The role of natural killer cells in the immune response against CMV infection in immunosuppressed patients

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Thesis

Reference

The role of natural killer cells in the immune response against CMV infection in immunosuppressed patients

DE RHAM, Casimir

Abstract

Natural Killer (NK) cells are part of the innate immune system and represent 5 to 15% of the total lymphocytes in a healthy individual. At their surface different families of inhibitory and activating receptors are expressed, where the killer immunoglobulin-like receptors (KIR) are of special interest. NK cells reactivity will depend on the binding of their receptors and their specific ligands, the major histocompatibility complex I (MHC-I). This KIR-MHC-I interaction plays an important role in transplantation. As transplanted patients are immunosuppressed, they represent an easy target to opportunistic infections, such as cytomegalovirus (CMV).

Specific T-cells can clear CMV infection, but immunosuppressive drugs, which help to tolerate the graft, inhibit the activity of these T-cells. Interestingly, NK cells seem not affected by these drugs. The aim of this thesis is to investigate the anti-viral role of the NK cells during CMV infection after solid organ transplantation.

DE RHAM, Casimir. The role of natural killer cells in the immune response against CMV infection in immunosuppressed patients. Thèse de doctorat : Univ. Genève, 2011, no. Sc. 4321

URN : urn:nbn:ch:unige-166973

DOI : 10.13097/archive-ouverte/unige:16697

Available at:

http://archive-ouverte.unige.ch/unige:16697

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département d'Anthropologie FACULTÉ DES SCIENCES Professeure Alicia Sanchez- Mazas

FACULTÉ DE MÉDECINE Département de Pathologie et Immunologie Professeur Shozo Izui Département de Médecine Interne Docteur Jean Villard

The Role of Natural Killer Cells in the Immune Response against CMV Infections in

Immunosuppressed Patients

THÈSE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention biologie

par

Casimir de Rham de

Giez (Vaud)

Thèse N° 4321

GENÈVE

Atelier d'impression ReproMail Uni Mail

2011

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UNIVERSITÉ DE GENÈVE

FACULTÉ DES SCIENCES

Doctorat ès sciences Mention biologie

Thèse de

Monsieur Casimir de RHAM

intitulée:

"The Role of Natural Killer Cells in the Immune Response against CMV Infections in Immunosuppressed Patients"

La Faculté des sciences, sur le préa vis de Messieurs Sh. Il Ul, professeur ordinaire et directeur de thèse (Faculté de médecine, Département de pathologie et immunologie), J. VILLARD, docteur et codirecteur de thèse (Faculté de médecine, Département de médecine interne), de Mesdames A. SANCHEl-MAlAS, professe ure ordinaire et codirectrice de thèse (Département d'anthropologie et écologie), V. BRAUD, professe ure (Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire, Sophia Antipolis, Valbonne, France) et de Monsieur L. KAISER, professeur associé (Faculté de médecine, Département de médecine interne), autorise l'impression de la présente thèse, sans exprimer d'opinion sur les propositions qui y sont énoncées.

Genève, le 8 avril 201 1

Thèse - 4321 

, Jean-Marc TRISCONE

N.B. - La thèse doit porter la déclaration précédente et remplir les conditions énumérées dans les "Informations relatives aux thèses de doctorat à l'Université de Genève".

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UNIVERSITÉ DE GENÈVE

Département d'Anthropologie FACULTÉ DES SCIENCES Professeure Alicia Sanchez- Mazas

FACULTÉ DE MÉDECINE Département de Pathologie et Immunologie Professeur Shozo Izui Département de Médecine Interne Docteur Jean Villard

The Role of Natural Killer Cells in the Immune Response against CMV Infections in

Immunosuppressed Patients

THÈSE

Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention biologie

par

Casimir de Rham de

Giez (Vaud)

Thèse N° 4321

GENÈVE

Atelier d'impression ReproMail Uni Mail

2011

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Remerciements

Je remercie très chaleureusement le Professeur Shozo Izui d'avoir accepté, "au pied levé", d'être mon directeur de thèse.

D'autre part, je tiens à remercier tout particulièrement le Professeur Jean-Michel Dayer de son soutien et de m'avoir accueilli par deux fois dans son département.

