The role of human Natural Killer cells (NK) in anti-tumour immune responses

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The role of human Natural Killer cells (NK) in

anti-tumour immune responses

Giulia Fregni

To cite this version:

Giulia Fregni. The role of human Natural Killer cells (NK) in anti-tumour immune responses. Im-munology. Université Paris Sud - Paris XI, 2011. English. �NNT : 2011PA11T068�. �tel-01078862�

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Université Paris-Sud XI

Faculté de Médecine

École Doctorale « Cancérologie - Biologie - Médecine – Santé » (CBMS)

Spécialité : Immunologie

Thèse

pour obtenir le grade de

Docteur de l’Université Paris-Sud XI

The role of human Natural Killer cells (NK)

in anti-tumour immune responses

Présentée et soutenue publiquement par

Giulia FREGNI

Le 28 Octobre 2011

Jury

Pr Antoine DURRBACH

Président

Dr Vincent VIEILLARD

Rapporteur

Dr Francesco COLUCCI

Rapporteur

Pr Marie-Françoise AVRIL

Examinateur

Dr Philippe BOUSSO

Examinateur

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Acknowledgments

I thank Pr Antoine Durrbach for having accepted to preside my dissertation committee.

I am very grateful to Dr Francesco Colucci and Dr Vincent Vieillard for having agreed to referee my thesis and for the precious comments on how to improve this manuscript.

I am thankful to Dr Philippe Bousso for having accepted to be examiner of my thesis.

My sincere gratitude to Pr Marie-Françoise Avril for the last four years of collaboration and for having accepted to examine my thesis. Without your participation this work could never have been accomplished.

My deep and sincere thanks to my thesis supervisor Dr Anne Caignard. Anne, merci beaucoup de m’avoir accueillie à l’IGR pour mon premier stage « Leonardo da Vinci », de m’avoir donné la possibilité de préparer ma thèse sous votre direction, de m’avoir aidée à développer mon indépendance, pour votre esprit toujours positif, pour votre encouragement, pour la confiance que vous m’avez témoignée… Anne, merci pour tout!

My research was supported by a three year-fellowship from “Cancéropôle Ile de France”. One supplemental year of fellowship has been funded by “Ligue Nationale contre le Cancer”, allowing me to achieve my thesis.

Un remerciement spécial va à la co-directrice de l’équipe Armelle Blondel et à tous les autres membres, pour avoir partagé avec moi ces quatre dernières années ou seulement une partie de celles-ci: Renée pour toutes les réponses aux questions techniques que je vous ai posé, Laetitia et Sarra pour les moments éclatants que vous m’avez fait passer, Marylène pour votre disponibilité et gentillesse, Nadège pour ton apport scientifique et pour tout le reste, Maxime et Jonathan pour les mois pendant les quels nous avons partagé le bureau, Raouf pour le prélèvements « difficiles » que tu as récupéré, Farida pour ma première expérience d’encadrement, Emma et Meriem pour prendre le relai du projet Immumela...

Aurélie, je ne t’ai pas oubliée... tu m’as manqué cette année au labo!!! Un grand merci à toi parce que depuis le début tu m’as beaucoup aidée… je me rappelle encore la soirée que tu as passé avec moi à l’IGR pour améliorer ma demande de financement. Merci pour les longues et nombreuses journées de manips passées ensemble en L2, pour tes explications sur la bureaucratie française, pour ton support dans les moments difficiles, mais surtout pour ton amitié…Merci beaucoup!

A huge thanks to patients, for having accepted to be included in this study.

My sincere gratitude to all the medical and nursing staff of Bichat, Cochin, Curie and Foch Hospitals for their participation in this project: Dr E. Maubec, Dr E. Marinho, Dr L. Deschamps, Dr S. Albert, Dr C. Guedon, C. Deschamps, I. Scheer, Dr S. Jacobelli, Dr F. Boitier, Dr N. Franck, Dr I. Gorin, Dr N. Wallet-Faber, Pr N. Dupin, Pr B. Couturaud, Dr V. Fourchotte, Dr X. Sastre, Dr D. Mitilian.

A sincere thanks to all the collaborators with whom I have had the pleasure to work: Dr I. Cremer, Pr L. Zitvogel, Dr N. Delahaye, Dr S. Rusakiewicz, Pr N. Gervois, Dr E. Donnadieu, Dr H.-J. Garchon, Dr C. Capron, Dr A. Maresca, Dr C. Fauriat, Dr S. Caillat-Zucmann, Dr N. Rouas-Freiss.

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Gianfranco, un pensiero speciale per te. Grazie per avermi introdotto alle NK e per avermi incoraggiata a rimanere a Parigi.

Un grand merci à tous les membres présents et passés des équipes « Hosmalin » et « Lucas » et de la plateforme d’« Immuno-biologie », que j’ai eu le plaisir de connaitre et qui ont rendu agréable mon travail au 8ème étage du bâtiment G. Roussy. Charly, un remerciement particulier à toi. Merci de m’avoir initiée à la cytométrie 8 couleurs mais surtout de m’avoir dédié ton temps même pendant des moments très difficiles pour toi. Grazie ad Annalisa, per la tua allegria, per le serate trascorse insieme e per il pomeriggio in bateau.

Merci à Véronique et Magali, per avermi presentato Alessia, per le chiacchierate in italiano e i consigli sul post-doc. Alessia, é stato un piacere conoscerti! Un grosso in bocca al lupo per il proseguimento del tuo dottorato!

Merci aux autres doctorants et jeunes chercheurs que j’ai connu à Cochin: Pablo, Rania, Jérôme, Nathalie, Ana, Hélène, Quitterie, François, Florent… Raluca, merci pour ton amitié et pour les nombreux diners après le labo. Shufang, thank you for letting me training my english!

Maria, un remerciement particulier à toi. Merci pour ton amitié, pour tous les bons et les mauvais moments que nous avons partagé à Cochin et au dehors…de m’avoir conseillé Milos pour les vacances, trop bien ! J’espère que nos expériences de post-doc ne nous éloigneront pas trop ! Thanks to all the people who let me enjoy my stay in Paris!

Grazie alle persone con cui ho condiviso il « Leonardo », perché hanno contribuito a rendere unica la mia iniziale esperienza parigina.

All’Eugi, che nonostante le nostre comuni origini, il destino mi ha fatto incontrare qui a Parigi. Grazie per i tre anni che abbiamo condiviso e per l’amicizia che ne è nata.

Grazie ai “logici” che ho conosciuto, per le cene e le serate in compagnia: Giulio, Alberto, Laura, Giulio, Beniamino.

Un pensiero speciale va alle persone a me più care che ogni volta che rientro in Italia continuano a farmi sentire come se non fossi mai partita: Giuli, Matte, Lara, Luca, Chiari, Ele, Ia, Laura, Sara, Giaco, Marco, Jules, Biagio, Leo, Dani e alle espatriate come me: Iri e Gio.

Eli, grazie per la nostra amicizia, per come si è trasformata e rinnovata. Grazie per la dolce notizia che mi hai dato via mail perché non potevi aspettare… un grosso in bocca al lupo per la nuova vita che sta(i) per iniziare!

Eugi, con te ho condiviso i primissimi anni di laboratorio e tante altre esperienze che hanno fatto nascere e maturare la nostra amicizia. Grazie per i soggiorni qui a Parigi, per i weekend a sciare con Diego, e per aver sognato insieme l’organizzazione delle vacanze...chissà che l’estate prossima non si riesca a partire davvero? Grazie perché il sapore del nostro legame, delle nostre risate e lunghe chiacchierate non é mai cambiato.

