Production of IFN- γ by neonatal Natural Killer cells in response to Trypanosoma cruzi and cross-talk

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Faculté de Médecine

Laboratoire de Parasitologie

Production of IFN- γ by neonatal Natural Killer cells in response to Trypanosoma cruzi and cross-talk

with monocytes

Aline Guilmot

Thèse de doctorat présentée en vue de l’obtention du grade académique de Docteur en Sciences Biomédicales et Pharmaceutiques

Promoteur : Dr Carine Truyens

Co-Promoteur : Pr Yves Carlier

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Acknowledgments

This text is the final point of a long journey sowed with traps and difficulties and now it is finished, it is time to thank all the people who made this possible by helping and supporting me.

First of all, I would like to express my heartfelt gratitude to Professor Carlier who let me carry out my phD thesis in his laboratory and gave me the opportunity to be his assistant.

I would also like to thank my promoter Doctor Carine Truyens who assisted me the whole way in this thesis. She brought me valuable practical and theoretical help and was always present when needed.

I also thank all the present and previous Parasitology Lab staff, Patty, Italo, Eric, Carl, Sabrina, Nicolas, Loes, Magda, Pascale and Maria for their help and support. I want to thank Alain in particular for the many cups of coffee and tea that we drunk when my experiments did not want to work.

A special thanks goes to all the students I supervised and followed, Julie, Tiago, Saria, Rodriguo and Philippe who made me realise that I love teaching and supervising.

Particularly, I want to say a big thank you to Maxime for the many lunch breaks that we spent discussing everything and to Elisée for the confidence she had in me and for lighting up my days.

Finally, I want to thank my friends and family who supported me these long years and I want to dedicate this work to my grand-father who was obsessed by knowledge and would have been proud of this achievement if he were still here.

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Résumé

La maladie de Chagas, due au protozoaire Trypanosoma cruzi, est un important problème de santé publique en Amérique latine. Le parasite peut se transmettre à l’homme via un vecteur de la famille des triatomes, par transfusion sanguine ou transplantation d’organe et congénitalement de la mère à son fœtus. Le Laboratoire de Parasitologie s’est particulièrement intéressé à la maladie de Chagas congénitale. Dans le cadre de cette thématique, le Laboratoire a montré que les nouveau-nés congénitalement infectés par T. cruzi développent une forte réponse lymphocytaire T CD8⁺ spécifique semblable à celle des adultes, accompagnée d’une production d’interféron-gamma (IFN-), et ce en dépit de l’immaturité bien connue du système immun néonatal. En effet, le système immun néonatal est naturellement orienté vers le développement de réponses immunes Th2 tandis que la réponse Th1 est inhibée. De multiples mécanismes sont à l’origine de cette déviation en début de vie.

Certaines déficiences au niveau des cellules du système immun inné y contribuent, dont la difficulté des cellules dendritiques (DCs) à produire de l’IL-12, cytokine clé dans l’induction d’une réponse Th1.

Les multiples déficiences du système immun en début de vie rendent les nouveau-nés et jeunes enfants particulièrement sensibles aux pathogènes et limitent l’efficacité des vaccins administrés en début de vie.

Afin de mieux connaître les mécanismes par lesquels T. cruzi induit cette forte réponse immune de type 1 chez les nouveau-nés, nous nous sommes intéressés à l’activation de la réponse immune innée par le parasite. De nombreuses cellules peuvent être impliquées dans la mise en place d’une réponse de type 1, dont les cellules dendritiques (DCs), les monocytes et les cellules NK. Le Laboratoire de Parasitologie a montré que T. cruzi activait in vitro les DCs néonatales, les rendant capables d’induire une réponse lymphocytaire T plus orientée vers la production d’IFN-. D’autres données obtenues chez les nouveau-nés congénitalement infectés par T. cruzi suggèrent que les cellules NK ont été activées in utero quand le parasite a été transmis par la mère infectée. Nous nous sommes ici intéressés à la capacité des cellules NK néonatales à produire rapidement de l’IFN- en réponse à T. cruzi. Une telle production précoce est en effet un élément contribuant à orienter une réponse immune de type 1.

Nous avons effectué des co-cultures de cellules mononucléaires de sang de cordon de nouveau-nés sains (CBMC) ou de sang périphérique adulte (PBMC) avec des trypomastigotes vivants de T. cruzi.

Nos résultats montrent qu’en présence d’IL-15, T. cruzi induit une forte production d’IFN- par les CBMC. Cette réponse est précoce et est accompagnée d’une production de TNF- mais pas d’IL-10.

Les cellules NK CD56^brightCD16^/low et CD56^dimCD16^ sont les meilleures productrices d’IFN- dans les deux groupes d’âges. La réponse des cellules NK néonatales est substantielle mais reste légèrement inférieure à celle des cellules adultes. Nous avons par ailleurs observé un déficit de production précoce d’IFN- par les cellules T CD3⁺CD56⁺ (NK-like) et CD3⁺CD56^ (« classiques ») néonatales par rapport aux cellules adultes.

La réponse IFN- par les cellules NK est proportionnelle aux concentrations de parasites et d’IL-15 et accompagnée d’une activation phénotypique des cellules NK. Il est bien connu que des cellules

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accessoires telles que les cellules dendritiques et les monocytes contribuent généralement à activer indirectement les cellules NK. Des expériences de déplétion cellulaire indiquent que la production d’IFN- par les cellules NK néonatales sensibilisées par l’IL-15 fait intervenir les monocytes mais pas les DCs myéloïdes, et qu’un contact avec les monocytes est nécessaire. De plus, elle requiert un contact du parasite vivant avec les CBMC et implique l’engagement des TLR2 et TLR4, ainsi qu’une production endogène d’IL-12.

Enfin, nous avons observé que les monocytes, et non les DCs myéloïdes, sont la source précoce de l’IL-12p70. Les parasites sont capables d’induire la synthèse de cette cytokine importante pour l’initiation d’une réponse de type 1 en l’absence de cytokines additionnelles, aussi bien dans les monocytes néonataux qu’adultes. La synthèse d’IL-12 par les monocytes s’accompagne d’une augmentation de l’expression de molécules co-activatrices CD40, CD80 et CD83 à leur surface. Ces dernières pourraient dès lors jouer un rôle supplémentaire dans l’activation indirecte des cellules NK néonatales par le parasite.

Cet ensemble de résultats montre que T. cruzi active les cellules néonatales du système immunitaire et plus particulièrement la production d’IL-12 par les monocytes et d’IFN- par les cellules NK. Cette voie d’activation monocytes – IL-12 – cellules NK – IFN- pourrait contribuer à la levée de l’immaturité du système immun des nouveau-nés congénitalement infectés décrite plus haut. Ces observations ont d’importantes implications pour la compréhension des mécanismes de protection en début de vie et pourraient aboutir à la mise au point d’un nouvel adjuvant vaccinal permettant de réduire la polarisation Th2 physiologique des nouveau-nés.

