-ve use of OXPHOS and glycolysis by target cell lines.

  103

Figure 42 .

 42 Figure 42. A proposed schemaLc representaLon of the steps involved in the NK killing process.Effector cells recognize target cells by ac@va@ng and inhibi@ng membrane receptors which engage various signaling pathways, all integrated in the "decision to kill" (inputs). The effector response (outputs), possibly via a metabolic control, is in the form of cytokine produc@on, granule exocytosis, death receptor engagement and our described unconven@onal cytotoxicity pathway.

  Figure 43. Comparison of effect sizes for response to REH (and RS4;11, and their difference) for NKAES-NK cells from controls (n = 4 per condiLon) and a X-CGD paLent.

Figure 44 ,

 44 Figure 44, related to figure 42. Proposed models for NK cell effector responses at the single cell level: a metabolic fingerprinLng?Cytotoxic cell output can engage an effector response (Figure42) in the form of cytokine produc@on, granule exocytosis, death receptor engagement and our unconven@onal cytotoxicity pathway. At the single cell level, one may propose two alterna@ve models: a plas@c and permissive model (A) which postulates possible switches between effector responses, and a commihed and restric@ve model (B) which proposes that dis@nct NK clones engage each type of response. Whatever the model, we hypothesize the metabolic state to be a central checkpoint, possibly through a crosstalk with epigene@c regula@on.

  109

Figure 45 .

 45 Figure 45. Experimental design for a genome-scale CRISPR/Cas9-based screening.Sensi@ve and resistant pre-B ALL cell lines are infected at low mul@plicity of infec@on (MOI) to ensure that blasts receive only a single viral par@cle (here, a strategy using a two-plasmid system is depicted;[START_REF] Shalem | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells[END_REF]. For each cell line, a first group is exposed to NKAES-NK cells. The second group of cells, ac@ng as the nega@ve control group cultured under the same condi@on, is not exposed to effector cells. Following incuba@on, the genomic DNA is extracted and sgRNA amplified with specific primers.

  Figure S2, related to Figure 27. Pre-B ALL cell lines are sensiHve to TRAIL mediated apoptosis. (A-C) Overlay histograms showing FAS (A), DR4 (B) and DR5 (C) surface expression by 697, NALM6 and REH cell lines (isotype control is depicted in gray).(D-G) Percentages of specific apoptosis among APO-1 (100 and 250 ng/mL; Jurkat cell line and PBMCs were used as posi,ve controls) and SuperKillerTRAIL (250 ng/mL, with or without 0,1 μg/mL cycloheximide to poten,ate killing effect; Jurkat cell line was used as posi,ve control) treated 697, NALM6 and REH cell lines. (H) Specific cytotoxicity of WT and hFADD-DN transduced K562, NALM6, REH and 697 cell lines following 4h and 25h incuba,on with SuperKillerTRAIL (250 ng/mL).

  implique une reconnaissance et une réponse immune poten3elle entre donneur et receveur, et réciproquement. Elle se manifeste ainsi par deux effets étroitement corrélés et liés à la présence de cellules immunocompétentes dans le greffon : l'effet délétère du greffon contre l'hôte (ou GvHD pour gra$-versus-host disease) et celui bénéfique du greffon contre la tumeur (ou GvT pour gra$-versus-tumor). Les lymphocytes T et les cellules NK sont deux popula3ons cellulaires du greffon contribu3ves de ces effets. Concernant les cellules NK, la base de leur alloréac3vité repose sur l'engagement ou non de récepteurs présents à la surface de ces cellules et capables de reconnaître les déterminants polymorphiques des molécules du complexe majeur d'histocompa3bilité (CMH) de classe I. Par ailleurs, des récepteurs ac3vateurs entrent également en jeu et c'est la balance entre signaux ac3vateurs et signaux inhibiteurs qui déterminent la réponse effectrice des cellules NK. En 2002, Loredana Ruggeri et al. ont rapporté le rôle important joué par les cellules NK dans