Grazie alla mia numerosa famiglia, per la calorosa accoglienza che mi riservate ogni volta che torno. Un ringraziamento particolare va alle mie cugine Chiara e Raffaella e a Vania (ormai cugina acquisita), per i weekend in cui siete venute a trovarmi… e alla nonna Evolle, per gli ottimi pranzi domenicali che mi prepari ogni volta che torno.

Ai miei genitori e a mio fratello Matte. Grazie per avere sempre creduto in me e per essermi stati vicini nonostante la lontananza. Non ci sono parole per esprimervi la mia gratitudine.

Dulcis in fundo… a Mattia. Per tutto l’amore, l’appoggio e la pazienza che mi hai dimostrato in

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Abstract

Natural Killer cells are cytotoxic lymphocytes involved in the immune response against tumours and infections.

We investigated the NK-mediated functions in response to clear-cell renal cell carcinoma (RCC) and metastatic melanoma, two human immunogenic tumours. We showed that certain

VHL mutations increased RCC cell susceptibility to NK lysis. VHL loss of function correlated

with lower expression levels of membrane HLA-I molecules on VHL-mutated RCC and a decreased triggering of inhibitory NK receptors compared to RCC with a functional VHL. In stage IV melanoma patients, we showed that blood NK cells displayed a unique NKp46dim/NKG2Adim phenotype and high lytic potential towards melanoma cells. Following chemotherapy, NK cell function was reduced and the phenotype modulated. To study melanoma-infiltrating NK cells, we have set up experimental conditions to characterise NK cells in metastatic LNs from stage III melanoma patients. Our preliminary data show that, compared to normal LNs, NK cells from metastatic LNs are altered.

Our findings suggest that oncogenic-dependent immunogenicity, tumour-associated NK alterations and chemotherapy are important factors that must be taken into account in the choice of immunotherapeutic protocols based on NK cells.

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Résumé

Les cellules Natural Killer (NK) sont des effecteurs cytotoxiques impliqués dans la réponse immune contre les infections et les tumeurs.

Pendant ma thèse j’ai étudié la fonctionnalité des cellules NK humaines en réponse à des lignées cellulaires de carcinome rénal à cellules claires (RCC) et de mélanome métastatique, deux tumeurs immunogènes. Nos résultats montrent que certaines mutations de VHL augmentent la susceptibilité des lignées RCC à la lyse NK. La perte de fonction de VHL corrèle avec une expression membranaire diminuée des molécules HLA-I par les lignées RCC mutées pour VHL. Chez les patients atteints de mélanome métastatique de stade IV, nous avons décrit un phénotype particulier des NK sanguines (NKp46dim/NKG2Adim) qui leur confère une forte activité antitumorale. Après traitement des patients par chimiothérapie, la fonctionnalité NK était réduite et le phénotype modifié. Pour étudier les cellules NK infiltrant les mélanomes, nous avons mis au point des conditions expérimentales pour caractériser les cellules NK de ganglions métastatiques de patients de stade III. Nos résultats préliminaires montrent que, par rapport aux ganglions sains, les NK des ganglions métastatiques présentent un phénotype altéré et un potentiel fonctionnel diminué.

Nos résultats suggèrent que d’une part l’immunogénicité dépendante des oncogènes et d’autre part les altérations NK induites par la tumeur et/ou par la chimiothérapie sont des facteurs importants à considérer dans le choix des protocoles d’immunothérapie basés sur les cellules NK.

Mots clés : cellules NK, mélanome métastatique, carcinomes rénaux, immunogénicité des

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Abbreviations

5-FU: 5-fluorouracil

ADC: adoptive cell therapy

ADCC: antibody-dependent-cell-cytotoxicity AICL: activation-induced C-type lectin AJCC: American Joint Committee on Cancer ALL: acute lymphoblastic leukaemia

AML: acute myeloid leukaemia AN: absolute numbers

APC: antigen presenting cell

BAT3: HLA–B-associated transcript 3 BiMAb: bispecific monoclonal antibody BM: bone marrow

CD: cluster of differentiation

CLA: cutaneous lymphocyte-associated antigen CLP: common lymphoid progenitors

CML: chronic myelogenous leukemia CRC: colorectal carcinoma

CSC: cancer stem cell

CTC: circulating tumour cells CTL: cytotoxic T lymphocytes

CTLA4: cytotoxic T-lymphocyte–associated antigen 4 DNAM-1: DNAX accessory molecule-1

dNK: NK cells in maternal deciduas DTIC: dacarbazine

EBV: Epstein-Barr Virus

EGFR: epidermal growth factor receptor ELP: early lymphoid precursors

FCS: foetal calf serum FcRI: Fc receptor I 

FDA: Food and Drug Administration FGFR1: fibroblast growth factor receptor 1 Flt3L: fms-like tyrosine kinase-3 ligand GIST: gastrointestinal stromal tumours

GM-CSF: granulocyte-macrophage colony-stimulating factor GVHD: graft versus host disease

GvL: graft-versus-leukaemia HA: hemagglutinin antigens HCMV: human cytomegalovirus HIF: hypoxia inducible factor HLA: human leukocyte antigen HPC: hematopoietic precursor cells

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HSC: hematopoietic stem cells

HSCT: hematopoietic stem cell transplantation HSPG: heparan sulphate proteoglycans

ICs: immunocytokines

ICAM-1: intracellular adhesion molecule-1 IFN-b: interferon alpha-b

IFN:interferon-

Ig: immunoglobulin

ILT: immunoglobulin-like transcripts iNK: immature NK-cells

ITAM: immunoreceptor tyrosine-based activating motif ITIM: immunoreceptor tyrosine-based inhibitory motif KIRs: Killer Immunoglobulin-like receptors

KLRB1: Killer cell lectin-like receptor subfamily B member 1 LAMP-1: lysosomal-associated-membrane protein-1

LDH: lactate-dehydrogenase

LFA-1: lymphocyte function associated-antigen 1 LIF: leukaemia inhibitory factor

LIRs: leukocyte Ig-like receptors

LMP2: latent membrane protein 2 of Epstein-Barr virus LN: lymph node

LRC: leukocyte receptor complex mAb: monoclonal antibody MCA: methylcholanthrene

MCMV: murine cytomegalovirus mDC: myeloid DC

MFI: mean flouorescence intensity MHC: major histocompatibility complex MICA: MHC-class I-related chain A

MIP-1:macrophage inflammatory protein-1

MIRs: macrophage Ig-like receptors MLTA: malignant lung tissue area mRCC: metastatic RCC

N-CAM: neural cell adhesion molecule NCR: natural cytotoxicity receptors NHL: non-Hodgkin’s lymphoma NK: Natural Killer

NKP: NK cell precursors

NSCLC: non small cell lung cancer o/n: overnight

PB: peripheral blood

PBMC: peripheral blood mononuclear cells PC5: PE-Cy5

PC-PLC: phosphatidylcholine-specific phospholipase C pDC: plasmacytoid DC

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PDGF: platelet-derived growth factor PI3K: phosphatidylinositol 3-kinase PMA: phorbol 12-myristate 13-acetate pro-NK: NK cell progenitors

Pt: patient

PVR: poliovirus receptor

Rag: recombination-activating gene RCC: renal cell carcinoma

rhuIL2: recombinant human interleukin 2

RITA: Reactivation of p53 and Induction of Tumour cell Apoptosis RT: room temperature

SCF: stem cell factor sHLA: soluble HLA

siRNA: small interfering RNA SLT: secondary lymphoid tissue

STAT1: signal transducer and activator of transcription factor-1 STRA13: stimulated by retinoic acid-13