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List of Figures

1 CD4⁺ T cell subsets development and function . . . 8

2 Model of development of human NK cells in lymph node . . . 12

3 The family of cytokines sharing the common γ-chain in their receptor com- plexes . . . 18

4 Interaction of IL-2 with its receptor contrasted to that of IL-15 and its receptor 19 5 IL-12 family of cytokines and their receptors . . . 21

6 TIR domain Receptor family and their ligands . . . 22

7 Interactions of NK cells, accessory cells and pathogens . . . 26

8 Signals passed from accessory cells to NK cells . . . 34

9 Current estimated global population infected byTrypanosoma cruzi . . . 41

10 Parasitological forms ofT. cruzi . . . 42

11 Schematic view of the various phases of the interaction of T. cruzi with ver- tebrate cell . . . 44

12 Development of the innate immune response inT. cruziinfection . . . 50

13 Production of IL-12 by neonatal and adult monocytes in response to LPS and IFN-γ . . . 63

14 Production of IL-12 by neonatal and adult monocytes in response toT. cruzi 65 15 Production of IL-12 by neonatal and adult monocytes in response toT. cruzi 67 16 Modeling of thein vitro activation of neonatal NK cells byT. cruziand IL-15 79 17 Gating strategy for discrimination of the different CD56⁺ sub-populations . . 90

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List of Tables

1 Receptors and effectors molecules on/in the different blood resting NK subsets 13 2 NCR and their microbial ligands [82] . . . 27 3 Production of cytokines by cord and adult blood cells in response toT. cruzi 69 4 Antibodies used in flow cytometry . . . 88 5 Primers used in qPCR . . . 92

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Abbreviation List

ADCC: Antibody-dependent cellular cytotoxicity

AICL: Activation induced C-type lectin AP: Activator protein

APC: Antigen presenting cells Bcl-2: B cell lymphoma - 2 BCR: B cell Receptor CB: Cord blood

CBMC: Cord blood mononuclear cells CD: Cluster of Differentiation

CD40L: CD40 ligand

CpG: Cytosine - phosphate - Guanine CTL: cytotoxic T lymphocytes

DAP: DNAX activation protein DC: Dendritic cells

DNA: Deoxyribonucleic acid

EBI: Epstein–Barr virus-induced gene product

ERK: Extracellular signal-regulated kinases

FcR: Fragment crystallizable receptor FimH: Fimbrial type 1 pilli H

FMO: Fluorescence minus one GBS: group B Streptococcus

G-CSF: Granulocyte colony-stimulating factor

GIPL: Glycoinositolphospholipids GM-CSF: Granulocyte macrophage CSF gp: glycoprotein

GPI: Glycosylphosphatidylinositol HLA: Human leukocytes antigen IFN: Interferon

Ig: Immunoglobulin IL: interleukin

iNOS: inducible NO Synthase

IRAK: Interleukin-1 receptor-associated kinase

IRF: IFN regulatory factor

ITAM: Immunoreceptor tyrosine-based activating motif

ITIM: Immunoreceptor tyrosine based inhibitory motif

JAK: Janus-activated-kinase

KIR: Killer immunoglobin-like receptors

KO: Knock-out

LAK: Lymphokine activated killers LPS: Lypoplolysaccharide

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MALP: Monocyte activating lipopep- tide

MAPK: Mitogen-activated-protein- kinase

mDC: myeloid dendritic cell

MHC: Major histocompability complex MIC: MHC-class-I-related chains

(m)RNA: (messenger) ribonucleic acid MyD88: Myeloid differentiation primary response gene product

NCAM: neural cell-adhesion molecule NCR: Natural cytotoxicity receptor NF-κB: Nuclear factorκB

NK cells: Natural killer cells

NKp46: NK cell p46-related protein NMR: NOD-like receptor

NO: Nitric oxide

NOD: Nucleotide-binding oligomerization domain

PAMPs: pathogen-associated molecular

patterns

PBMC: Peripheral blood mononuclear cells

PRR: Pathogen Recognition Receptor Ras: Rat sarcoma

ROS: Reactive oxygen species

SHP-1: Src homology region 2 domain- containing phosphatase-1

STAT: Signal transducer and activator of transcription

TCR: T cell Receptor TGF: Tumor growth factor Th cells: T helper cells

TIR: Toll/interleukin-1 receptor TLR: Toll-like receptors

TNF: Tumor necrosis factor

TRIF: TIR-domain-containing adapter- inducisng interferon-β

ULBP: Unique long-16-binding proteins

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Introduction

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Introduction

1 The immune response to pathogens

Throughout evolution, living organisms developed mechanisms to protect themselves against pathogens. This protection was at first somewhat primitive, though already based on recognition of microbial common patterns as non-self or danger signals.

In mammals, innate cells still use this global recognition system, which has evolved to more complexity. In addition, other mechanisms have appeared to respond more specifically and more robustly to the distinct pathogens, which is called the adaptive response. These two systems coexist and interact. They bring into play different cells expressing distinct surface molecules recognizing pathogens (T and B cell receptors, Toll-like receptors, . . . ) or mediating activation (CD3, CD40,. . .) and producing diverse effector molecules like chemokines (involved in cell migration and homing), cytokines (activation or inhibition of the immune response) or cytotoxic compounds [1].

1.1 The innate immune response

The innate immune system is the first line of defense against pathogens after me- chanical barriers, constituted by the skin and mucosal layers, and acts immediately when a pathogen invades the host. It involves ready-to-act soluble molecules (complement system, natural antibodies, antimicrobial peptides and some acute phase proteins) and cellular compounds. The principal actors of innate immunity are monocytes/macrophages, granulocytes, mast cells, natural killer (NK) cells, innate lymphocytes and dendritic cells (DCs). These cells possess pathogen recognition receptors (PRRs) that sense common conserved molecules of pathogens (called PAMPs for pathogen-associated molecular patterns) or molecules abnormally expressed by infected cells [2–4]. When they encounter pathogens, innate cells contribute to elim-

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inate them by phagocytosis and production of intracellular and extracellular toxic compounds. They also process microbial molecules to generate peptides in order to present them to T cells. These different cells can play one or several roles cited above. For instance, macrophages are simultaneously phagocytic cells, effector cells and antigen presenting cells (APCs) (see § 4). This response leads to a quick first vague of elimination of pathogens while the release of chemokines and cytokines and presentation of antigen to T lymphocytes allows the setup of the subsequent adaptive immunity [1].

1.2 The adaptive immune response

The adaptive immune system is so called because it occurs as a specific adaptation to an invading pathogen. This response is antigen-specific and generally confers a long-lasting protective immunity. In order to mount an efficient specific response, re- quiring a sufficient number of activated effector cells, the adaptive immune response needs a preparation time and is thus delayed in regard to the innate response. Two major cell types, both lymphocytes, are the actors of the adaptive immune response.

T cells, characterized by surface expression of T cell receptors (TCR) and of CD3 molecule, possess cytotoxic properties and release cytokines. B cells, characterized by the expression of B cell receptors (BCR, which are a membrane-bound form of the further secreted antibodies) and of CD19 molecule, act by releasing antigen-specific- antibodies. The particularity of these lymphocytes is that each TCR or BCR possesses a different antigen-binding site that specifically recognizes a unique epitope. This multitude of TCR and BCR allows the recognition of virtually all existing molecular structures. The huge number of diverse antigen-binding sites is generated by several mechanisms of gene rearrangement. When B and T cells are activated, they proliferate, inducing an expansion of clones, each specifically recognizing one antigenic epitope of the pathogen. T lymphocytes are subdivided into CD4⁺and CD8⁺subsets. They recognize antigenic epitopes presented by class II and class I molecules of the major his-

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tocompatibility complex (MHC), respectively. CD4⁺ T cells, also named T helper (Th) cells, assist other effector immune cells in their maturation or activation by expressing co-activating surface molecules like CD40 ligand (CD40L) and by secreting cytokines.