Je tiens à exprimer ma profonde gratitude au Docteur Jean Villard, mon co-directeur de thèse, ainsi qu'à la Doctoresse Sylvie Ferrari-Lacraz de m'avoir en premier lieu accueilli dans leur laboratoire, puis de m'avoir soutenu et permis d'élaborer cette thèse. En espérant que je fus à la hauteur de leurs attentes, je les remercie de leur confiance.

Je remercie la Professeure Alicia Sanchez-Mazas (Université de Genève), ma répondante pour la Faculté des Sciences, ainsi que la Professeure Véronique Braud (CNRS, Sofia Antipolis, Valbonne) d'avoir accepté de faire partie du jury de thèse.

Merci au Professeur Laurent Kaiser pour, d'une part, avoir accepté de prendre part au jury de thèse, et d'autre part, m'avoir permis de travailler dans le laboratoire de virologie. Merci également à Delphine Garcia et Maria Scanzi pour la préparation régulière des fibroblastes.

Je remercie en outre le Professeur Shozo Izui et le Docteur Bertrand Huard, mes parrains de thèse.

Merci à toute l'équipe du FACS, grâce à laquelle je sais démonter et remonter un FACS Aria jusque tard dans la nuit.

Au Docteur Jean-Marie Tiercy pour ses lumières sur le HLA et les récepteurs KIR, ainsi qu'au LNRH, pour leur bonne humeur.

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Aux membres passés et présents du laboratoire: Souad, Sabrina, Delphine, Jérôme, Grégory, Joël et Yannick.

A toutes les personnes passées et présentes du 4^e étage: Danielle, Rachel, Lyssia, Caroline, Alexandra, Nicolas, Christophe, Karim, Montsé, Elisa, Nicolo, Amandine et tous les autres, pour tous leurs conseils, leurs expériences et les bons moments passés hors-laboratoire.

Un merci tout particulier à Renata, Rakelita et Marie-Elise pour les corrections, commentaires, critiques et suggestions concernant l’élaboration de cette thèse.

Ce projet de recherche a été rendu possible grâce au soutien financier du Fond National Suisse de la Recherche; Fonds: CRSI33-125405.

A Joëlle, pour sa présence, sa patience et sa gentillesse lors de la rédaction de cette thèse.

Je tiens, pour terminer, à remercier vivement toute ma famille pour leur soutien depuis toujours et pour toujours, MERCI.

Success is how high you bounce when you hit bottom.

General George Patton Jr.

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Abbreviation List

7-AAD 7-Aminoactinomycin D

ADCC Antibody Dependent Cellular Cytotoxicity AIDS Acquired Immunodeficiency Syndrome APC Antigen Presentation Cells

Asn Asparagine

Arg Arginine

BM Bone Marrow

Bp Base Pair

CMV Cytomegalovirus

CsA Cyclosporin A

CTL Cytotoxic T-Lymphocytes

DAP-10 DNAX-activating protein of 10KDa DAP-12 DNAX-activating protein of 12Kda DCs Dendritic Cells

Dexa Dexamethasone

DNA Deoxyribose Nucleic Acid EBV Epstein-Barr Virus

FL Flt3 Ligand

G-CSF Granulocyte Colony-Stimulating Factor GvH Graft versus Host

GvHD Graft versus Host Disease GvL Graft versus Leukemia

hES cells Human Embryonic Stem cells HCV Hepatitis C Virus

His Histidine

HIV Human Immunodeficiency Virus HLA Human Leukocyte Antigen

HSCT Haematopoietic Stem Cell Transplantation HS cells Haematopoietic Stem cells

HSV Herpex Simplex Virus HvG Host versus Graft

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ICP0 Infected Cell Protein 0

IDs Immunosuppressive Drugs

IFN-γ Interferon-γ

IFNAR 1/2 Interferon α/β Receptor alpha chain

Ig Immunoglobulin

IL-2 Interleukin 2 IL-12 Interleukin 12 IL-15 Interleukin 15 IL-21 Interleukin 21

Ile Isoleucine

ITAM Immune Tyrosine-based Activating Motifs ITIM Immune Tyrosine-based Inhibitory Motifs

JAK Janus Kinase

Kb Kilo base pair (1'000bp)

kDA Kilo Dalton

KIR Killer Immunoglobulin-like Receptor

KL c-Kit Ligand

LAIR Leukocyte-Associated Immunoglobulin-like Receptor LRC Leukocyte Receptor Complex