SRRs: SLAM-related receptors TCR: T-cell receptor

TGFtransforming growth factor 

TIL: tumour infiltrating lymphocytes TNFtumour necrosis factor-

TNM: tumour node metastasis TNM: tumour node metastasis

TRAIL: TNF-related apoptosis-inducing ligand Treg: regulatory T cells

UCB: umbilical cord blood

UC-MSC: umbilical cord mesenchymal stem cells

UISS: University of California Integrated Staging System ULBP: UL16-binding proteins

VEGF: vascular endothelial growth factor VHL: von Hippel Lindau

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List of figures and tables

Figure 1. Flow cytometry characterization of CD3-CD56+ NK cells from blood ... 5

Figure 2. Natural cytotoxicity ... 8

Figure 3. NK cell receptors ... 15

Figure 4. Pattern of expression of human NK cells during maturation ... 26

Figure 5. Model of NK development in secondary lymphoid tissues ... 28

Figure 6. Models proposed to explain the process of NK cell education ... 32

Figure 7. NK cell education in bone marrow and “re-education” in periphery ... 34

Figure 8. NK and DC interactions at the site of inflammation... 40

Figure 9. Proposed model for NK cell memory: generation, maintenance and reactivation ... 43

Figure 10. NK cell-based cancer immunotherapy ... 51

Figure 11. Control of HIF complex by VHL... 56

Figure 12. Gating procedure of immune cells on LN cell suspensions ... 102

Figure 13. Characterization of paired blood and LN NK cells in stage III melanoma patients . 104 Figure 14. Proportion of immune cells (CD45+) in LNs from donors and patients ... 105

Figure 15. Comparison of NK phenotype in LN from patients and donors ... 107

Figure 16. Functional potential of LN NK cells ... 108

Table 1. Killer Ig-like receptors ... 22

Table 2. TNM Staging Categories for Cutaneous Melanoma ... 59

Table 3. Characteristics of Stage III melanoma patients ... 99

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1

PREAMBLE

Tumorigenesis is a multistep process that allows cancer cells to gain functional capabilities enabling their growth and dominance in a local tissue environment. Resistance to apoptosis, survival, proliferation, and dissemination are among the characteristic features acquired by different tumour types via distinct sequential mechanisms and at various time during tumour progression. Genomic instability and random mutations endow cancer cells with these specific properties and are essential hallmarks for tumour development and progression.

Several clinical evidences suggest that immune system plays an important role in tumour control. On one hand, in immunocompromised individuals, the risk to develop tumours is higher than in immunocompetent individuals. On the other hand, patients displaying high tumour infiltration by immune cells have a better prognosis than patients exhibiting poor infiltration by cytotoxic lymphocytes (Pages et al., 2010).

Along tumour progression, certain cancer variants acquire an advantage through the selection of cells expressing low levels of molecules recognised by immune cells (immunoediting) or through their ability to counteract and anergize the immune response (immunosuppression). Thus, mechanisms of immune escape favour the survival and development of tumours and are now considered as emerging hallmarks in cancer biology (Hanahan and Weinberg, 2011). In the last decades, different immunotherapeutic approaches have been developed aiming at boosting and/or recovering an efficient immune response against tumour cells. Some complete and objective responses have been obtained in patients resistant to conventional treatments indicating that immunotherapy is a promising strategy for the cure of cancer patients (Pardoll, 2011).

The anti-tumour effect of the immune system relies on the activation of cytotoxic lymphocytes. Among immune cells, Natural Killer (NK) are cytotoxic effectors participating in the immune response against tumours. Therapies based on these cells could constitute a valuable approach for the adjuvant treatment of cancers. However, for a rational usage of such NK-based immunotherapies, it is important to characterise the biology of NK cells and to determine the relevant parameters that modulate and interfere with their capacity to lyse cancer cells. It is known that NK activation depends on the intricate balance between activating and inhibitory signals derived from receptors that recognise ligands on tumour cells. Thus, the functional status of NK cells from cancer patients and the expression of ligands by tumour cells are important factors to consider.

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2 During my thesis, I investigated several mechanisms involved in the recognition and lysis of two immunogenic tumours by NK cells. Renal cell carcinoma (RCC) and melanoma are highly metastatic cancers responsive to immunotherapy by IL2 and IFN. For these tumours, new treatments targeting tumour-related signalling pathways are currently developed, showing higher rates of responses than conventional treatments by chemotherapy and radiotherapy. However, only few complete or sustained responses are achieved and adjuvant immunotherapy remains a valuable strategy to ameliorate and enhance the treatment of these cancers.

In the first part of my work, I analysed the role of VHL mutation (a frequent and crucial oncogenic event in RCC development) on RCC susceptibility to NK lysis. In the second part, I characterised the phenotype and function of circulating NK cells from stage IV melanoma patients in relation with the presence of the tumour and with the treatment of patients by chemotherapy. Finally, to further explore the role of melanoma on NK modulation, I set up experimental conditions to study the phenotype and function of NK cells in regional metastatic lymph nodes, a crucial site for melanoma dissemination. Preliminary results on NK cells from metastatic lymph nodes are presented.

Our findings on these two models of immunogenic tumours give insights into certain important factors that need to be considered for more efficient NK-based therapeutic strategies.

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3

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4

1. NATURAL KILLER CELLS (NK)

1.1. Introduction

In the beginning of the 1970s a new lymphocyte subpopulation has been described in mice for its spontaneous cytotoxic capacity against tumour cells without prior sensitization and without MHC (Major histocompatibility complex) restriction. These cells were named “Natural Killer” cells or, in brief, “NK” cells (Herberman et al., 1975; Kiessling et al., 1975).

Natural Killer cells have been defined as large granular lymphocytes of innate immunity because, unlike T and B lymphocytes, they do not rearrange T-cell receptor or Immunoglobulin genes from their germline configuration. However, due to the recent evidences on NK cell biological functions traditionally attributed to adaptive immunity like NK “memory” (O'Leary et

al., 2006; Sun et al., 2009), the rigid classification of Natural Killer cells in innate immunity is

currently discussed (Sun et al., 2011; Vivier et al., 2011).

Natural Killer cells participate in anti-tumour and anti-viral immune host defence being efficient for the elimination of cancer and infected cells. They also display immunomodulatory functions through secretion of cytokines, like Interferon-IFNand Tumour Necrosis Factor- (TNF), and chemokines. The activation of NK cells depends on an intricate balance between activating and inhibitory signals that determines if a target will be susceptible to NK-mediated lysis. To “see” and discriminate between normal and transformed cells, NK cells express activating and inhibitory membrane receptors that recognise ligands at the surface of target cells. The interactions between NK receptors and their ligands are complex and remain partially understood.

NK cells represent 5-20% of circulating lymphocytes, but they are widely present in other tissue compartments. In particular, they are abundant in secondary lymphoid tissues (lymph node, spleen, tonsils) and in inflamed tissues (Gregoire et al., 2007).

Among the lymphocyte population, they are defined by the positive expression of CD56 and the lack of expression of the T cell receptor and the CD3 complex (Figure 1). CD56 expression is not restricted to NK cell population: it represents the 140 kDa isoform of the neural cell adhesion molecule (N-CAM), and it is also expressed by a T cell subtype and some cancer cells (Lanier et al., 1989). Although this molecule is used for human NK cell phenotypic definition and selection, its role on NK cell function is still unknown. The expression of another

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5 NK cell surface molecule, the low-affinity receptor for the Fc portion of IgG CD16 (FcRIIIA), is also used to define NK cells; like CD56, its expression is not restricted to these cells. The definition of NK cells by a unique marker is still debated. Vivier and colleagues proposed the Natural Cytotoxic Receptor NKp46 as the “NK signature” through the species (Walzer et al., 2007). However, a cytotoxic T cell subpopulation expressing NKp46 has been found (Meresse et

al., 2006) and it also exists a minority of CD3-CD56+ NK cells not expressing this molecule. Nowadays, no membrane molecule has been found to be selectively and exclusively expressed by all Natural Killer cells.