Different subsets of Th cells, producing different cytokines, differentiate from naïve T cells, or Th precursors, according to the immune environment established by innate cells after pathogen contact (cf. §1.3, Figure 1). For instance, Th1 response, charac- terized by interferon (IFN)-γ production is triggered by intracellular pathogens, Th2 response, characterized by interleukin (IL)-4, IL-5, IL-10 and IL-13 production is induced by helminths while Th17 response, characterized by IL-17, IL-21 and IL-22 production is preferentially boosted against extracellular bacteria and fungi on mucosal surfaces. These different responses can be counter-regulated by other CD4⁺ T cells named regulatory T cells (Figure 1) [1; 5–8]. CD8⁺T cells, also named cytotoxic T lymphocytes (CTLs), act by lysing infected cells in a MHC class I–restricted way.

Briefly, a cytotoxic T cell recognizes an infected cell, makes a junction with the target cell thanks to adhesion surface molecules and induces a programmed cell death or apoptosis. Apoptosis is mainly induced by preformed cytotoxic molecules (perforin, granzymes, granulysin) released by degranulation of vesicles. Perforin polymerizes to form a pore in the target cell membrane through which granzymes and granulysin can pass. Once they are in the cytoplasm, these proteins can induce apoptosis by activating the caspase cascade. CTL can also induce target cell apoptosis through Fas-FasL interactions which also lead to the activation of the caspase cascade. Furthermore, in addition to their lytic activities, CTLs also release cytokines as IFN-γ and tumor necrosis factor (TNF)-α, which greatly contribute to host defense [1].

1.3 Interactions between innate and adaptive systems

Innate and adaptive immune responses are strongly interconnected: the innate immune system is essential to activate adaptive immune responses while cytokines and antibodies produced by adaptive lymphocytes improve the response of innate cells.

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Indeed, the activation, proliferation and differentiation of a pathogen-specific clone of adaptive T cells is dependent on i) the presentation of microbial antigens by APCs through MHC molecules, ii) contact-dependent co-activation signals and iii) the cy- tokinic environment set up by innate immune cells. The different CD4⁺T cell subsets are induced by different cytokines produced by innate cells: the Th1 response is induced by IL-12p70 and IFN-γ, produced respectively by APCs and NK cells, the Th2 response is induced mainly by IL-4, the Th17 response is induced by IL-6 and IL-23

Figure 1 – CD4⁺T cell subsets development and function, adapted from [6]

Differentiation into different effector CD4⁺ T cell lineages, T helper (Th) 1, Th2, Th17, and regulatory T cells (Treg) is initiated through an interaction of dendritic cells with naïve CD4⁺ T helper cells (Thp). The effector cell types are characterized by their synthesis of specific cytokines and their immuno-regulatory functions, as indicated on the right. The differentiation along different lineages involves different cytokines and the activation of distinct signaling cascades and transcription factors.

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while Treg differentiation is induced by tumor growth factor (TGF)-β and IL-2 (Fig- ure 1)[1; 5–8]. Conversely, cytokines produced by T cells improve the innate response.

For example, IL-2, predominately produced by activated CD4⁺ T cells, contributes to NK cell proliferation and activation (see § 2.5.1.1) while IFN-γ primes and activates monocytes, macrophages and DCs (see § 1.4). Finally, antibodies produced by plas- mocytes, the activated form of B cells, improve pathogen clearance by innate cells by several ways. They firstly coat pathogens by binding to surface antigens (opsoniza- tion), allowing the activation of the complement. Secondly, opsonized pathogens are more easily recognized and uptaked by phagocytic cells. Finally, innate cells including NK cells, neutrophils and eosinophils perform what is called antibody-dependent cellular cytotoxicity (ADCC) [1; 9].

1.4 Importance of interferon-gamma in the immune response

IFN-γ is a type 1 cytokine produced by several innate and adaptive cells, among them NK cells, different subsets of T cells (CD4⁺ Th1 cells, CD8⁺ T cells, γδ T cells, iNKT cells, CD3⁺CD56⁺ NK-like T cells), innate lymphocytes and, to a lesser extent, monocytes and DCs [4; 10–13]. The IFN-γ receptor is expressed by nearly all cell types including monocytes, macrophages, DCs but also platelets or epithelial cells. It is composed of a double heterodimer of two subunits IFNGR1 and IFNGR2. The ligation of IFN-γto its receptor induces a Janus-activated-kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, leading to the translocation of STAT-1 phosphorylated homodimers to the nucleus. This transcription factor then binds to the promoter of interferon responsible genes such as those encoding inducible Nitric Oxide Synthase (iNOS), some interferon responding factors (IRF) and IL-12 [11; 14].

IFN-γ is central in the immune response against pathogens as shown by the high susceptibility of IFNGR^-/- KO mice in different models of infections. IFN-γ induces

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monocytes/macrophages to produce cytokines like IL-12p70 and large amounts of microbicidal molecules as reactive oxygen species (ROS) which leads to the destruction of intracellular pathogens. IFN-γ also enhances the expression of MHC class II molecules, which allows a better presentation of pathogen antigens to CD4⁺ T cells [11]. Finally, IFN-γis highly important in the priming of monocytes and macrophages [15](see § 4.4).

1.5 The memory of the immune system

During the development of the adaptive response, some clones of T and B lymphocytes differentiate into memory cells which confer a long-lasting protective immunity [1]. For long, memory has been thought to be an exclusive adaptive immune fea- ture. However, recent studies show that innate cells like macrophages or NK cells also possess a sort of memory called “trained immunity”. On the contrary to adaptive immunity, this “training” is not pathogen-specific since “trained” cells display a

“cross-protection” against other related-microbes [16]. The phenomenon described in innate cells is more a heightened state of activation which allows cells to be more effective towards further invasions. This heightened activation state is characterized by an upregulation of activating receptors on the cells allowing an earlier and stronger response to stimuli. This training in contact to microbes could explain phenotypic differences observed between innate cells of different age populations. This also high- lights a crucial difference between pathogen-free experimental models and infections in humans [16]. In addition to this sort of non-specific memory, NK cells have recently been shown to also display pathogen-specific memory [17–20]. These new concepts temper the frontier between innate and adaptive immune systems.

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2 Natural killer cells

2.1 Definition

NK cells are large lymphocytes phenotypically characterized, in humans, by surface expression of CD56 (also named Leu-19 or NKH1), an isoform of the neural cell- adhesion molecule (NCAM) whose function on NK cells is still unknown, and the absence of CD3 and CD19. Once activated by an association of signals (cytokines and/or other ligands, see further), NK cells act as cytotoxic effector cells and immune- modulators (cytokine release). Since they are rapidly activated, do not require gene rearrangement and lack TCR or BCR, NK cells are commonly classified as innate cells. However, as mentioned above, recent findings show that they also display some adaptive immune features. Indeed, they can mount a form of pathogen-specific immunologic memory which, in case of reinfection, allows a more robust IFN-γ and cytotoxic response than resting NK cells. Moreover, some cells displaying specific receptors proliferate preferentially in some infections, mimicking clonal expansion of adaptive lymphocytes. These new findings lead some authors to consider that NK cells rather belong to adaptive than to innate immune response [10; 17; 19].