LN Lymph Nodes

LPS Lipopolysaccharides

Lys Lysine

Mb Mega base pairs (1'000'000bp) MCMV Mouse Cytomegalovirus

MHC-I Major Histocompatibility Complex I MHC-II Major Histocompatibility Complex II MIC-A MHC-I Chain related protein A MIC-B MHC-I Chain related protein B

ML Missing Ligand

mRNA Messenger RNA

N-CAM Neural Cell Adhesion Molecules NCR Natural Cytotoxic Receptors

NFAT Nuclear factor of activated T-cells

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NKC Natural Killer Complex NK cells Natural Killer Cells

NPC Neuronal Progenitor Cells

PAMP Pathogens Associated Molecular Pattern PBL Peripheral Blood Lymphocyte

PCR Polymerase Chain Reaction POLY I:C Polyinosinic : Polycytidylic Acid

RA Rheumatoid Arthritis

RNA Ribonucleic Acid

SCID Severe Combined Immuno-Deficiency

SIGLEC Sialic acid binding Immunogobulin-like Lectins SOT Solid Organ Transplantation

STAT Signal Transducer and Activator of Transcription

Tacro Tacrolimus

TAP Transporter Associated with antigen Processing

TCR T-Cell Receptor

Th1 Type 1 Helper T-cells Th2 Type 2 Helper T-cells

Thr Threonine

TNF Tumor Necrosis Factor

TYK Tyrosine Kinase

ULBP UL-16 Binding Protein

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RESULTS ... 56 1. The Proinflammatory Cytokines IL-2, IL-15 and IL-21 Modulate the Repertoire of Mature Human Natural Killer Cell Receptors ... 56 2. Natural Killer Cell Receptor Repertoire and their Ligands, and the Risk of CMV Infection after Kidney Transplantation ... 72 3. The role of KIR3DL1 and activating KIRs expressed by NK cells of

immunosupressed patients to eliminate human CMV-infected fibroblasts... 83 4. Neural progenitors derived from human embryonic stem cells are targeted by allogeneic T and natural killer cells ...115 5. Interaction of ES cell derived neural progenitor cells with NK cells and

cytotoxic T cells ...131 DISCUSSION AND CONCLUSION...148 REFERENCES ...156

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RESUME DU TRAVAIL DE THESE

Introduction

Le système immunitaire d'un individu est composé de deux groupes cellulaires qui peuvent agir séparément l'un de l'autre, ou de manière concertée, face à une intrusion d'un pathogène. La première ligne de défense est composée par le système immunitaire inné, qui est constitué de cellules dendritiques, de monocytes-macrophages, et de cellules NK (natural killer). La réponse immune donnée par ces cellules est directe, rapide, courte et sans activation préalable. La deuxième ligne de défense, où l'on trouve les lymphocytes T et B, forme la réponse immunitaire acquise. Cette réaction est plus lente à se mettre en place, du fait que ces lymphocytes doivent passer à travers différents stades de maturation pour êtres opérationnels, mais son effet est extrêmement efficace et dure plus longtemps. La combinaison de ces deux réponses permet une réponse sûre contre toutes sortes d'allergènes, de bactéries, ou de virus.

Les cellules NK font partie du système immunitaire inné, et représentent entre 5 et 15% des cellules immunes chez un individu sain. Les cellules NK ne secrètent pas d'anticorps comme le font les lymphocytes B. N'exprimant pas le CD3 mais le CD56 en grande quantité, les cellules NK sont définies d'après leur phénotype, CD3^- /CD56⁺. Suivant la densité du CD56 exprimé, 2 sous-populations de cellules NK peuvent être définies, les CD56^dim (CD3^-/CD56⁺/CD16⁺) et les CD56^bright (CD3^- /CD56⁺/CD16^-), chacune ayant un phénotype et une fonctionnalité bien définie. Les cellules CD56^dim ont un profil cytotoxique, et sécrètent très peu de cytokines. En revanche, les CD56^bright secrètent plusieurs types de cytokines telles que l' IL-10, l' IFN-γ ou le TFG-β, et possèdent un profil plutôt immuno-régulateur. Les cellules NK expriment à leur surface, toute une série de récepteurs activateurs et inhibiteurs, qui peuvent êtres regroupés en plusieurs familles, telles que la famille des récepteurs lectines de type C (NKG2A, NKG2C et NKG2D), la famille des récepteurs cytotoxiques naturels (NKp30, NKp44 et NKp46) et la famille des récepteurs KIR (killer immunoglobulin-like receptor). C'est grâce à ces récepteurs activateurs et inhibiteurs que les cellules NK peuvent détruire des tumeurs ou des cellules infectées. Pour être totalement effective, la cellule NK doit passer par une

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étape de maturation; le récepteur inhibiteur exprimé par la cellule NK est "activé"

ou éduqué par son propre MHC-I, exprimé par une cellule mature spécialisée. Une fois ce signal acquis, la cellule NK est non seulement opérationnelle mais aussi tolérante face à des cellules qui expriment le MHC-I du soi.