  Nos résultats ont permis de démontrer que les LAL pré-B ne sont pas intrinsèquement résistantes à l'ac3on cytotoxique des NK. Une exposi3on prolongée des blastes leucémiques (de lignées ou primaires) à des cellules NK amplifiées et ac3vées in vitro (NKAES-NK) permet de surmonter ceKe résistante de façon efficace. Surtout, nous avons constaté que ceKe cytotoxicité ne reposait pas sur des mécanismes classiques tels que l'exocytose de granules cytotoxiques ou l'engagement de la voie des récepteurs de mort cellulaire mais sur un démontrent que les cellules NK expriment bien au niveau transcriptomique mais aussi protéique, p22 phox et gp91 phox , mais pas le flavocytochrome b558 exprimé par les phagocytes et les lymphocytes B. Le complexe moléculaire précis présent dans les cellules NK reste ainsi à caractériser de façon approfondie. Notre étude s'est poursuivie par une analyse transcriptomique des cellules NK non ac3vées ainsi que NKAES-NK de contrôles sains et de pa3ents aKeints d'une granulomatose sep3que chronique liée à l'X (due à un déficit en gp91 phox ), éventuellement post-exposi3on à une lignée LAL pré-B sensible (REH) ou résistante (RS4;11) à l'ac3on cytotoxique des NKAES-NK. Les résultats suggèrent des modifica3ons métaboliques importantes parmi les NKAES-NK exposées ou non aux différentes cibles leucémiques. Nous avons pu le confirmer par une étude fonc3onnelle de la glycolyse et de la phosphoryla3on oxida3ve des cellules NKAES-NK, révélant une signature métabolique dis3ncte selon l'engagement des voies effectrices par les cellules NK. Lorsque les cellules ont été exposées à la lignée K562 (où la voie de l'exocytose des granules cytotoxiques prédomine), les modifica3ons métaboliques sont dis3nctes de celles mesurées après coculture avec des LAL pré-B. Dans ce dernier cas, une u3lisa3on accrue de la glycolyse est observée. De façon intéressante, les cellules NKAES-NK de pa3ents déficitaires en gp91 phox ont montré un défaut de ceKe hyperglycolyse, suggérant une altéra3on de la capacité d'adapta3on métabolique de ces cellules. Dans leur ensemble, les résultats présentés dans ceKe thèse remeKent en cause le dogme de la résistance des LAL pré-B à l'ac3on cytotoxique des cellules NK, ainsi que l'absence d'expression ou d'un rôle de la protéine gp91 phox dans ces mêmes lymphocytes cytotoxiques. La poursuite de ces travaux devrait permeKre une meilleure compréhension des mécanismes moléculaires contribuant à la nouvelle voie de cytotoxicité que nous décrivons, et en corollaire, au type de mort cellulaire induit dans les cellules de LAL pré-B. Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Cellules Natural Killer et leucémies aiguës lymphoblastiques pré-B : éléments de preuve d'une voie de cytotoxicité non conventionnelle Mots clés : Cellules NK ; leucémie aiguë lymphoblastique ; cytotoxicité ; mort cellulaire ; granulomatose septique chronique ; déficits immunitaires Résumé : Les cellules Natural Killer (NK) représentent une population de cellules innées lymphoïdes aux fonctions anti-infectieuses et antitumorales. Les leucémies aiguës lymphoblastiques pré-B (LAL pré-B) constituent le cancer de l'enfant le plus fréquent et ont été décrites comme résistantes à la cytotoxicité médiée par les NK bien que les bases moléculaires demeurent inconnues. L'objectif de ces travaux a été de caractériser cette résistance. En développant un essai de cytotoxicité par cytométrie en flux et en utilisant des cellules effectrices activées in vitro, nous avons établi la sensibilité retardée des LAL pré-B à la cytotoxicité NK : initialement résistantes après 4h d'incubation, elles sont fortement tuées après 25h.

TABLE OF CONTENTS 2.14. SEAHORSE ANALYSIS . ................................... p 2.15. RNA-SEQUENCING . ..................................... p 2

 OF

.15.1. EXPERIMENTAL DESIGN ............................

p 2.15.2. RNA EXTRACTION, LIBRARY PREPARATION AND SEQUENCING .