2.2 Ontogeny of NK cells

NK cells are known to be derived from common CD34⁺ hematopoietic progenitor cells. However, their site of development remains uncertain since not all the develop- mental intermediates can be isolated from the bone marrow nor the thymus, as it is the case for B and T lymphocytes respectively. On the other hand, it is well accepted now that IL-15, cross-presented by accessory cells (cf. § 2.5), is required for several steps of NK cell maturation [21]. In accordance with those facts, lymph nodes and tonsils are plausible sites of development of NK cells. Indeed, we can find there an abundance of CD34⁺CD45RA⁺ pre-NK cells, CD56^bright NK cells and DCs and other

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APCs that express membrane-bound IL-15. Figure 2shows a model of NK development, including the steps of differentiation from the pro-NK cells to the CD56^dim NK cells [10].

Figure 2 – Model of development of human NK cells in lymph node

(1) Bone marrow-derived CD34⁺CD45RA⁺HPCs circulate in the blood and (2) ex- travasate across lymph node high endothelial venules to enter the parafollicular space.

There, (3) pro-NK cells are activated to progress through distinct stages of maturation (far right) to create both CD56^brightand CD56^dimNK cells. Maturing CD56^dimNK cells return to the circulation via the efferent lymph (4), whereas some CD56^brightNK cells remain within the secondary lymphoid tissue to interact with DCs (5) [10].

2.3 Sub-populations

Mature human NK cells are commonly divided into two sub-populations based on cell-surface density of two proteins: CD56 and CD16 (or FcγRIII). The CD56^brightCD16⁻^/low and the CD56^dimCD16⁺ NK cell subsets are thus distinguished. If CD56^dimCD16⁺ NK cells are predominant in blood (±90% of adult blood NK cells), CD56^brightCD16⁻^/low are preponderant in secondary lymphoid organs [22; 23]. Some authors are going further in the categorization of sub-populations, separating CD56^bright NK cells in CD56^brightCD16⁻ and CD56^brightCD16^low NK cells and adding a CD56^dimCD16⁻ subpopulation [24–27]. Since no actual functional differences have currently been reported between both CD56^brightsubsets, we have not distinguished them in our work.

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On the contrary, CD56^dimCD16⁻NK cells functionally differ from other subsets(Table 1). In this manuscript, the name CD56^dimNK cells will refer to the major CD56^dimCD16⁺ NK cell subset.

Table 1. Receptors and effectors molecules on/in the different blood resting NK subsets

NK cell subsets differentially express various receptors (Table 1). These differences have functional implications. For instance, the constitutive expression of IL-2Rα(CD25)

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by CD56^brightand CD56^dimCD16⁻allows higher proliferation rate and better activation of these subsets following IL-2 stimulation. Conversely, the higher expression of killer immunoglobin-like receptors (KIRs) and the lower expression of NKG2A inhibitory receptor allow a more efficient activation of CD56^dimCD16⁺NK cells. Furthermore, the differential expression of chemokine receptors and adhesion molecules indicates differential homing of the subsets. Indeed, CD56^brightNK cell subset expresses CCR7 and CD62L, which allow trafficking into secondary lymphoid organs through endothelial venules whereas CD56^dimCD16⁺ NK cell subset express CXCR1, CXCR2, CXCR4 and CX3CR1 which allow trafficking towards the site of inflammation [28–31].

CD56^bright NK cells are commonly described to be preferentially cytokine producers whereas CD56^dimCD16⁺ NK cells rather perform cytotoxicity [10; 22; 23]. How- ever, recent studies have challenged this dichotomy. Indeed, CD56^dim NK cells may quickly but transiently produce IFN-γafter in vitrostimulation by target cell recognition [32; 33] (see § 2.4.2).

It is noteworthy to mention that the CD56 molecule is not expressed by murine NK cells. Murine NK cell subsets are therefore differentially identified. Classification however greatly varies between authors [34; 35] and it is not easy to compare these sub- populations with those in humans. Since NKp46, a member of the highly conserved natural cytotoxicity receptor (NCR) family receptors is present in both species (and others) on almost each NK cells, it has recently been suggested that this molecule best defines NK cells across mammalian species and could be used to compare both models [35].

2.4 Functions

NK cells have been first described as cytolytic effector cells which can directly induce the death of target cells. Later on, they additionally have been recognized as modulators of immunologic responses. They are indeed responsible for an early production of substantial amounts of cytokines, chemokines and growth factors, further impacting

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the immune response [19].

2.4.1 Cytolytic activities

NK cells can lyse infected or tumoral target cells or pathogens in the absence of prior antigen-presentation [10; 37]. They mediate target cell lysis by the same mechanisms than CTL(§ 1.2). The induction of these cytotoxic functions requires the formation of an immunological synapse between the NK cell and its target cell, wherein cytotoxic effector molecules are delivered, ensuring precise targeting of the cytolytic process [38].

CD56^dim NK cells are better armed to perform cytotoxicity than CD56^bright NK cells.

This relates to their higher content of cytotoxic granules and to the expression of activating receptors (Table 1)able to trigger degranulation. Furthermore, ADCC mediated by NK cells requires the recognition of antibody-coated targets by the CD16 receptor, highly expressed by CD56^dimNK cells subset [23; 38; 39].

2.4.2 Production of soluble mediators

Depending on the stimulus (cf § 2.5), NK cells can produce a large number of cy- tokines, both proinflammatory (as IFN-γ or TNF-α) and immunosuppressive (as IL- 10), of growth factors (as granulocyte colony-stimulating factor [G-CSF] or granulocyte macrophage [GM]-CSF) and of chemokines. The secretion of chemokines and growth factors is a very early event (1h after target recognition) [40] and is important for assuring the co-localization of NK cells with other hematopoietic cells, such as DCs, in inflamed tissues or secondary lymphoid organs [19].

The most documented NK produced cytokine is IFN-γ (see § 1.4). As mentioned earlier, CD56^bright NK cells are the most potent IFN-γ producers following cytokine stimulation [10; 23]. It has however recently been shown in some in vitromodels that CD56^dimand not CD56^brightNK cells may produce IFN-γin a very early and transient way [32; 33]. Importantly, the kinetics and NK subsets producing IFN-γ depend on

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the nature of the stimulus [32; 41–43].

2.5 Mechanisms of NK cell activation

The first mechanism of activation described for NK cells was the “missing self” recognition, based on the detection of the loss or deficiency of MHC class I molecules on target cells by inhibitory KIRs. Many other receptors have since then been shown to be implied in NK cell activation, as cytokine receptors, NCRs and PRRs. Activation of NK cells is consequently dependent on the balance between inhibitory and activating signals received by the cell. It is in addition admitted that, firstly, several concomitant signals are needed to activate NK cells and, secondly, NK cells have to be primed by IL-15 to be activated [10; 19; 21].

2.5.1 Cytokine-mediated activation

NK cell functions are regulated by diverse cytokines, most of them being produced by mononuclear phagocytes and APCs. At least part of resting NK cells constitutively express several cytokine receptors including IL-1R [23], IL-2Rβγ [44; 45], IL-10R [46], IL-12R [47], IL-18R [47; 48] and low levels of IL-15Rα [49]. As already mentioned in § 2.3, the CD56^bright and CD56^dimCD56⁻ NK cell subsets uniquely express IL-2Rα [23; 25; 26; 44]. The expression of these receptors can be enhanced upon activation.