Les récepteurs KIR, et plus particulièrement, les KIR inhibiteurs possèdent comme ligand la molécule du complexe majeur d'histocompatibilité I (MHC-I), alors que le ligand spécifique pour les récepteurs KIR activateurs n'est, pour l'heure, toujours pas connu. Alors que pour certains récepteurs activateurs, le ligand est un peptide issu de certain pathogène, par exemple, un protéine du CMV, pp65, qui se lie avec le NKp30. La réponse de la cellule NK dépend donc de la liaison récepteur-ligand, et plus particulièrement du récepteur KIR inhibiteur. En effet, les cellules NK sont capables de détruire toute cellule n'exprimant pas le MHC-I à sa surface.

Autrement dit, tant qu'il n'y a pas l'implication d'un récepteur KIR inhibiteur, la cellule NK est activée et lyse toute cellule MHC-I négative. Mais il existe une situation, ou même lorsque le MHC-I est exprimé par une cellule, celle-ci est attaquée par les NK. Cette situation, appelée absence du KIR ligand, apparaît lorsqu'une cellule-cible exprime des molécules MHC-I qui ne sont pas spécifiques pour les récepteurs KIR inhibiteurs exprimés pas la cellule NK.

Objectif du travail de thèse

Cette interaction KIR-MHC-I joue un rôle important dans certaines maladies, ainsi que dans le domaine de la transplantation. En effet, dans le cas d'une transplantation de cellules souches hématopoïétiques, les cellules NK du donneur peuvent lyser les cellules leucémiques en raison de l'absence de l'expression du MHC-I. Dans ce cas, on a affaire à une réaction "greffe contre leucémie" (GVL).

Ainsi le rôle des cellules NK avec l'interaction KIR-MHC n'est pas négligeable lors d'une transplantation.

Un autre problème majeur qui survient après une transplantation est l'infection au cytomegalovirus (CMV). Le CMV est un virus, de la famille des virus de l'herpès, qui est latent chez un individu immunocompétent, mais qui devient virulent chez des patients immunodéficients. Environ 60% des patients transplantés ont, soit une infection, soit une réactivation du CMV après une transplantation. Pour contrer ce

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virus, il existe des anticorps spécifiques contre le CMV, ainsi que les lymphocytes T cytotoxiques exprimant un antigène spécifique pour le CMV. Mais ces lymphocytes T cytotoxiques sont inhibés par les immunosuppresseurs, avec lesquels sont traités les patients, afin de tolérer la greffe. Ainsi, ces cellules T cytotoxiques sont impuissantes à détruire le CMV. Or il se trouve que les cellules NK ne sont pas affectées par ces immunosuppresseurs, et que même, leurs activités cytotoxiques semblent être augmentées. Le but de ce travail est donc d'étudier plus en détail le rôle anti-viral des cellules NK dans l'infection CMV chez des patients ayant subi une transplantation d'organe solide.

Résultats

Après avoir mieux caractérisé le phénotype et la fonctionnalité des cellules NK en présence de différentes cytokines pro-inflammatoires (IL-2, IL-15 et IL-21), nous nous sommes intéressés à connaître plus en détail la fonction et le phénotype des cellules NK lors d'une infection CMV après chez des patients transplantés.

Dans un premier temps, nous avons mis en évidence, au niveau génétique, que chez les patients transplantés, non seulement l'absence du KIR ligand, mais aussi le nombre de KIR activateur jouent un rôle important dans la diminution de l'infection à CMV. En parallèle, nous avons aussi analysé le phénotype (NKG2A, NKG2C et NKG2D) ainsi que la fonctionnalité (sécrétion d'IFN-γ) des cellules NK issues de patients transplantés à différents moments de l'infection CMV; au pic de l'infection (jour 0), à la fin du traitement anti-viral (jour 20), et six mois après l'infection (jour 180). Nous avons mis en évidence qu'au pic de l'infection, les cellules NK étaient incapables de produire de l'IFN-γ, par contre, l'expression de NKG2C et NKG2D restait élevée.