ILCs) Innate Lymphoid Cells

  plas.city has been demonstrated among ILCs (Ar.s and Spits, 2015); for instance, local TGF-β can drive conversion of NK cells into ILC1s with defec.ve cytotoxic func.on, thus contribu.ng to tumor immunoevasion

	helper cells,
		IL-12 IL-15 IL-18	IL-25 IL-33 TSLP	IL-1β IL-23	Tissue signals
	Group 1 ILC	Group 2 ILC	Group 3 ILC
		ILC1	ILC2	ILC3
	CD8 cytotoxic T cells	Th1 CD4 + T cells	Th2 CD4 + T cells	Th17 CD4 + T cells	AdapFve Lymphocytes
	cytotoxicity perforin granzymes TRAIL IFN-γ TNF-α	IFN-γ TNF-α	IL-4 IL-5 IL-9 IL-13 Areg	IL-17A IL-22 GM-CSF LT-α1β2 IFN-γ	Effector molecules
	Immunity to
	viruses Tumor surveillance Chronic	Immunity to bacteria Inflamma.on	Immunity to helminth Allergic diseases Metabolism	Immunity to bacteria Intes.nal homeostasis Chronic inflamma.on Lymphoid development	Func.ons
	inflamma.on

Table 1 . CharacterisFcs of geneFc disorders associated with occurrence of HLH.

 1

	Subtype	GeneFc defect	Affected protein
	FHL type 1	unknown	unknown
	FHL type 2	PRF1	Perforin
	FHL type 3	UNC13D	Munc13-4
	FHL type 4	STX11	Syntaxin 11
	FHL type 5	STXBP2	Munc18-2
	Griscelli syndrome type 2	RAB27A	RAB27A
	Chediak-Higashi syndrome	LYST	LYST
	Hermansky-Pudlak syndrome type 2	ADTB3A	AP-3
	XLP-1	SH2D1A	SAP
	XLP-2	XIAP	XIAP

FHL and XLP stand for familial hemophagocy.c lymphohis.ocytosis and X-linked lymphoprolifera.ve disease, respec.vely.

Table 2 . Pa7ents and cytotoxicity assays characteris7cs. Pa7ent ID Recrui7ng center Disorder Gene7c defect Muta7on Sample's type Cell lines tested in cytotoxicity assay

 2

	P01	CHUSJ	X-linked lymphoprolifera2ve (XLP1) disease, Pur2lo syndrome	SH2D1A		PBMCs blood from fresh	K562, REH
	P02	CHUSJ	Type 2 familial hemophagocy2c lymphohis2ocytosis (FHL2)	PRF1	[c.116C>A ] +[c.445G>A], [p.Pro39His] +[p.Gly149Ser]	PBMCs from fresh blood	K562, NALM6, REH, 697
	P03	CHUSJ	NEMO deficiency syndrome	IKBKG		PBMCs blood from fresh	K562, REH
	P04	CHUSJ	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.252G>A, p.Ala84Ala	Frozen PBMCs	K562, NALM6, REH
	P05	CHUSJ	Autosomic recessive chronic granulomatous disease (AR47-CGD)	NCF1	[c.75_76delGT] +[c.75_76delGT], [p.Tyr26HisfsX26] +[p.Tyr26HisfsX26]	PBMCs from fresh blood	K562, NALM6, REH
	P06	CHUSJ	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.252G>A, p.Ala84Ala	PBMCs from fresh blood	K562, NALM6, REH
	P07	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.217C>T, p.Arg73X	PBMCs from fresh blood	K562, NALM6, REH
	P08	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.1610_1611delGT, p.Cys537TrpfsX3	PBMCs from fresh blood	K562, NALM6, REH

Table 2 . (Con7nued.) Pa7ent ID Recrui7ng center Disorder Gene7c defect Muta7on Sample's type Cell lines tested in cytotoxicity assay P09 CCHMC