Figure 1. Flow cytometry characterization of CD3-CD56+ NK cells from blood

CD56dim are the dominant NK cell subset, while CD56bright represent about 10% of NK cells in healthy donors.

According to the density of CD56 and CD16 surface markers, two subpopulations of NK cells are defined in humans: the CD56dim population, expressing low amount of the CD56 molecule but highly expressing the CD16, and the CD56bright NK population, that highly expresses the neural cell adhesion molecule and lacks, or faintly expresses, the FcRIIIA (Figure

1). This phenotypic distinction between the two NK cell subtypes also reflects different functions

and maturation state. The CD56dim population represents the terminally mature and classical cytotoxic NK cell subset. In contrast, CD56bright NK cells are considered a less mature CD56dim -precursor, they produce high amount of cytokines, like IFN and TNF thus playing a primary role in immune regulation and only exert marginal cytotoxic activity. Interestingly, a CD56 -CD16+ NK subpopulation, rare in healthy individuals, has been found expanded in HIV patients (Mavilio et al., 2005).

The distribution of CD56dim and CD56bright subsets is different according to the organ (spleen, tonsil, lymph nodes, etc...); in blood, the CD56dim subpopulation is dominant and represents 90% of NK cells (Poli et al., 2009).

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1.2. NK cell function

1.2.1. Effector function

Natural killer cells are able to lyse virus-infected and transformed cells without any priming and are particularly activated towards targets that display low levels of major histocompatibility complex (MHC) class I molecules via two main cell-to-cell contact dependent mechanisms: the Antibody Dependent Cell Cytotoxicity (ADCC) and the Natural Cytotoxicity.

The binding to a target cell is accompanied by the formation of a complex structure at the cell–cell interface, named immunological synapse. Following the recognition of a target cell, NK cytotoxic granules containing perforin (a membrane-disrupting protein) and granzymes (serine proteases) are secreted by a Ca2+-dependent exocytosis in the immunological synapse. This mechanism is also known as degranulation. Perforin polymerizes and forms a transmembrane pore that allows the delivery of granzymes to the cytosol where they cleave and activate different caspases leading to the apoptosis of the target cell (Trapani and Smyth, 2002). Lining the membrane of the lytic granules is the lysosomal-associated-membrane protein-1 (LAMP-1, or CD107a). Alter et al., demonstrated that the up-regulation of this molecule on NK cell membrane is a marker of NK cell activation and strongly correlates with both cytokine secretion and NK cell-mediated lysis of target cells (Alter et al., 2004).

Perforin is very important for the clearance of tumour cells. Interestingly, perforin deficient mice exhibit increased sensitivity to methylcholanthrene (MCA)-induced fibrosarcomas (van den Broek et al., 1996). Otherwise, mice deficient for a single granzyme protein displayed a phenotype similar to wild-type mice, probably due to function redundancy of granzyme isoforms.

NK cell cytotoxicity can be mediated by another mechanism that involves the direct binding between NK cell molecules belonging to the Tumour Necrosis Factor (TNF) family, like FasL and the soluble TNF-related apoptosis-inducing ligand (TRAIL), and their receptors expressed by target cells (Fas, and TRAIL-R). This second pathway is less implicated in the host defence against pathogens in vivo but is important for the elimination of auto-reactive lymphoid cells and homeostasis. Fas (CD95) is expressed by numerous cells and belongs to the TNF receptor family. This receptor contains a conserved intracytoplasmic “death domain” that, following the interaction with Fas-L, indirectly activates the cleavage of caspases and induces apoptosis. Two additional members of this family, DR4 (TRAIL-R1) and DR5 (TRAIL-R2), also transduce apoptotic signals upon TRAIL binding.

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7

1.2.1.1. Antibody dependent cellular cytotoxicity (ADCC)

ADCC is an important immune effector mechanism by which antigens of tumour or infected cells coated by IgG antibodies are recognised by Fc receptors present on effector cells. Thus, antibodies mediate contacts between effector and target cells allowing the subsequent killing of target cells.

Natural Killer cells are one of the major effector cells involved in this mechanism due to their expression of CD16, the FcRIIIA. In particular, CD56dim NK cells, highly expressing this receptor, are more implicated in ADCC than CD56bright NK cells. The interaction between CD16 and its ligand induces NK cell degranulation with the exocytosis of perforin and granzymes at the immune synapse.

This mechanism has been well studied and is now exploited in mAb-based cancer immunotherapy. The production of antibodies directed against specific antigens overexpressed by tumour cells is an active field in immunotherapy. Trastuzumab and Rituximab are two humanized monoclonal antibody agents used to treat breast cancer and non-Hodgkin’s lymphoma respectively. Trastuzumab interferes with the p158HER-2/neu oncoprotein, while Rituximab is directed against CD20 on B cells, both inducing apoptosis and tumour inhibition. Beside these direct effects, in a mouse experimental model it has been shown that the engagement of Fc receptors on effector cells plays a dominant role in the in vivo anti-tumour activity of these antibodies (Clynes et al., 2000). This work also showed that, in monocytes and macrophages, ADCC is controlled by the inhibitory FcRIIB receptor, suggesting that, for an optimal anti-tumour activity, antibodies have to bind preferentially to the activating receptor FcRIIIA. NK cells do not constitutively express FcRIIB.

Moreover, CD16 polymorphism can influence the efficacy of antibody therapy. In particular, it has been shown that the therapeutic response of Rituximab is more efficient in patients homozygous for the FcRIIIA/158V allotype than in FcRIIIA/158F carriers, in agreement with a higher affinity for IgG1 and an increased ADCC in vitro (Cartron et al., 2002). Recently, it has also been shown that the strong ADCC in patients carrying the FcRIIIA/158V allotype is due to the higher CD16 expression at NK cell membrane (Hatjiharissi et al., 2007).

1.2.1.2. Natural Cytotoxicity

Natural cytotoxicity refers to the capacity of Natural Killer cells to lyse a target cell without prior sensitization and without antibody-mediated recognition.

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8 In 1990, Klas Karre proposed “the missing self” hypothesis to explain how NK cells are capable to lyse target cells lacking or displaying low expression of MHC class I molecules (Ljunggren and Karre, 1990). This theory derived from the numerous experimental observations that tumour cells deficient for MHC class I molecules were efficiently lysed by NKs, conferring them a role in the control of NK cell activation. The missing self hypothesis was proposed before the discovery of the receptors implicated in the recognition of MHC-I molecules (KIRs, CD94/NKG2 and ILT). The last two decades have brought experimental confirmations of the hypothesis and new findings to better understand NK cell regulation. Through the identification of activating and inhibitory NK cell receptors, the missing self theory has evolved. It is currently accepted that NK cell activation depends on an intricate balance between positive and negative signals derived from membrane receptors (Figure 2). In particular, it is now known that a lack of inhibition per se is not sufficient to induce NK activation and that a positive signal is always needed, thus explaining why some cells could be targeted by NK cells despite the presence of high amount of MHC class I molecules (Leibson, 1997).

Figure 2. Natural cytotoxicity

General diagram recapitulating the interplay between activating and inhibitory receptors in the regulation of NK cell activation in presence of normal and transformed cells.

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9 Early studies on resting circulating NK cells showed that the CD56dim subtype is more cytotoxic against tumour cells than CD56bright. After in vitro stimulation with IL2, CD56bright cells show a higher proliferating potential and acquire a cytotoxic activity comparable to that of CD56dim cells (Nagler et al., 1989).