Suite à ce projet, nous avons continué à analyser le rôle anti-viral des cellules NK pendant une infection à CMV, mais cette fois-ci au niveau du phénotype. C'est-à- dire, savoir s'il existait un récepteur KIR spécifique qui serait impliqué dans la lyse des cellules infectées par le CMV. Pour cela, nous avons mis au point un modèle in vitro, dans lequel des fibroblastes infectés par le CMV deviennent des cellules cibles pour les cellules NK. Par analyse FACS, nous avons pu non seulement mettre en évidence la lyse, par les cellules NK, des fibroblastes infectés, mais aussi

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étudier la fonctionnalité et le phénotype KIR de ces mêmes cellules NK. En corrélant l'expression des différents récepteurs KIR avec les fibroblastes infectés détruits, nous avons mis en évidence que le récepteur KIR3DL1 joue un rôle important dans la lyse de ces cellules infectées par le CMV. Puis en utilisant un anticorps (CD158e1e2; clone Z27), qui nous permet de discriminer la forme inhibitrice, KIR3DL1, de la forme activatrice, KIR3DS1, et en le combinant avec un marqueur de dégranulation, le CD107a, nous avons pu déterminer le niveau d'activation des cellules NK exprimant KIR3DS1 ou KIR3DL1, qui ont été en contact avec des fibroblastes infectés par le CMV. Ainsi, nous avons pu confirmer que les cellules NK positives pour KIR3DS1 expriment plus de CD107a que les cellules NK positives pour KIR3DL1. Nous avons pu mettre en évidence que le récepteur KIR3DS1 joue un rôle primordial dans la lyse des fibroblastes infectés par le CMV.

Conclusion

Le rôle des récepteurs KIR activateurs a été récemment mis en évidence, au niveau génétique, dans la diminution de l'infection CMV. Cette étude met en lumière le rôle d'un récepteur KIR activateur spécifique, KIR3DS1. Ce récepteur KIR exprimé par les cellules NK isolées de patients transplantés, joue un rôle dans l'élimination des cellules infectées par le CMV. Ce n'est pas la première fois que KIR3DS1 est impliqué dans la diminution d'une infection virale. Il a aussi été démontré que KIR3DS1 diminue la progression des cellules infectées par le VIH.

En combinant d'autres marqueurs spécifiques pour les cellules NK, notamment avec un marqueur de dégranulation comme le CD107a, nous avons pu déterminer le niveau d'activation des cellules NK, exprimant KIR3DS1 et KIR3DL1. Nous avons constaté que les cellules NK positives pour le récepteur activateur KIR3DS1 expriment plus de CD107a comparées aux cellules NK exprimant KIR3DL1, le récepteur inhibiteur.

Les récepteurs KIR sont exprimés à la surface de la cellule NK de manière complètement aléatoire, et chaque cellule NK exprime un phénotype bien précis et différent par rapport à sa voisine. Les cellules NK qui expriment KIR3DS1 et KIR3DL1 semblent être complémentaire. Si le récepteur activateur KIR3DS1 semble être plus efficace dans la lyse des cellules infectées au CMV, le récepteur

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inhibiteur KIR3DL1 joue son rôle dans le processus d'éducation et de tolérance de la cellule NK.

Le fait, que des cellules NK exprimant KIR3DS1 possèdent un rôle anti-viral contre des cellules infectées par le CMV, permettent d'entrevoir des perspectives de thérapies cellulaires. En effet, des cellules NK exprimant un récepteur spécifique pourraient être isolées, amplifiées, activées in vitro et ré-infusées chez un patient transplanté souffrant d'une sévère infection à CMV.

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ABSTRACT

Natural Killer (NK) cells are part of the innate immune system and represent between 5 to 15% of the total peripheral blood lymphocytes in a healthy individual.