 2

			Type 3 familial hemophagocy2c lymphohis2ocytosis (FHL3)	UNC13D	[c.2346_2349delGGAG] +[c.3065T>C], [p.Arg782fs] +[p.Leu1022P]	Frozen PBMCs	K562, NALM6, REH, 697
	P10	CCHMC	Type 3 familial hemophagocy2c lymphohis2ocytosis (FHL3)	UNC13D	[c.154-1G>C]+253kb inversion	Frozen PBMCs	K562, NALM6, REH, 697
	P11	CHUSJ	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.469C>T, p.Arg157*	Frozen PBMCs	K562, NALM6, REH
	P12	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB complete gene dele2on	PBMCs from fresh blood	K562, NALM6, REH
	P13	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.676C>T, p.Arg226*	PBMCs from fresh blood	K562, NALM6, REH
	P14	NCK	Autosomic recessive chronic granulomatous disease (AR47-CGD)	NCF1	[c.75_76delGT] +[c.75_76delGT], [p.Tyr26HisfsX26] +[p.Tyr26HisfsX26]	PBMCs from fresh blood	K562, NALM6, REH
	P15	HCL	Protein kinase C δ deficiency	PRKCD	[c.1528G>A]+[c. 1528G>A], [p.Gly510Ser]+ [p.Gly510Ser]	PBMCs from fresh blood	K562, NALM6, REH, 697
	P16	HSL	GATA2 haplo-insufficiency GATA2	[c.1076T>C], p.Leu359Ser	PBMCs from fresh blood	K562, NALM6, REH, 697
	P17	HCL	X-linked chronic granulomatous disease (X-CGD)	CYBB complete gene dele2on	Frozen PBMCs	K562, NALM6, REH
	P18	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.252G>A, p.Ala84Ala	Frozen PBMCs	K562, NALM6, REH
	P19	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB complete gene dele2on	Frozen PBMCs	K562, NALM6, REH
	P20	NCK	X-linked chronic granulomatous disease (X-CGD)	CYBB	c.481C>T, p.Arg157X	PBMCs from fresh blood	K562, NALM6, REH
			X-linked chronic			PBMCs
	P21	HCL	granulomatous disease	CYBB	c.375G>A, p.Trp125X	from fresh	nc
			(X-CGD)			blood
			Autosomic recessive		[c.204-2A>G]	PBMCs	K562,
	P22	NCK	chronic granulomatous	CYBA	+[c.204-2A>G],	from fresh	NALM6, REH,
			disease (AR22-CGD)		splicing site destruc2on	blood	697
	P23	NCK	X-linked agammaglobulinemia	BTK	c.1573C>G, p.Arg525Gly	Frozen PBMCs	K562, NALM6, REH, 697
	P24	NCK	X-linked immunodeficiency with hyper-IgM (XHIGM)	CD40LG c.682C>T, p.Gln221X	Frozen PBMCs	K562, NALM6, REH,

Table 2 . (Con7nued.)

 2 denotes CHU Sainte-Jus2ne, NCK Hôpital Necker-Enfants Malades, CCHMC Cincinna2 Children's Hospital Medical Center, HCL Hospices Civils de Lyon, HSL Hôpital Saint-Louis and PBMCs peripheral blood mononuclear cells.

	34

CHUSJ

Table 3 . List of oligonucleo7des related to sgRNA cloning.

 3

	Gene target	Expression system Cloning vector	Primer	Sequence (5'→3')
	CTRL	Uniplex	len2Guide Puro	CTRL_sgE0_Fw1	CACCGACGGAGGCTAAGCGTCGCAA
	CTRL	Uniplex	len2Guide Puro	CTRL_sgE0_Rv1	AAACTTGCGACGCTTAGCCTCCGTC
	CASP9	Uniplex	len2Guide Puro	CASP9_sgE3_Fw1	CACCGGGACACAGGCCAGGACATGC
	CASP9	Uniplex	len2Guide Puro	CASP9_sgE3_Rv1	AAACGCATGTCCTGGCCTGTGTCCC
	CTRL	Mul2plex	pmU6-sgRNA	CTRL_sgE0_Fw2	TTGTTTGCGCTTCCGCGGCCCGTTCAA
	CTRL	Mul2plex	pmU6-sgRNA	CTRL_sgE0_Rv2	AAACTTGAACGGGCCGCGGAAGCGCAA
	CTRL	Mul2plex	phU6-sgRNA	CTRL_sgE0_Fw3	CACCGATCGTTTCCGCTTAACGGCG
	CTRL	Mul2plex	phU6-sgRNA	CTRL_sgE0_Rv3	AAACCGCCGTTAAGCGGAAACGATC

Table 5 . Differen7al expression contrasts retrieved from Model 2.

 5

Table 6 . Tested geneVc defects with published NK cell deficiency. GeneVc defect Disorder Selected references of interest (relevant for NK cell cytotoxicity deficiency)

 6

Table S1 , related to Figure 36. Number of hits using the Emma nested models 1 and 2.