1.2.2. Cytokine secretion

Natural Killer cells are an important source of cytokines and chemokines. They thus display immunoregulatory functions in the innate immune response within inflamed tissues and control the induction and amplitude of the adaptive immune response in secondary lymphoid organs.

NK cells produce high amount of cytokines in response to monokine stimulation, such as IL12, IL15 and IL18, three proinflammatory and immunomodulatory cytokines produced by activated macrophages in response to infection (Trinchieri, 1995; Dinarello et al., 1998; Fehniger and Caligiuri, 2001).

IFN is the most abundant cytokine produced by NK cells and plays multiple immunoregulatory functions: it is critical for innate and adaptive immune responses against viral and intracellular bacterial infections and it is involved in tumour control enhancing the immunogenicity of cancer cells and stimulating the immune response against transformed cells. In addition, IFN upregulates both MHC class I and class II expression. It also contributes to macrophage activation increasing phagocytosis and priming the production of proinflammatory cytokines. Moreover, it controls cellular proliferation and apoptosis and the differentiation of naïve CD4 T cells into Th1 effectors (Schoenborn and Wilson, 2007).

NK cells also secrete TNF TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL5, IL8, IL10, macrophage inflammatory protein-1 (MIP-1MIP-1 and IL13 (Smyth et al., 1991; Warren et al., 1995; Bluman et al., 1996; Cooper, Fehniger and Caligiuri, 2001).

A different pattern of cytokine secretion is observed in CD56bright and CD56dim NK subsets in resting state or in response to in vitro stimulation by different recombinant monokines: IL12, IL15, IL18, and IL1 alone and in combination with IL12 or IL15. In particular, freshly isolated CD56bright cells are the principle source of cytokines and chemokines, whereas CD56dim subset produces negligible amounts of cytokines (Cooper, Fehniger, Turner et al., 2001).

Furthermore, under the in vitro stimulation by phorbol esters [e.g. phorbol 12-myristate 13-acetate (PMA)] and ionomycin, mediating a monokine receptor independent activation, the

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10 production of cytokines was still higher in CD56bright NK cell subset. In 2005, Trotta et al. provided a first molecular explanation of the distinct function of the two NK cell subsets under monokine stimulation. They showed that the production of IFN is regulated in vitro and in vivo by the phosphatase SHIP1 whose expression is higher in CD56dim cells (Trotta et al., 2005).

The CD56bright NK subset generally needs two signals to produce IFN, and one of these almost always includes IL12. The second can be IL1, IL2, IL15 or IL18, or the engagement of an NK activating receptor such as CD16 or NKG2D (Bryceson et al., 2006). Interestingly, the production of chemokines by CD56bright cells is dependent on the precise monokine combination used for the stimulation. For example, while the optimal stimulus for IFNγ production is “IL12 plus IL18”, the combination “IL15 plus IL18” results in optimal production of GM-CSF, whereas “IL12 plus IL15” induces the highest levels of IL10, MIP-1MIP-1and TNF. These data suggest that the quality and the quantity of monokines present at the site of infection are important in determining the cytokine response of CD56bright NK cells (Fehniger et al., 1999).

Whereas numerous studies investigated the secretion of cytokines and chemokines by NK cells under monokine stimulation, less is known about the secretion of cytokines by NK cells following target cell recognition. Surprisingly, Fauriat et al. recently reported that, conversely to the response to monokine stimulation, the CD56dim NK subtype was the main producer of IFN TNF, MIP-1 and MIP-1 following stimulation by K562 cells, a classical NK target (MHC class I negative) (Fauriat, Long et al., 2010). Furthermore, using a model of Drosophila cells (S2) expressing single or combined NK receptors, they found that the unique engagement of CD16, NKG2D or 2B4 was sufficient to induce a rapid secretion of chemokines (MIP-1 and MIP-1, while TNF and IFN production occurred later and required the simultaneous engagement of multiple receptors. Strikingly, no production of IL5, IL10, IL13 or GM-SCF was found following K562 stimulation.

Thus, the secretion of cytokines and chemokines is highly regulated in NK cells and is dependent on the type of stimulation: monokine-dependent or target-induced.

1.2.3. Proliferation

Unlike T and B cells, NK cells do not undergo sustained proliferation in vitro and, as for the other NK cell functions, differences could be appreciated between the two NK cell subsets in response to proliferative stimuli.

CD56bright cells express the high affinity IL2receptor (IL2R and expand tenfold more than CD56dim subset upon stimulation with low-doses (pM) of IL2. CD56dim cells,

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11 expressing the intermediate affinity IL2 receptor (IL2R), exhibit only low expansion rate even in response to high doses of IL2 treatment (nM) but exert enhanced cytotoxity. At high concentrations (nM), IL15 also induces signal through the IL2Rreceptor and is implicated in CD56bright proliferation (Carson et al., 1994). Thus, IL2 or IL15 alone are sufficient to induce NK cell proliferation but their effect can be enhanced by other cytokines or stimuli (Robertson et al., 1993). In particular, the presence of target cells improves the IL2-induced proliferation of NK cells (Baume et al., 1992). At low concentrations (0.5 to 5 ng/mL), IL10 also significantly augments the IL2-induced proliferation of CD56bright NK via the high-affinity IL2 receptor (Carson et al., 1995).

Various methods have been developed to improve NK cell differentiation/proliferation and to evaluate the role of expanded and activated NK cells in cancer cell therapy. In presence of cytokines, irradiated umbilical cord mesenchymal stem cells (UC-MSC) efficiently induce cord blood NK cell expansion (Boissel et al., 2008). Fujisaki et al. showed that a high in vitro expansion (21.6 fold) of NK cells could be obtained by a seven days co-culture of PBMC (peripheral blood mononuclear cells) with an irradiated K562 leukaemia cell line genetically modified to express a membrane-bound (mb) form of IL15 and 41BB ligand (K562-mb15-41BBL). In this condition, NK cell proliferation was superior to that induced with IL2, IL15, IL12 and/or IL21 and did not result in T cell expansion. These NK cells were also more cytotoxic (Fujisaki et al., 2009). Combined activation through NKp46 and CD2 receptors by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days. Furthermore, Somanchi et al. recently described rapid NK proliferation without senescence using K562 cells expressing membrane-bound IL21; a 21000-fold expansion of NK cells was achieved in 21 days (Somanchi

et al., 2011). Surprisingly, in mice, genetically reprogrammed NK-like cells have been obtained

from a thymocyte precursor (DN3, CD4 CD8 double-negative T cell precursor undergoing TCR gene rearrangement and TCR-dependent selection) upon the deletion of the T cell specific transcription factor bcl11b. These induced-T to Natural Killer (ITNK) cells displayed phenotype and function comparable to conventional NK cells. Moreover, they were characterised by a high proliferative potential: starting from one DN3 thymocyte 500000 ITNK cells were obtained upon IL2 stimulation (Li et al., 2010). Due to the remarkable expansion of these NK-like cells, this approach displays fascinating potential for translation in humans and subsequent clinical application in cell-based therapies. Nonetheless, the usage of genetically reprogrammed cells is potentially dangerous and several investigations are required to assess the safety of their infusion in humans.

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12

1.3. NK cell receptors and ligands

The activation of NK cell function depends on an intricate balance between positive and inhibitory signals derived from receptors. The next paragraphs describe the activating and inhibitory receptors involved in this process.