At their surface different families of inhibitory and activating receptors are expressed. Among them, the killer immunoglobulin-like receptors (KIR) have are of special interest. Whereas the ligand of inhibitory KIR is the major histocompatibility complex I (MHC-I) molecule, ligands for activating KIR are still unknown. NK cells reactivity will depend on the binding of their receptors and their specific ligands. Moreover, they are able to kill cells, which do not express MHC-I, or down-regulate its expression. This KIR-MHC-I interaction plays an important role in transplantation, where donor NK cells can exert a graft versus leukemia (GvL) effect in the recipient. As transplanted patients are immunosuppressed, they represent an easy target to opportunistic infections, such as cytomegalovirus (CMV). Specific cytotoxic T-lymphocytes (CTL) can clear CMV infection, but immunosuppressive drugs, which are given to patients in order to tolerate the graft, inhibit the activity of these CTL. Interestingly, NK cells seem not affected by these drugs. The aim of this project is precisely to investigate the anti-viral role of NK cells during CMV infection after solid organ transplantation (SOT).

We already highlighted, on the genotype level, the KIR-HLA-I interaction and the role of the activating KIR in reducing CMV infection after transplantation. From this, we wanted to investigate, if a specific KIR receptor is involved in the killing of CMV-infected cells. To achieve this work, we set up an in vitro model, in which CMV-infected fibroblasts were used as target cells. With this technique, we were able to analyze, by FACS, simultaneously the killing of CMV-infected fibroblasts and the KIR phenotype and the functionality of the involved NK cells. Then, by correlating KIR receptors and the killing of CMV-infected fibroblasts, we noticed a positive correlation with the KIR3DL1 receptor. To further investigate role of KIR3DL1 and its activating counter part KIR3DS1, we used a antibody, which could discriminate the inhibitory KIR3DL1 from the activating KIR3DS1. Combining this antibody with a degranulation marker, CD107a, which is expressed by activating NK cells, we were able to show that KIR3DS1 is significantly involved in the killing of CMV-infected fibroblasts comparing to KIR3DL1.

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INTRODUCTION

Among the hundred thousand billion (10¹⁴) estimated cells (1), which compose the human body, the cells responsible for the defense of the organism against the external environment, is defined as immune cells. These immune cells include several distinct cell populations. These cells originated from a common hematopoietic stem cell (HS cells) progenitor, which is derived to either a lymphoid progenitor and gives rise to T- or B-lymphocytes and NK cells, or in a myeloid progenitor producing granulocytes, monocytes, macrophages or dendritic cells. The same common myeloid progenitor is also the source of erythrocytes and platelets, which are not members of the immune cell populations.

All these immune cells have a well-defined role during an infection. They interact in order to activate or to inhibit the immune response. To fight pathogens with efficacy the immune system uses 2 different strategies. The first one is the innate immune response and the second is the acquired immune response.

In this introduction, I will briefly describe the innate and the acquired immune system, and then I will go into more details about NK cell biology; their different receptors, their development, their deficiencies and their role in transplantation.

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1. The Innate Immune System

The innate or natural immune system is the first response against infections. This response is fast and efficient over a short period of time. This reaction is not specific even if the cells of the innate immune system recognize different specific structures of pathogens like lipopolysaccharides (LPS), single or double strand ribonucleic acid RNA, or pathogens associated molecular pattern (PAMP).

Macrophages, dendritic cells (DCs), and natural killer (NK) cells are the main actors of this response. The main goal of the innate immune response is to contain infections, in order to avoid propagation throughout the entire organism. The method of killing pathogens differs among these cells.

Macrophages destroy microorganisms by phagocytosis. After engulfment of a pathogen, various intra-cellular toxic molecules, such as superoxide anions, hydroxyl radicals, nitric oxide or hypochlorous acid digest the pathogen (2). This process allows the macrophages to clean the organism of dead cells, and helps to avoid an inflammatory response triggered by these dead bodies. Moreover, macrophages express receptors for carbohydrates, such as mannose, that are not normally expressed by vertebrate's cells. This allows macrophages to distinguish between self and non-self molecules (3). In addition to macrophages, granulocytes that include neutrophils, basophils and eosinophils play also a critical role in the first line of the immune defense. Neutrophils are very efficient phagocytic cells, basophils and eosinophils secreted several very potent inflammatory mediators (4).

DCs play an important role in this immune response. They are very important for two reasons, first they are able to endocytose extra-cellular antigens, and secondly, when DCs are activated, they act as antigen presenting cells (APC) to the T-cells (5). Once the antigen is captured by the DCs, it is degraded in the specific vesicules into small peptides, which will fit inside the groove of the MHC molecules and will be expressed at the cell surface of the DCs. Then, activated DCs migrate to the lymph nodes (LN), in order to present the antigen to the T-cells. The role of MHC molecules will be described in the next chapter.