It is known for long that cytokines such as IL-1, IL-2, IL-12, IL-15, IL-18 and type I IFNs activate NK cells [23; 48; 50; 51], and that others such as IL-10 or TGF-β[23; 52]

modulate their activity. Furthermore, the constant discovery of novel cytokines like IL- 27 [53], IL-33 [54] or IL-35 [55] increase the spectrum of soluble molecules that regulate NK cell immunobiology. In this introduction, we will focus on several cytokines that have been described to induce IFN-γ response by NK cells, that are members of the IL-2, IL-12 and IL-1 (super)families [10; 23; 48; 51].

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2.5.1.1 Members of the IL-2 superfamily

The IL-2 superfamily comprises for now IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. The receptor for each cytokine is composed on the common γ-chain (γ_c or CD132) which, except for IL-2 and IL-15, dimerizes with a cytokine-specific high affinity α subunit which participates to the transduction of the signal. By contrast, the IL-2 and IL-15- specific α chains are involved in the high affinity recognition of each cytokine while the signal transduction is mainly provided by a common receptor core formed by the dimeric association of CD132 and IL-2Rβ(CD122), which is already a low-affinity receptor. The final high-affinity receptor is thus a heterotrimer composed by IL-2Rβγ and the specific IL-2Rα (CD25) and IL-15Rα respectively (Figure 3). Since all these cytokines share CD132, they display some functional redundancy in the regulation of immune responses but have also specific functions. They all have a potential role in NK cell activities (reviewed in [51]). We will however here focus on the action of IL-2 and IL-15, known to be of particular importance for NK cells.

IL-2 and IL-15 are monomeric proteins of respectively 15.5 and 15 kDa [56] presenting similar functional properties, since their receptors share the βγ heterodimer and common signaling pathways. Once engaged, the βγ-chains induce the phosphory- lation of STAT-3 and STAT-5 via the JAK-1 and JAK-3 respectively. Phosphorylated STATs then dimerize and translocate into the nucleus, where they function as transcription factors. Binding of these dimers to STAT-sensitive regulatory elements con- trols the transcription of several interleukin-inducible genes including genes mediating differentiation and proliferation of NK cells [44; 51]. In addition to JAK/STAT signaling, IL-2/IL-15 binding to their receptor initiates other signaling events such as the induction of Bcl-2, a protein that promotes cell survival [57]. Finally, it triggers a Ras/Raf/mitogen-activated-protein-kinase (MAPK) cascade that culminates in activation of the activator protein (AP)-1 and the nuclear factor (NF)κB, that are transcription factors both known to induce Ifng (IFN-γ gene) and PRF1 (perforin gene)

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transcription [13; 44; 58; 59].

Figure 3 – The family of cytokines sharing the common γ-chain in their receptor complexes

Each cytokine binds to a specific α chain, which form a receptor complex with γ_c. In case of IL-2 and IL-15, trimeric high affinity complexes, which include common IL-2Rβ and γ_c, can be formed. Each receptor complex mediates signal transduction through JAK1 and/or JAK3 and different STAT molecules. Phosphorylated STAT dimers regulate transcription of specific cytokine-sensitive genes [51].

Despite their common transducing chains, IL-2 and IL-15 also display specific functions thanks to their distinct cellular origin and the different cellular distribution and functional properties of their α chains [44]. Indeed, whereas IL-2 is mainly produced by activated T cells [56], IL-15 is produced by monocytes/macrophages and dendritic cells but also by fibroblasts, epithelial cells or nerve cells. Furthermore, soluble IL-15 is rarely detected except in pathogenic situations. Indeed, IL-15 is mainly produced in an IL-15Rα-bound form at the surface of producing cells and is preferentially trans- presented in this form to IL-2R/IL-15Rβandγc receptor-bearing NK and T cells(Fig- ure 4)[56; 60; 61]. These differences have a huge impact for the role of both cytokines.

Indeed, IL-15, particularly produced in lymph nodes and tonsils (see §2.2), plays a

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major role in NK differentiation and prevails in vivoon IL-2 in inducing survival and proliferation of NK cells [21; 44].

Figure 4 – Interaction of IL-2 with its receptor contrasted to that of IL-15 and its receptor

The high-affinity IL-2R is a heterotrimeric complex composed of the common cytokine receptorγsubunit (γc), a βsubunit (IL-2R/IL-15Rβ) that is also shared with the IL-15 receptor (IL-15R), and a privateαsubunit (IL-2Rα). All three subunits are expressed on activated NK, T and other immune cells. Receptor specificity is conferred by theαsub- unit of each cytokine (IL-2Rαor IL-15Rα) and signal transduction is mediated through IL-2R/IL-15Rβ and γc. For IL-2, all three subunits (IL-2Rα, IL-2R/IL-15Rβ, and γc) are expressed by the same cell and as a complex directly binds to soluble IL-2. By contrast, the interaction of IL-15 with its receptor is more complex. Little IL-15 appears to be expressed in a free soluble state. Instead, IL-15 is presented in trans pre-associated with IL-15Rα by antigen-presenting cells to the IL-2R/IL-15Rβ and γc receptor sub- units expressed on NK and T cells. IL-15 is expressed by monocytes/macrophages and dendritic cells and is stabilized by being associated with IL-15Rα in the endosomal–Golgi apparatus. The IL-15/IL-15Rα complex is secreted and expressed on the surface of monocytes and dendritic cells to present IL-15 to the IL-2R/IL-15Rβandγc subunits expressed on NK and CD8⁺T cells [60].

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2.5.1.2 Members of IL-12 family

IL-12p70 is a cytokine of particular interest since it plays a key role in the induction of CD4⁺ Th1 cell differentiation and is preponderant in the induction of IFN-γ production by T and NK cells. Other related cytokines (IL-23, IL-27 and IL-35) have also been described, which are all composed of two disulphide-linked subunits. IL-12 and IL-23 share a common chain, p40, which is respectively associated with the p35 and p19 chains. IL-27 and IL-35 are composed of the shared Epstein–Barr virus-induced gene product (EBI)-3, which is a homolog of p40, respectively associated with the p28 (related to p35) and p35 chains. Despite their structural similarities, these cytokines have distinct functions. Indeed, even if all these cytokines transduce their signal trough a dimeric receptor, inducing a JAK/STAT cascade, different STAT predominances occur, inducing different responses (Figure 5). IL-12 thus induces IFN-γ production by NK and T cells and the subsequent type 1 response while IL-23 induces IL-17 production by T cells. IL-27 and IL-35 have rather immunosuppressive activities [62–65].

IL-12 is important for the induction of IFN-γproduction by NK cells [65–67]. Since this cytokine has a critical role in driving type 1 immune responses, it is tightly regulated at the transcriptional, post-transcriptional, translational and post-translational levels [13; 66; 68]. The genes encoding the two IL-12 subunits are independently regulated.

Indeed, a basal ubiquitous expression of p35is observed while p40is only expressed by IL-12p70 producing cells (DCs, monocytes/macrophages, neutrophils and, to a lesser extent, B cells) and need less microbial activating signals to be induced. Even if p35 mRNA is ubiquitously observed at low levels, the protein is rarely produced and released due to the presence of an inhibitory ATG in the 5’-untranslated region [66]. A study also reports that a preformed bioactive form of IL-12 is stored in a membrane- bound state, allowing its release within minutes after intracellular pathogen-contact [69]. However, since it has not been further investigated or confirmed by others, this information has to be taken with caution. Finally, the action of IL-12p70 synthesized

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by APCs on NK cells requires the formation of a synapse, allowing the direct use of IL-12 by NK cells [67; 70]. IL-12R engagement induces a JAK2/STAT4 phosphorylation cascade which leads to homodimerization of STAT4 and its recruitment to the Ifng promoter (Figure 5), inducing transcription of the gene and further production of IFN-γ [62; 66].