1.3.1. ADCC receptor: CD16

CD16 is a type I transmembrane receptor also known as FcRIIIA and it contains two extracellular immunoglobulin-like (Ig-like) domains. As mentioned before, CD16 is the low-affinity receptor of the Fc portion of immunoglobulin G (IgG1) involved in the lytic process ADCC. The cross-linking of CD16 on NK cells results in signal transduction via the Fc receptor I  (FcRI) and T-cell receptor (TCR) chains with the subsequent increase of intracellular Ca2+, leading to NK cell degranulation (Vivier et al., 1991).

In addition to its role in ADCC, CD16 exerts a direct cytotoxic function through the binding of appropriate ligands on target cells (Mandelboim et al., 1999). Even more, the engagement of CD16 alone was sufficient to activate antibody-dependent redirected lysis in resting NK cells, while all other receptors tested (NKp46, NKG2D, 2B4, DNAM-1, or CD2) required the engagement of another receptor for NK cell activation (Bryceson et al., 2006).

CD16 is highly expressed by circulating NK cells, especially by the CD56dim subset (Figure 1). A low expression is found in tissue NK cells where the CD56bright subset is dominant. CD16 expression is regulated by the phosphatidylcholine-specific phospholipase C (PC-PLC). The direct engagement of the receptor or the in vitro stimulation of NK cells by PMA induce the shedding of CD16 from NK cell membrane (Masilamani et al., 2009).

1.3.2. Natural cytotoxicity receptors (NCR)

Three NK cell triggering surface receptors that display a critical role in the induction of NK cell-mediated non-MHC-restricted cytotoxicity of tumour and transformed cells have been identified. These important activating receptors for NK cell lytic function are named “Natural Cytotoxicity Receptors” (NCR). The cross-linking of NCR with specific mAb strongly increase the cytolytic activity in redirected killing assays, whereas blocking of NCR by mAbs inhibits NK cell cytotoxicity against most target cells. Furthermore, NCR surface density on NK cells correlates with the magnitude of cytotoxicity against NK-susceptible target cells (Sivori et al., 2000; Biassoni et al., 2001; Moretta et al., 2001).

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13

1.3.2.1. NKp46 (NCR1)

NKp46 is a 46 kDa molecule, member of the Ig superfamily and characterised by two C2-type Ig-like domains. The NKp46 gene is located on chromosome 19 and the protein is expressed by resting and activated NK cells. For the signal transduction, NKp46 is associated with the TCR  chain bearing a tyrosine-based activating motif (ITAM) (Figure 3). The downstream signalling leads to the phosphorylation of Lck and Zap70 (Mandelboim and Porgador, 2001).

NKp46 expression was initially described to be restricted to NK cells. However, in the last years its expression has been identified in intra-epithelial T cells from intestine of celiac patients, in cultured human umbilical cord blood CD8+ T cells, and more recently on a minute fraction of CD3+CD56+ cells in blood of healthy individuals and augmented in leukaemia patients (Meresse et al., 2006; Tang et al., 2008; Yu et al., 2011).

Except for the hemagglutinin antigens (HA) expressed on virus-infected cells, potential specific ligands of NKp46 on malignant cells are yet unknown (Figure 3), even if they might exist considering that NKp46 has been involved in the recognition of several malignancies (Mandelboim et al., 2001; Eisenring et al., 2010). In addition to its role in tumour eradication by NK cells and cytokine secretion, NKp46 has also been recently involved in the process of tumour immunoediting (Elboim et al., 2010).

1.3.2.2. NKp44 (NCR2)

NKp44 is a 44 kDa molecule belonging to the Ig superfamily. In contrast to other NCR receptors, NKp44 is associated with a different signal-transducing polypeptide, KARAP/DAP12 (Vitale et al., 1998) (Figure 3). NKp44 was first described to be exclusively expressed after NK cell activation and not by resting NK cells.

Like NKp46, NKp44 binds to viral hemagglutinins for the recognition of infected cells (Arnon et al., 2001). In 2005, it was shown that the expression of a NKp44 ligand was induced in CD4+ T cells from HIV patients by a gp41 peptide and correlated with the progressive reduction of CD4+ T cell number during HIV infection (Vieillard et al., 2005). In 2007 and for the first time, NKp44 was found involved in the direct recognition of bacterial pathogens by NK cells (Esin et al., 2008). Concerning tumour recognition, it was demonstrated that NKp44 binds to tumour cells in a heparan sulphate proteoglycans (HSPG)-dependent manner (Hershkovitz et

al., 2007). Expression of NKp44 ligands is variable on tumour cells and seems to be linked to

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14 susceptibility to NK cell-mediated lysis, and is reduced in cells arrested in G2/M phase (Byrd et

al., 2007).

Recently, a new NK cell subset named NK-22 has been discovered in mucosa-associated lymphoid tissues (tonsils, not lymph nodes) to constitutively express NKp44 and produce IL22 (Cella et al., 2009). This unique NK cell subset will be described more precisely in the “Natural Killer cell compartments” section.

1.3.2.3. NKp30 (NCR3)

NKp30, the last NCR discovered, is a 30 kDa molecule and, like NKp46, is expressed by resting and activated NK cells. It belongs to Ig superfamily and is associated with the TCR  chain (Pende et al., 1999) (Figure 3).

NKp30 is involved in the NK-mediated recognition and lysis of multiple tumour cell lines and primary tumour cells (carcinomas, neuroblastomas, and myeloid and lymphoblastic leukemias) but NKp30 ligands expressed by tumour cells are poorly defined. Recently, a new member of the B7 family has been described to be a NKp30 ligand restricted to malignant tissues, B7H6 (Brandt et al., 2009). Only few years before, two ligands binding to NKp30 but not involved in the recognition of tumour cells were reported: the HLA–B-associated transcript 3 (BAT3), a nuclear protein released upon heat shock treatment, and pp65, a cytomegalovirus tegument protein (Arnon et al., 2005; Pogge von Strandmann et al., 2007).

In addition to its role in triggering anti-tumour NK cytotoxicity and in cytokine secretion, NKp30 was found to be important in NK-DC interactions in tissues. NK cells can recognise and kill immature DCs through NCR3, while mature DCs can modulate NK cell proliferation and activation via this receptor (Ferlazzo et al., 2002). The expression of NKp30 is downregulated by TGF1, thus affecting both anti-tumour and DC-lytic NK cell functions (Castriconi et al., 2003).

NCR3 gene is located on chromosome 6 in the highly polymorphic telomeric end of HLA class III region. Different splice variants are transcribed and three isoforms of NKp30 (NKp30 a b or c) can be expressed at the NK membrane depending on which isoform of exon 4 is translated (Neville and Campbell, 1999). Recently, it has been shown that the three isoforms exert different functions. NKp30a and NKp30b are immunostimulatory: NKp30a is the only isoform triggering cytotoxicity, while both a and b isoforms stimulate Th1 cytokine release. In contrast, NKp30c transfectants exert immunosuppressive functions inducing the production of significant amount of IL10 upon co-cultures with B7H6 tumour transfectants or iDC.

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15 Furthermore, the NKp30c isoform is preferentially expressed in GIST (gastrointestinal sarcoma) patients and correlates with a negative prognosis (Delahaye et al., 2011).

Figure 3. NK cell receptors

Main activating and inhibitory receptors implicated in NK cell triggering and inhibition. Molecules and domains involved in activating signalling are in green; those inducing inhibitory signals in red.

Adapted from: (Moretta, Bottino et al., 2006)

1.3.3. C-type lectin-like NKG2 receptor superfamily

Natural-Killer group 2 (NKG2) receptors belong to the C-type lectin-like receptor superfamily, i.e. receptors containing a carbohydrate-binding protein domain, known as a lectin, and requiring calcium for binding (C-type). Activating and inhibitory NK receptors belonging to this family have been identified. Their expression is not restricted to NK cells and they are also expressed by T cell subsets.