The third largest lymphocyte families, which play an important role in the innate immune system, are NK cells. This cell family will be described in Chapter 3.

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2. The Acquired Immune System

This second type of immune response is slower to react, but is more effective over the long time. This reaction is driven by T- and B-lymphocytes, which need to be previously activated.

Bone marrow (BM) is the place where B-cells originate but also maturate. The role of B-cells is to secrete antibodies. Antibodies are made up of immunoglobulin (Ig) and carbohydrates, which maintain the structure of the antibody together.

Antibodies are made of 2 identical heavy chains and 2 identical light chains, bound together with disulfide bonds (6). The N-terminal of each chain consists of a variable domain, which will bind to antigens. On the other hand, the C-terminal of the heavy and light chain forms the constant region (Fig. 1). This constant region determines the class of the antibody. There are five

classes of immunoglobulins; IgG, IgA, IgM, IgD and IgE (Fig. 2).

Fig. 1. Schema of an IgG. In grey, the constant region; in red, the variable region with the antigen-binding site in blue.

Surrounded with a thin black line, are the light chains, the heavy chain is surrounded by a thick black line.

The orange line represents disulfide bonds.

These Ig can be secreted or fixed to the B-cell

membrane. This means that the antibody expresses a hydrophobic trans-membrane domain, which allows it to attach to the B-cells membrane and act as a B-cell receptor (7, 8).

Fig. 2. View of the five different Ig classes. The light chain is in yellow, and the heavy chain is in a different color, which represents the sub-class (γ, α, µ, δ, or ϵ). The green line represents the disulfide bonds.

From (Freeman, 2002).

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Next to B-cells, T-cells are the main lymphocytes of the acquired immune system.

All T-cells receptors are bound to the T-cell surface and are not secreted. The T- lymphocytes originate from the BM and maturate in the thymus, an organ located behind the sternum, and divided into several distinct functional areas, the cortex, the cortico-medullary junction and the medulla. Progenitor T-cells issued from bone marrow reach the thymus through blood vessels at the cortico-medullary junction. Once there, they proliferate, differentiate and migrate to the cortex. In the cortex, the double negative CD4/CD8 thymocytes mature into double positive CD4/CD8 thymocytes. These double positive CD4/CD8 thymocytes encounter the cortical thymic epithelial cells, which express a peptide-MHC complex. The surviving T-cells are those, expressing functional T-cell receptors and are able to bind this peptide-MHC complex. Once this first selection is completed, a second selection is made in the cortico-medullary junction, where the double-positive T-cells mature into single positive. These single positive T-cells return to the medulla, where they leave the thymus for the periphery. (9, 10).

The masterpiece, which regulates the immune system, is the major histocompatibility complex (MHC) molecule, or human leukocyte antigen (HLA) in humans. The MHC is encoded by chromosome 6 and contains over 200 genes (11).

The MHC genes encode 2 different MHC proteins; each has its own structure and its own function (Fig. 3). The MHC-I gene codes for the α-chain and the β-chain, called β2-microglobulin, of the molecule, is coding by a gene situated on chromosome 15.

There are around 20 MHC-I genes and 3 of them, HLA-A, HLA-B and HLA-C are the so-called the classical HLA-I, and are expressed by all somatic cells (12, 13). MHC-I is expressed constitutively on the surface of most of cells. MHC-II includes 3 locus, HLA-DP, HLA-DQ and HLA-DR, the class II gene encode for the α-chain and β-chain of the molecule. MHC-II is expressed constitutively by a limited number of immune cells, such as B-cells, DCs, macrophages and activated T-cells, and under stimulation of different cytokines, MHC-II can be expressed by many cell types.

Fig. 3. Left: structure of MHC-I;

right: structure of MHC-II.(14)

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The function of MHC-I and MHC-II is to present a short part of a peptide issued from a pathogen, and to trigger the acquired immune response. But the processing of MHC-I and MHC-II happens in different cell compartments, and the origin of the presented peptide is different. MHC-I presents peptides issued form the cytosol.