Figure 5 – IL-12 family of cytokines and their receptors (adapted from [62])

Members of the IL-12 family of cytokines comprise a helical subunit (blue ovals) and a receptor-like β-chain. IL-12p70 and IL-23 share a common chain (p40) and their receptors share the IL-12 receptor β1 chain. IL-12 induces the secretion of IFN-γ by CD4⁺and CD8⁺T cells and by NK cells. IFN-γ suppresses the development of IL-17- secreting T cells and thereby limits IL-17-mediated inflammatory events. IL-23 (com- prising p40 and p19) promotes IL-17 production by several T-cell types including the T helper 17-cell subset. IL-17 is a potent pro-inflammatory cytokine that induces tissue damage at least in part through neutrophil recruitment. IL-27 and IL-35 shares homology with IL-12p70 and IL-23 and signals through a receptor that shares the gp130 chain with the IL-6 receptor. They both have more immunosuppressive activities, even if IL-27 can promote Th1-cell differentiation. Signalling pathways that induce IL-12 family members include the JAK–STAT. Predominant STAT molecules that are known to be activated are indicated in bold [62].

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2.5.1.3 IL-1 superfamily

Cytokines of the IL-1 superfamily belong to an ancient protein family, related to the Toll-like receptor (TLR) family, which is largely conserved in mammals. Only three of these cytokines display an extracellular form, IL-1β, IL-18 and IL-33.

Figure 6 – TIR domain Receptor family and their ligands [73]

Toll-like Receptors and IL-1 cytokine family receptors belong to the same TIR domain family. They thus share the TIR domain but also the further MyD88/IRAK cascade, inducing an overexpression of many pro-inflammatory genes.

IL-1β and IL-18 present the particularity to be synthesized as biologically inactive precursor molecules inside cells before being cleaved by caspase-1 to become active.

Caspase-1 is activated by a cytosolic multiprotein assembly, called the inflammasome, whose activation is triggered by a variety of danger signals, including disrupted

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phagocytosis or intracellular infection [71; 72]. These cytokines induce different NK cell responses after signaling through myeloid differentiation primary response gene product (MyD)88/Interleukin-1 receptor-associated kinase (IRAK) cascade(Figure 6).

On the one hand, IL-1βis a pro-inflammatory cytokine. On the other hand, IL-18 and IL-33 rather act as general amplifiers of inflammation. For instance, in the presence of IL-12, IL-18 potentiates IFN-γproduction by NK cells by stabilizing IFN-γtranscripts while it potentiates IL-13 production in the presence of IL-2 [48; 50; 71; 72].

2.5.2 Activation through contact-dependent signals

NK cells possess a large variety of activating and inhibitory receptors in addition to cytokine/chemokine receptors. The most important ones will be described here below.

2.5.2.1 The killer immunoglobulin-like receptors family

KIRs are a multigenic family of polymorphic proteins, members of the immunoglobulin (Ig) superfamily of receptors, that bind to MHC class I molecules on host cells.

Since every cell in the body expresses MHC class I molecules, NK cells can recognize all self-healthy cells thanks to them. There are at least 17 KIR genes or pseudo- genes giving, with the allelic diversity, a huge NK cell diversity between individuals.

KIRs are separated in two groups, depending of the length of their cytoplasmic tail.

Long cytoplasmic tail receptors induce inhibitory signals thanks to their immunoreceptor tyrosine based (IT) inhibitory motifs (ITIMs), while the short tailed KIRs activate NK cells by binding to DNAX activation protein 12 (DAP12) adaptor protein which contains an IT activating motif (ITAM) [10; 74]. Engagement of inhibitory KIRs prevents NK cell activation by recruiting Src homology region 2 domain-containing phosphatase (SHP)-1 molecules through their ITIM. SHP-1 can dephosphorylate proteins such as ITAM-bearing activating receptors, disabling activation cascades. Each

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activating KIR can induce different functions. For instance, KIR2DL4 have been shown to specifically induce IFN-γ production and not cytotoxicity [75; 76]. In resting NK cells, the KIR inhibitory signal quite dominates over any activating signals and prevents the NK cell from killing self-cells [77]. Interestingly, each NK cell expresses only a fraction (0 to 4) of the available KIRs, resulting in a varied NK cell repertoire capable of rapidly sensing any changes in MHC class I expression [78; 79].

2.5.2.2 The NKG2 receptors

NKG2A, NKG2C and NKG2D are expressed by NK cells and some other innate populations such as γδ T cells or NK-like T cells (see § 3). NKG2A and NKG2C are re- spectively inhibitory and activating receptors that dimerize with CD94 molecule and recognize non-classical MHC molecule HLA-E. After binding with HLA-E molecule, CD94/NKG2A recruits SHP-1 whilst CD94/NKG2C is associated with DAP12, like inhibitory and activating KIRs respectively (§ 2.5.2.1). When there is a similar ex- pression of NKG2A and NKG2C, it seems that the inhibitory NKG2A/CD94 dimer predominates thanks to its slightly better affinity for HLA-E molecules.

NKG2D have numerous ligands going from polymorphic MHC-class-I-related chains (MIC)-A and MIC-B to unique long-16-binding proteins (ULBPs) which are expressed by infected and tumoral cells. Activation of NKG2D triggers a signaling cascade implying JAK2, STAT5, extracellular signal-regulated kinases (ERK) and MAPK. These interactions induce NK functions including IFN-γproduction [80–82].

2.5.2.3 The natural cytotoxicity receptors

NCRs are a family of activating receptors which gather together NKp30, NKp44 and NKp46 molecules. They were first described in NK cells but are also present in other cells including NK-like T cells, γδT cells and type 1 innate lymphoid cells [4; 83; 84].

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The ligands of these molecules are multiple, going from viral hemagglutinin and hemagglutinin-neuraminidase (see §2.5.3) to abnormal heparan sulfates on tumoral cells. Once triggered, NCRs activate p38 MAPK and ERK cascades which induce NK cytotoxicity against a broad spectrum of tumor and virus-infected cells and IFN-γ production [32; 80; 84; 85].

NKp80 is a C-type lectin-like homodimeric receptor expressed by all NK cells and a part of NK-like and γδ T cells. NKp80 binds to activation-induced C-type lectin (AICL) proteins on target cells including TLR-stimulated monocytes. This signal is reported to be important in monocyte-NK crosstalk(see § 5.)[82; 86].