1.3.3.1. CD94/NKG2 heterodimers

NKG2A and its four molecular variants B, C, E, and H have been shown to bind to the transmembrane-anchored glycoprotein CD94 forming disulfide-linked heterodimers. This association is important for the translocation of NKG2 receptors to cell surface and for ligand recognition. NKG2 receptors share a common structure composed of a C-type lectin-like domain, a transmembrane domain and a cytoplasmic segment. According to the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains and to their binding with adaptor molecules, they were classified as inhibitory or activating receptors. NKG2 and CD94 genes are located in a region of chromosome 12 known as “Natural Killer

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16 complex”. NKG2A and the alternative spliced form B contain two ITIM motifs, form a heterodimer with CD94 molecule and are thus inhibitory receptors. After interaction with the ligand, the tyrosine residue in each ITIM domain is phosphorylated leading to the recruitment and activation of SHP-1 and SHP-2 tyrosine phosphatases and finally blocking the NK cell activation cascade (Borrego et al., 2005; Lieto et al., 2006) (Figure 3). In contrast, NKG2C, E and H variants lack ITIM domains and are associated with the ITAM-bearing DAP12 adaptor molecule. Thus, CD94/NKG2C (E) molecules form heterodimeric activating receptors (Borrego

et al., 2006) (Figure 3).

All CD94/NKG2 receptors are specific for the non-classical MHC class I molecule HLA-E (Figure 3). The affinity of NKG2 receptors with ligands was investigated and seemed to be different for each receptor: in particular, CD94/NKG2A displayed a higher affinity for HLA-E molecules compared to CD94/NKG2C. Interestingly, binding affinity was dependent on the peptide sequence and not on HLA-E allelic differences (Braud, Allan, O'Callaghan et al., 1998; Brooks et al., 1999; Kaiser et al., 2005).

Cell surface expression of HLA-E is regulated by the expression of other classical class I molecules, as they are the major source of HLA-E binding peptides in normal cells. In particular, signal sequence peptides from HLA-I molecules (HLA-A, B and C as well as the non-classical HLA-G molecule) bind to HLA-E allowing its surface expression (Braud, Allan, Wilson et al., 1998). Consequently, CD94/NKG2 receptors, through the recognition of HLA-E, let NK cells indirectly survey the global expression of class I molecules, frequently altered in transformed or infected cells. The decrease of classical HLA molecules at the surface of tumour cells is a major mechanism involved in tumour immune escape from cytotoxic T effectors while it leads to NK cell activation. Conversely, non-classical HLA-E and HLA-G molecules are frequently overexpressed by cancer cells participating in tumour immune escape through the interaction with inhibitory receptors and the subsequent inhibition of NK and cytotoxic T cell (CTL) activation (Algarra et al., 2004). The production of soluble HLA molecules (sHLA; classical and non-classical) have been described as another potential mechanism involved in cancer immune escape and in vitro studies demonstrated that sHLA could induce inhibition and apoptosis of NK and CD8+ T lymphocytes. Increased levels of sHLA have been found in serum of patients with malignant diseases (Contini et al., 2003; Campoli and Ferrone, 2008). Derre et al. showed that a significant fraction of melanomas in vivo expressed HLA-E. They also demonstrated that the expression of membrane and soluble HLA-E could be in vitro induced on melanoma cell lines treated with IFN(Derre et al., 2006). In collaboration with this group, we recently participated in the validation of an ELISA assay for the quantification of sHLA-E in biological fluids of

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17 cancer patients. We demonstrated that serum sHLA-E is significantly increased in melanoma patients compared to healthy individuals (Allard et al., 2011) (Appendix 1).

Because CD94/NKG2 heterodimeric receptors can bind the same ligand, it is interesting to understand the functional implication resulting from the co-expression of activating and inhibitory receptors. Small subsets of NK and T cells are found to co-express NKG2A and NKG2C receptors in human PBMC. To study the functional effect of NKG2A/NKG2C co-expression, a larger population was obtained upon in vitro induction of NKG2A by IL12 on NKG2C+ cells. In NKG2A+NKG2C+ cells, the inhibitory signal was dominant and CD94/NKG2A receptor regulated the response of CD94/NKG2C following the interaction with HLA-E molecules (Saez-Borderias et al., 2009). Recently Béziat et al. provided the first in vivo evidence that the expression of NKG2A on NKG2C+ cells prevents the autoreactivity against self-HLA-E+ cells (Beziat et al., 2011).

1.3.3.2. NKG2D

NKG2D and F, two other receptors of the C-type lectin-like family, do not form dimers with CD94. These are activating receptors and they associate with different adaptor molecules: NKG2D binds to DAP10 (Figure 3) and NKG2F to DAP12. NKG2F does not translocate to the cell surface (Kim et al., 2004). In addition, NKG2I, a novel NKG2 family member, has been discovered in mice to act as an activating receptor in bone marrow allograft rejection. No human orthologue of this receptor was found (Koike et al., 2004).

NKG2D is one of the best characterised activating NK cell receptors. It works both as an activating and co-stimulatory receptor. As for the other NKG2 members, NKG2D gene is located on chromosome 12 within the NK gene complex. It is expressed as an homodimer. Stechiometry studies showed that one NKG2D homodimer assembles with four DAP10 molecules to form a hexameric receptor complex. The association with DAP10 is essential for signal transduction. Following NKG2D/ligand interaction, DAP10 is phosphorylated in the intracellular motif YxxM and induces the binding of the receptor complex to the phosphatidylinositol 3-kinase (PI3K) Grb2-Vav1 signalling pathway (Burgess et al., 2008; Lopez-Larrea et al., 2008).

NKG2D expression can be positively or negatively regulated by cytokines. In particular, it can be induced by IL2, IL12, IL15, IL18, TNF and IFN and diminished by TGF (Castriconi et al., 2003; Burgess et al., 2008; Zhang, Zhang, Niu and Tian, 2008; Zhang, Zhang, Niu, Zhou et al., 2008).

The specific ligands of this receptor are characterised. NKG2D binds to two different families of ligands: MHC-class I-related chain A (MICA) and B (MICB), and the

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18 cytomegalovirus UL16-binding proteins 1 to 6 (ULBP1-6) (Figure 3). MICA and B are highly polymorphic: their genes are encoded in the MHC class I region of chromosome 6 and share properties with HLA molecules. They are composed of three extracellular domains but they do not require 2-microglobulin for their surface expression and do not present peptides. ULBP1 and 2 were the first ligands identified and are the only ULBP members to effectively bind to cytomegalovirus glycoprotein UL16. Based on their sequence homology, four additional members were found: ULBP3, ULBP4, RAET1G (or ULBP5), and RAET1L (or ULBP6) (Champsaur and Lanier, 2010).

NKG2D ligands are rarely detected at the surface of normal cells, except for MICA that is constitutively expressed by intestinal epithelial cells (Groh et al., 1996). Their expression is induced on “stressed” cells following different stimuli like malignant transformation, viral infection and classical heat shock but the exact mechanisms for their induction are globally unknown. Nevertheless, the DNA-damage-pathway was found to induce NKG2D ligands in a p53-independent manner (Gasser et al., 2005). A recent report showed that ULBP1 and ULBP2 are target genes of the transcription factor p53. The reactivation of wild-type p53 (but not of mutant p53) in tumour cell lines led to the enhanced membrane expression of these NKG2D ligands (Textor et al., 2011).