 S1

	Contrast	Number of hits FDR<0.1 (0.05)
	1.	differen,al expression between pa,ents and controls for res,ng NK cells	317 (176)
	2.	disease effect on NKAES cells	197 (108)
	3.	disease effect on NKAES cells exposed to REH	128 (67)
	4.	disease effect on NKAES cells exposed to RS4;11	637 (425)
	5.	ac,va,on effect on controls	9723 (8489)
	6.	ac,va,on effect on pa,ents	10480 (9434)
	7.	differences in ac,va,on effects between pa,ents and controls	464 (289)
	8.	REH effect in controls' NKAES's	3713 (2875)
	9.	REH effect in pa,ent' NKAES's	981 (752)
	10.	Difference between REH effect in pa,ent minus controls' NKAES's	902 (682)
	11.	RS4;11 effect in controls' NKAES's	3140 (2464)
	12.	RS4;11 effect in controls' NKAES's	1219 (881)
	13.	Difference between RS4;11 effect in pa,ent minus controls' NKAES's	1257 (941)
	14.	RS4;11 minus REH effect in controls' NKAES's	410 (324)
	15.	RS4;11 minus REH effect in pa,ent' NKAES's	531 (434)

FDR stands for false discovery rate.

Table S2 , related to Figure 37. Enrichment of GO Biological process terms.

 S2 For each contrast, + or -stand for up-regulated or down-regulated genes respec,vely.

	Contrast	FDR threshold	Number of genes in target	Number of terms
	8(+)	0.01	874
	8(-)	0.01	1033
	11(+)	0.01	742
	11(-)	0.01	898
	14(+)	0.2	321
	14(-)	0.2	217
	1(+)	0.2	272
	1(-)	0.2	405

Table S3 , related to Figure 37. Enrichment of GO Cellular components terms.

 S3

	Contrast	FDR threshold	Number of genes in target	Number of terms
	8(+)	0.01	874
	8(-)	0.01	1033
	11(+)	0.01	742	3
	11(-)	0.01	898
	14(+)	0.2	321	6
	14(-)	0.2	217	9
	1(+)	0.2	272
	1(-)	0.2	405

LIST OF FIGURES

Appendix 4 and Figure 13) to derive stably expressing Cas9n (D10A mutated nickase) cell lines arer blas2cidin selec2on (5 µg/mL).

For the final sgRNAs expression vector, we modified the protocole from Dr. Charles Gersbach lab ( [START_REF] Kabadi | Mul=plex CRISPR/Cas9-based genome engineering from a single len=viral vector[END_REF] and Figure 13). We created the len2Guide-Puro mul2plex loxP vector (Appendix 10) by PCR-cloning the Gloden Gate Assembly compa2ble casseZe from the pLV hUbC-Cas9-T2A-GFP vector (Addgene #53190, Appendix 9) using the following primers: [Mul2plex/Fw] 5'-GATGCCACATGCGTGTGCGCCAATTCTGCAGAC-3' and [Mul2plex/ Rv] 5'-GAGTACCCGGGgctgcacccTTAatTAATTGTGGAG-3' and then AleI / XmaI digested to be inserted into the XmaI digested len2Guide-Puro. The forward orienta2on of the DEST casseZe was confirmed by restric2on mapping. The final len2Guide-Puro mul2plex loxP vector was generated by replacing the WPRE casseZe with a loxP-WPRE one.

According to the published protocol [START_REF] Kabadi | Mul=plex CRISPR/Cas9-based genome engineering from a single len=viral vector[END_REF], the appropriate sgRNAs were cloned into pmU6-sgRNA (Addgene #53187, Appendix 5), phU6-sgRNA (Addgene #53188, Appendix 6), phH1-sgRNA (Addgene #53186, Appendix 7) and ph7SK-sgRNA (Addgene #53189, Appendix 8). The final expression vector was created by Golden Gate assembly of each of these single vectors with the len2Guide-Puro mul2plex loxP vector using a BsmBI diges2on, and then used to transduce Cas9n-expressing cell lines. Arer puromycin selec2on

(5 µg/mL), single clones were isolated by FACS sor2ng into 96 wells plates.

SGRNA CLONING

We listed the primers used for sgRNA cloning in both the uniplex and mul2plex expression systems (Table 3).