2.5.2.4 The Toll-like receptors family

TLRs are glycoproteins, members of the IL-1R family, which recognize PAMPs and play a pivotal role as sensors of pathogens and in host antimicrobial defenses. Ten TLRs have been described in humans that recognize a broad spectrum of conserved microbial ligands. Some of them are situated on the cytoplasmic membrane, sensing extracellular PAMPs (TLR1, 2, 4, 5, 6 and 10) and others are endosomal (TLR3, 7, 8 and 9) and recognize internalized PAMPs. TLRs are highly expressed by monocytes, macrophages and DCs [87] and are more lightly expressed by NK cells [88–91], T cells [92; 93], B cells [94] and epithelial cells [94]. The recognition of microbial ligands by their dedicated TLR induces in NK cells, like in other cells, a signaling pathway through their toll/interleukin-1 receptor (TIR) intracellular domain, implying MyD88 and/or TIR-domain-containing adapter-inducing interferon-β(TRIF) adaptor proteins (Figure 6). Through serial phosphorylation of proteins and activation of the transcrip- tion factor NF-κB, TLRs induce transcriptional and post-transcriptional activation of several genes involved in a broad spectrum of responses going from cell proliferation to pro- and anti-inflammatory cytokine production [87; 94]. These receptors were for long thought to be quite inefficient on NK cells but some in vitro studies show an induction of NK cytotoxicity and IFN-γ production by TLR 2 [88; 91], 3 [89; 90], 4

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[95; 96], 5 [91], 8 [97] and 9 [89] but not TLR 7 or 8 [90] ligation.

2.5.3 Pathogen-mediated activation

Pathogens provide a wide range of signals to NK cells, directly and indirectly. Indi- rect signals are provided by APCs through cytokine production (IL-12 p70, IFNα/β, IL-18. . . ) and contact-dependent co-stimulation (Figure 7).

Figure 7 – Interactions of NK cells, accessory cells and pathogens

NK cells generally require signals from accessory cells in order to respond to pathogens. Accessory cells become activated following ligation of pathogen- recognition receptors by pathogen-encoded ligands. Both contact-dependent and soluble signals then induce activation of NK cells [67].

Accessory cells play a huge role in NK activation following infection, especially in protozoan infections [98–101]. The role of accessory cells in NK cell activation will be discussed later(§ 5)and we will hereunder describe how pathogens may activate NK

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cells without involvement of such accessory cells.

Pathogens can interact with numerous of the NK receptors previously cited (§ 2.5.2).

Firstly, KIRs and NKG2/CD94 can of course sense classical MHC class I and HLA-E perturbations, respectively, on target cells. Similarly, NKG2D ligands may be either induced or down-modulated during a viral infection, depending on the pathogen.

Finally, AICL can also be down-modulated in infected cells by proteins of the HHV-8 virus [82; 102].

Table 2. NCR and their microbial ligands [82]

Besides, pathogens have been described to directly interact with NK cells through TLRs. Indeed, Leishmaniapromastigotes and Mycobacterium bovis induce purified NK cells to produce IFN-γ and TNF-α in a TLR-2 dependent way [88; 103]. In addition, pathogenic but not non-pathogenic strains of E. coli induced IFN-γ and TNF-α production and cytotoxic activities by purified NK cells through interaction between TLR-4 and an adhesin only produced by pathogenic E. coli, FimH (fimbrial type 1 pilli H) [96]. Although TLRs can play a role in NK activation, this stimulation seems to be marginal since they are not brightly express on NK cells. There is to date no evidence for a direct interaction between NKG2D and pathogen molecules but other molecules, more specific of NK cells, seem to provide more potent signals. In particular, NCRs sense infected cells, through recognition of viral-encoded molecules

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expressed at the surface of infected cells, but may also directly recognize bacterial molecules. Hence, NCRs are important in several infectious diseases as summarized inTable 2[82; 104; 105].

3 NK-like T cells

3.1 Definition

CD3⁺CD56⁺ cells were first discovered in the early 1980s as a component of lymphokine activated killer (LAK) cells [106] and were described in blood in 1986 by two groups [107; 108]. They have been named differently throughout the past three decades: NKT cells (before NKT cells have been defined as a subset of T cells that express invariant TCRs), non-MHC-restricted cytotoxic T cells [109], natural T cells [110; 111], T cells with NK markers [112; 113] or NK-like T cells [42; 43; 84; 114–

118]. Furthermore, different authors gave all these names to cells expressing different NK receptors (CD56, NKG2D, CD161 . . . ), which make comparisons between studies quite difficult. Finally, it is to note that the terminology of NKT cells is still erro- neously used by some authors to name NK-like T cells.

NK-like T cells are a heterogeneous subset of non-MHC-restricted CD3⁺CD56⁺T cells which, similarly to NK cells, are rapidly effective after activation. These cells are characterized by the expression of TCR, CD3 and CD8 (in most cases) but also various NK markers, especially CD56, but also NKG2D, CD161, NCR or KIRs [84; 115; 119; 120].

After their discovery, the study of these cells has been intensified as components of LAK cells (blood mononuclear cells cultured for 5 days with diverse cytokines) and cytokine-induced killers (CIK) cells (cells cultured during 28 days with cytokines) for their interesting anti-tumoral properties in immunotherapies. On the contrary, in or ex vivo blood NK-like T cells have been less studied, though a regain of interest has occurred these last few years for their potential involvement in control of cancers and microbial diseases [42; 114; 115; 121].

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3.2 Functions of NK-like T cells

3.2.1 Functions of cultured NK-like T cells

As said above, NK-like T cells have first been studied in LAK and CIK cells, which are activated killer cells that are effective against tumoral cells. They are thus of promi- nent importance in the field of immunotherapy against cancers. These cultured cells contain a small proportion of NK cells and a majority of T cells (around 90%), com- prising around 35% of CD56⁺T cells [118; 119]. CD3⁺CD56⁺T cells highly express the CD8 molecule and genes encoding granzyme B, IFN-γ, TNF-α, MIP-1α, RANTES, IL- 12Rβ2 and caspase-1, which indicates a type 1 polarization [118]. NK-like T cells can recognize target cells in a MHC-dependent- and in a MHC-independent-way. Indeed, some tumoral cell lines (myeloma) have been shown to be recognized by NK-like T cells via NKG2D-mediated recognition of MIC-A and MIC-B on the target cells, without the need of TCR recognition [118; 119; 122].

3.2.2 Functions of blood NK-like T cells

Blood CD3⁺CD56⁺ T cells are a heterogeneous population composed of αβ and γδ T cells. Their percentage among total lymphocytes highly varies between donors from 1 to 15% [107; 115; 123]. NK-like T cells may express a large number of NK cell receptors in addition to CD56 such as NKG2D, KIRs, CD94/NKG2A or CD161 [115].

On the contrary to CD3⁺CD56⁺ found in LAK and CIK cells, they do not express CD16 [124]. The vast majority of these T cells are CD8⁺. Since NK-like T cells express a diverse TCR repertoire, which tends to oligoclonality [112; 125], and since the size of this subset expands with aging [113], it has been postulated that NK-like T cells are effector memory CD8⁺T cells, although the precise steps of differentiation of CD56⁺T cells from naive CD8⁺T cells are still unknown. Since they lack CD16, blood NK-like T cells cannot mediate ADCC but their high intracytoplasmic granzyme B and perforin

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content allows them to be good cytolytic effector cells [112; 115; 125]. Indeed, they can lyse NK-sensitive K562 cells, though on a less efficient way than NK cells [124], but also NK-resistant cells like Raji cells [112]. Although, at first, authors reported that NK-like T cells did not produce IFN-γ in response to IL-2 [124], recent studies show they do produce IFN-γ in response to some cytokines like IL-15 [112; 115], the combination IL-12/23 and IL-18 [43; 115] or pathogens like Salmonella [42; 114] or Mycobacteria [115]. It has also been reported that NK-like T cells also produce other cytokines such as TNF-α [110].