Several studies showed that the expression of NKG2D ligands on tumour surface rendered them susceptible to NK cell killing in vitro and resulted in the in vivo rejection of transplanted tumours (Nausch and Cerwenka, 2008). Furthermore, Guerra et al. recently showed that NKG2D-deficiency in TRAMP mice results in higher incidence of highly malignant prostate adenocarcinoma in comparison to control mice. They also showed that in NKG2D-deficient mice, tumours have an higher expression of ligands thus demonstrating that NKG2D is implicated in tumour immunoediting (Guerra et al., 2008).

Mechanisms of immune-escape involving NKG2D have also been described further supporting the important role of NKG2D in anti-tumour immune response. It was shown that the constitutive expression of ligands can induced the downregulation of NKG2D receptor leading to a reduced immunosurveillance (Oppenheim et al., 2005). Soluble forms of the ligands are found in serum of cancer patients and are implicated in the immune evasion process by precluding the binding of surface-expressed NKG2D ligands to the receptor. In particular, soluble MICA can be used as a diagnostic marker for cancer at early stages, whereas seric MICB levels are correlated with advanced cancer stages and metastasis (Holdenrieder et al., 2006; Holdenrieder et al., 2006).

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19 In addition to this role in anti-tumour immunity, NKG2D is implicated in other pathologies. A role of NKG2D has been described in different autoimmune diseases: celiac disease, rheumatoid arthritis and type I diabetes mellitus (Groh et al., 2003; Ogasawara et al., 2003; Caillat-Zucman, 2006; Stepniak and Koning, 2006). It has also been demonstrated that virus have evolved numerous mechanisms to escape from NKG2D-mediated recognition and clearance (like the downregulation of NKG2D ligands), highlighting the role of NKG2D in the control of viral infections (Lodoen et al., 2004; Lenac et al., 2006). Furthermore, emerging evidences suggest that NKG2D/ligand interactions can have negative consequences for organ transplants (Ogasawara et al., 2005; Seiler et al., 2007).

1.3.4. Co-receptors involved in NK cell cytotoxicity

Additional molecules appear to be important in the activation of NK cell cytotoxicity, among them DNAM-1, NKp80, 2B4 and NTB-A. These are considered as co-receptors since their function is dependent on the concomitant stimulation by true activating receptors.

1.3.4.1. DNAM-1 (CD226)

DNAX accessory molecule 1 (DNAM-1) is a leukocyte adhesion molecule involved in the induction phase of NK cell activation. DNAM-1 gene is encoded on chromosome 18 and is expressed by NK cells, T cells and monocytes. Its structure is characterised by an extracellular portion with two Ig-like domains and a cytoplasmic tail containing three tyrosine residues. Following DNAM-1 cross-linking, tyrosines are phosphorylated and NK cell cytotoxicity is triggered. Interestingly, DNAM-1 expression is dependent on the surface expression of lymphocyte function associated-antigen 1 (LFA-1) (Shibuya et al., 1999).

Two ligands of DNAM-1 have been identified: the poliovirus receptor (PVR; CD155) and Nectine-2 (CD112) (Figure 3). These molecules are strongly expressed by tumour cell lines of epithelial and neuronal origin like carcinomas, melanomas and neuroblastomas (Bottino et al., 2003). In association with NCR or NKG2D receptors, DNAM-1 is involved in the recognition and lysis of different types of cancer cells: myeloma (El-Sherbiny et al., 2007), melanoma (Lakshmikanth et al., 2009; Chan et al., 2010), ovarian carcinomas (Carlsten et al., 2007), etc. Its role in anti-tumour immunity is prominent when ligands for other activating receptors are poorly expressed (Gilfillan et al., 2008).

An altered DNAM-1 expression by NK cells was described in cancer patients. In ovarian carcinoma patients, DNAM-1 expression was reduced and the direct interaction with PVR ligand

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20 seemed to downregulate its expression (Carlsten, Norell et al., 2009). Recently, decreased DNAM-1 expression on NK cells was documented in acute myeloid leukaemia patients (Sanchez-Correa et al., 2011) and in lung carcinoma patients (Platonova et al., 2011).

Taken together, these observations confirm the important role of DNAM-1 in tumour recognition and lysis. However, a new protein called TIGIT has been recently described to bind to PVR with higher affinity than DNAM-1 and to display immunoregulatory functions. Although no T cell-intrinsic functions seem to be mediated by TIGIT, the interaction between this protein and its ligand led to IL10 secretion by PVR-expressing DC cells causing the subsequent suppression of T cell activation (Yu et al., 2009). In NK cells, TIGIT seems to transduce a negative signal providing an alternative mechanism that prevents NK cell cytotoxicity (Stanietsky et al., 2009). These new findings suggest that, when studying the anti-tumour function of DNAM-1, it is important to consider the co-expression of TIGIT on NK cells.

1.3.4.2. NKp80

NKp80 is an activating homodimeric C-type lectin-like receptor. It is expressed by all resting and activating NK cells and by a subset of CD56+ T cells. It is an activating receptor of 80 kDa that binds to activation-induced C-type lectin (AICL). It participates in NK cell cytotoxicity against malignant myeloid cells, promotes the cross-talk between NK and monocytes and augments responses of effector memory CD8+ T cells. NKp80 signals trough the atypical hemi-ITAM motif via the recruitment of Syk kinase (Vitale et al., 2001; Dennehy et al., 2011).

1.3.4.3. 2B4 (CD244) and NTB-A

2B4 and NTB-A are members of the SLAM-related receptors (SRRs), a subgroup of the CD2 family of Ig-like receptors (Stark and Watzl, 2006).

2B4 is expressed by all human NK cells, a subpopulation of T cells, basophils and monocytes. Its ligand is CD48, a glycosyl-phosphatidylinositol-anchored surface molecule of the CD2 family (Chuang et al., 2001). CD48 is expressed by leukocyte cells and is upregulated on Epstein-Barr Virus (EBV)-infected B cells. The engagement of 2B4 and NTB-A with their respective ligands induces cytotoxicity (Stark and Watzl, 2006) (Figure 3).

A crucial role of 2B4/CD48 interactions in the control of NK cell activation was discovered when patients with x-lymphoproliferative disease unable to control the EBV infection were studied. 2B4/CD48 interaction induced an inhibitory rather than activating signal leading to

The role of human Natural Killer cells (NK) in anti-tumour immune responses

HAL Id: tel-01078862

https://tel.archives-ouvertes.fr/tel-01078862

The role of human Natural Killer cells (NK) in

anti-tumour immune responses

Giulia Fregni

To cite this version:

Université Paris-Sud XI

Faculté de Médecine

École Doctorale « Cancérologie - Biologie - Médecine – Santé » (CBMS)

Spécialité : Immunologie

Thèse

pour obtenir le grade de

Docteur de l’Université Paris-Sud XI

The role of human Natural Killer cells (NK)

in anti-tumour immune responses

Présentée et soutenue publiquement par

Giulia FREGNI

Le 28 Octobre 2011

Jury

Pr Antoine DURRBACH

Président

Dr Vincent VIEILLARD

Rapporteur

Dr Francesco COLUCCI

Rapporteur

Pr Marie-Françoise AVRIL

Examinateur

Dr Philippe BOUSSO

Examinateur

Acknowledgments

Abstract

Résumé

Abbreviations

Contents

Acknowledgments

Abstract

Résumé

Abbreviations

List of figures and tables

PREAMBLE ... 1

INTRODUCTION ... 3

RESULTS ... 63

GENERAL DISCUSSION and PERSPECTIVES ... 112

APPENDICES ... 121

REFERENCES ... 145

List of figures and tables

PREAMBLE

1.

NATURAL KILLER CELLS (NK)

1.1. Introduction

1.2. NK cell function

1.3. NK cell receptors and ligands