Although resistant to SuperKillerTRAIL induced apoptosis (data not shown), caspases 8 and 10 DKO blasts were killed as efficiently as control cells (Figure 29A), thus confirming results

we obtained with hFADD-DN transduced targets (Figures 29C-29J) and ruling out death receptor pathway.

Interes-ngly, caspase 9 KO ALL target killing was not affected following 4h incuba-on but displayed a lower cytotoxicity at 25h (Figure 29B), sugges-ng that mitochondria could amplify the death signaling.

We assessed the role of mitochondrial depolariza-on with BAX/BAK DKO cells. Upon SuperKillerTRAIL treatment or in presence of effector cells, these ALL target cells did not depolarize anymore (data not shown) although they were s-ll killed by NK cells as efficiently as control pre-B ALL cells (Figures 29F-29K). Of note, a notable excep-on was observed with 697 cell line: following 25h incuba-on, DKO targets killing was significantly reduced, showing that, albeit MOMP was not a strict requirement to cell death it instead tended to accelerate it to some extent (Figure 29K).

Apoptosis is defined as a programmed cell death with several possible caspase ac-va-on routes but irrespec-vely, these pathways lead to ac-va-on of the major effector caspases, i.e. caspase 3, caspase 6 and caspase 7 [START_REF] Taylor | Apoptosis: controlled demoli=on at the cellular level[END_REF]. Caspase 6 is cleaved by caspases 3 and 7 but can also undergo self-ac-va-on in some serngs [START_REF] Inoue | Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway[END_REF][START_REF] Parrish | Cellular Mechanisms Controlling Caspase Ac=va=on and Func=on[END_REF].

Caspases 3 and 7 DKO cells have deep defects in their ability and kine-cs to apoptosis [START_REF] Lakhani | Caspases 3 and 7: key mediators of mitochondrial events of apoptosis[END_REF]: we studied cell death among these targets co-incubated with NK cells. Strikingly, caspases 3 and 7 DKO pre-B ALL targets were killed by NK cells as efficiently as their control counterparts, demonstra-ng that these terminal effector proteases were not required for late NK killing (Figures 29C-29E).

Taken together, our results showed that NK cells induced pre-B ALL cell death with detectable poly caspase ac-va-on and mitochondrial depolariza-on but neither one nor the other were strictly required since selected KO targets with defec-ve signaling pathway were s-ll efficiently killed.

78

(I-K) Pooled results from BAX BAK DKO targets (I, NALM6, n = 6 tested donors; J, REH, n = 3 tested donors; K, 697, n = 6 tested donors). Sta-s-cs: Wilcoxon matched-pairs signed rank test (* = p < 0.05, n.s. = non significant). For REH cell line, the sample size was not sufficient to perform sta-s-cs. K562 cytotoxicity was assessed as a posi-ve control (not shown). Control and (D)KO cell lines are respec-vely displayed in blue and in orange.

ROLE OF REACTIVE OXYGEN SPECIES (ROS)

Apart from a pivotal role of mitochondria during the intrinsic apoptosis, reac-ve oxygen species (ROS) produced by the mitochondria can be involved in cell death, as well as extramitochondrial ROS [START_REF] Galluzzi | Organelle-specific ini=a=on of cell death[END_REF][START_REF] Klamt | Oxidant-induced apoptosis is mediated by oxida=on of the ac=n-regulatory protein cofilin[END_REF]Marenez-Lostao et al., 2015;[START_REF] Tabas | Integra=ng the mechanisms of apoptosis induced by endoplasmic re=culum stress[END_REF].

We decided to explore a poten-al contribu-on to ROS in our killing by incuba-ng cells with the ROS scavenger N-acetylcystein (NAC). In our hands, experiments using NAC (whose pH once recons-tuted is acidic and has to be strongly buffered) were some-mes difficult to perform with a variability and a sensi-vity of pre-B ALL cell lines which could die of ROS scavenging, especially for long term incuba-ons. However, preliminary experiments suggested that ROS inhibi-on could increase blasts survival when exposed to NK cells (Figure 30). We then decided to explore oxida-ve metabolism in a systema-c way. CHAPTER 6. REFERENCES 6.

CHAPTER 7. RÉSUMÉ DE LA THÈSE EN FRANÇAIS