4 Monocytes

4.1 Definition

Monocytes are circulating blood mononuclear phagocytic cells which constitute around 10% of leukocytes in human blood. They are phenotypically defined by the expression of the cell surface receptor CD14 (associated with TLR-4 in the composition of the PRR for LPS) and are very heterogeneous from a morphological point of view (size, granularity . . .). Monocytes develop in the bone marrow from a myeloid precursor, are released in the bloodstream as non-dividing cells and can rapidly differentiate into macrophages or myeloid dendritic cells (mDCs) in inflammatory conditions, then entering tissues. Monocytes are therefore often considered as a huge permanent reser- voir of more differentiated phagocytic cells but they are also a first line of pathogen recognizing cells, ready to fight invading microbes [126; 127].

4.2 Sub-populations

Monocytes are commonly subdivided in two or three subpopulations according to their expression of CD14 and CD16: the majority of monocytes (80 to 90% of blood monocytes) belongs to the so called “classical” subpopulation which expresses CD14

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but not CD16 (CD14⁺CD16⁻); the other part of monocytes expresses CD16 and is sometimes further divided according to the level of CD14 expression (CD14^dimCD16⁺ and CD14⁺CD16⁺). These different sub-populations differ in function (see § 4.3) and in surface receptor expression [126–128].

4.3 Function of monocytes

In addition to their phagocytic properties, monocytes are antigen presenting cells and cytokine producers. They are thus accessory cells that can link the innate and adaptive immune system after pathogen recognition. When activated, monocytes can produce large amount of ROS, nitric oxide, cytokines, complement factors and other pro-inflammatory factors. They may also express co-stimulatory molecules such as CD40, CD80, CD83 and CD86. However, even if they express MHC class II molecules, monocytes have been found in most cases to be far less efficient than dendritic cells in antigen presentation to T cells [127; 129].

"Classical" monocytes display higher phagocytic properties but lower cytokine production capacities than the so called “pro-inflammatory” CD16⁺ subset [130]. Cy- tokine production is important for orienting further adaptive responses (see § 1.3).

Monocytes can produce a large spectrum of cytokines including type 1 IFNs, IL-6, IL-8, IL-15, members of IL-1, IL-10 and IL-12 family, TGF-βor TNF-α. These cytokines are all induced by LPS but display dramatic different kinetics, some being produced very early in less than 6h after activation (TNF-α, IL-8, IL-12, IL-15) while others are more lately produced after 24-48h of activation (IL-10, IL-18, IL-23, TGF-β) [87; 127].

4.4 Mechanisms of activation

Monocytes can be activated by diverse signals but it is now admitted that they must be primed by low concentrations of IFN, mostly IFN-γ. This priming is not sufficient to activate monocytes but will potentiate further early innate immune responses

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against invading pathogens [15]. Monocytes express many PRRs including TLRs and nucleotide-binding oligomerization domain (NOD)-like receptor (NLRs). When triggered, TLRs signal through MyD88 or TRIF-associated pathways (see § 2.5.2.4 and figure 6) leading to different biological responses according to the TLR. TLR ligation can induce the transcription of many genes including those encoding for pro- inflammatory cytokines such as type I IFNs, IL-6, IL-12, pro-IL-1β and pro-IL-18 or co-stimulatory proteins such as CD80 or CD86. They also favor the setup of the inflammasome involved in caspase-1 activation of IL-1 and IL-18, and enhances phagocytosis [94; 131]. Furthermore, the different PRRs synergize in order to induce potent pro-inflammatory cytokine production. Particularly, multiple signals such as a combination of TLR-4 and TLR-8 or a single TLR and a co-stimulatory signal such as IFN-γ are required for a strong induction of IL-12 [131; 132]. Monocytes also express FcγR of moderate and low affinity, namely CD32 and CD16. These receptors allow monocytes to lyse opsonized target cells by ADCC mechanisms. Furthermore, binding of antibodies to these receptors activate phagocytosis and cytokine production [133]. Fi- nally, monocyte functions are modulated by cytokines. The most potent activating cytokine is IFN-γthat induces ROS, nitric oxide (NO) synthesis and IL-12 production but monocytes possess a large range of cytokine receptors including IL-10R, IL-13R or TNF-αR.

4.5 Further differentiation of monocytes

As said earlier, monocytes differentiate into a subgroup of macrophages and into myeloid dendritic cells which are specialized phagocytes that play a major role as antigen presenting cells for activation of naïve T cells. These cells are of major importance in the constant close watch over pathogen invasion. Macrophages and mDCs display similar properties than monocytes but have a different receptor repertoire. In- deed, all myeloid cells express MHC II molecule but mDCs lose CD14 upon differentiation. Similarly, PRR expression differs between cell type. For instance, while they all

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express TLR1, TLR-2, TLR-4, TLR-5, TLR-6 and TLR-8, monocytes and macrophages specifically express TLR-9 and mDCs TLR3 and TLR7. These differences allow a differential spectrum of pathogen recognition [127; 134; 135].

5 Cross-talk between NK cells and monocytic cells

As mentioned earlier (§ 2.5.1), NK cells are activated by monocyte-produced cy- tokines. But this is far to be the only interaction between NK cells and accessory cells. Indeed, NK cells and monocytes, macrophages and mDCs display bidirectional interactions allowing activation and maturation of both counterparts [67; 136].

5.1 Chemotaxis and site of interaction

After pathogen recognition, accessory cells produce a large spectrum of chemokines in order to recruit other immune cells to the infected tissues or secondary lymphoid organs where they can communicate. These chemokines differs following the type of immune response induced by the pathogen and the site of infection. Moreover, different NK cell subsets respond differently to these mediators (see § 2.3). For in- stance, CD56^brightNK cells and DCs both express CCR7 [23; 137], which allows both to migrate to secondary lymphoid organs where they interact.

5.2 Activation of NK cells by monocytic cells

Monocytic cells can activate NK cells through cell-to-cell contact or cytokine production (Figure 8). However, this activation requires the setup of an immunological synapse where polarized cytokines (particularly IL-12) are secreted [38; 70]. As mentioned earlier, the major cytokines that activates NK cells are IL-1, IL-12, IL-15 and IL-18(§ 2.5.1). Besides, several studies have shown that MIC-A/B, CD40, AICL and

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CD80/CD86 on accessory cells can co-induce IFN-γ production by NK cells through respective binding to NKG2D, CD40L, NKp80, and CD28 [67; 86; 138; 139].

Figure 8 – Signals passed from accessory cells to NK cells, adapted from [67]

Accessory cells sense pathogen ligands through receptors located on the cell surface, in the cytoplasm or in endosomal compartments. Following recognition, accessory cells become activated and transmit signals to NK cells through various soluble or membrane-bound molecules. The degree to which each signal contributes to NK-cell activation in response to different pathogens remains to be clearly established.

5.3 Effect of NK cells on accessory cells

Activated NK cells release cytokines such as IFN-γand TNF-α which induce the maturation and activation of some accessory cells (see § 1.3 and 4.4)and the destruction of others. Indeed, NK cells specifically lyse immature DCs in an NKp30-dependent mechanism, which is called “NK cell-mediated editing of DCs”, allowing a selection of fit DCs. Since immature DCs induce abortive or tolerogenic T cell response, their destruction is of particular importance for the good setup of a productive adaptive response [136; 140]. It is worth noting that, similarly to NK activation, all these mechanisms are dependent of the setup of an immunological synapse [38].

Production of IFN- γ by neonatal Natural Killer cells in response to Trypanosoma cruzi and cross-talk

Faculté de Médecine

Laboratoire de Parasitologie