Proteasome cleave these intracellular antigens into peptides, and inside the Golgi apparatus, these peptides are bound to the MHC-I and export to the cell surface. In contrast, extra-cellular antigens are phagocyted, and cleaved into peptide in the phagolysosome. The MHC-II, present inside a vesicle, is transported to the phagolysosome where the peptide is bound to the MHC-II and exported to the cell surface (14).

The specific part of an antigen, which is recognized by a receptor, is called epitope. The epitope recognized by T-cells receptors are specific amino acid sequences of the pathogen peptide derived by intra-cellular proteolysis. T-cells receptors are associated with the CD3 complex, which helps to transmit the activating signal through the cells. Two different types of T-cells exist; CD4⁺ T- cells, which are mainly cytokines secreting cells, and CD8⁺ T-cells, which are mainly cytotoxic cells. CD4⁺ T-cells can be divided into several sub-populations; the type 1 helper T (Th1) cells, which secretes IL-2 and IFN-γ and the type 2 helper T (Th2) cells, which produces IL-4, IL-5, IL-6 and IL-10. Infected cells are destroyed by CD8⁺ cells or so called cytotoxic T lymphocytes (CTL). Once the viral peptide is bound to the MHC, the infected cells will express it, the CTL binds to this viral peptide-MHC complex and with the help of co-stimulatory molecules, kills the infected cells. To destroy the infected cells, CTL secretes perforins and granzymes;

perforins create pores into the infected cells, which allow granzymes to penetrate the cells and mediate apoptosis through the caspase pathway. The other way is the binding of Fas ligand expressed by the CTL to Fas receptors expressed by the infected cells and mediated apoptosis.

Two main characteristics render this acquired immune response more effective than the innate immune response. First, once the T- or the B-lymphocytes are activated, they are able to proliferate clonally, in order to be redundant with regard to the specific antigen of pathogens; this will increase the immune reaction.

Second, among these cells, some of them called memory T-cells, can be activated rapidly in case of a new infection by the same pathogen. In this case, the second

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response of the immune system will be much faster and stronger compared to the first one. This allows the destruction of the pathogen before it causes a disease (7, 8, 12, 13, 14).

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3. The Natural Killer Cells

NK cells represent between 5 and 15% of all lymphocytes in a healthy individual and are members of the innate immune system. They are the third major lymphocyte population after the T- and B-lymphocytes (15). The origin of their progenitor is still debated. Some studies suggest a common progenitor similar to the T- and B-cells nevertheless, a distinct progenitor has also been proposed.

NK cells are defined according to their phenotype, the missing of CD3 and the expression of CD56; CD3^-/CD56⁺ (Fig. 4). CD56 is an adherent molecule from the immunoglobulin super-family neural cell adhesion molecule (N-CAM), encoded by a single gene on chromosome 11. CD56 used to be the prototypic marker for human NK cells. While monocytes/macrophages, CD4⁺ and CD8⁺ T-cells also express CD56, several authors suggest another more specific marker for NK cells such as NKp46 (16, 17). But with using NKp46 alone, total NK cells will be detected, and not the two NK subpopulations, based on CD56. CD56 is also found in brain, in the cerebellum, in the cortex, and on certain leukemia. Whereas in immunology, the real function of CD56 is unknown, in the nervous system, CD56 is implicated in neural development. Interestingly, while CD56 is expressed in mice and in human brain, it is not expressed by mice NK cells (18, 19).

It appears that CD56 expression is not homogenous, 2 distinct populations of NK cells appear, related to the expression of CD56. NK cells, which express low density of CD56, are called CD56^dim and represent 90% of the circulating NK cells. The remaining 10%, are the CD56^bright, and express high density of CD56 and are CD16 low. These 2 types of NK cells possess their own properties (20).

Fig. 4. Typical FACS of NK cells (CD3^-/56⁺).

In blue; CD56^dim population;

In red CD56^brightpopulation (20).

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CD56^dim sub-populations, characterized by CD3^-/CD56⁺/CD16⁺ phenotype, have a cytotoxic profile, they express high amount of CD16 and KIR receptor. NKG2D is also present at their surface, but the other

C-type lectins are absent. CD56^dim produces low amount of cytokines and CCR7 is not expressed. The low affinity receptor for IL-2, IL-2Rαβ, is found on this cells surface. This explains why CD56^dim proliferate poorly in presence of IL-2.

Fig. 5. Schematic view of a CD56^dim cell (20).

The CD3^-/CD56⁺/CD16^- NK cell phenotype represents the CD56^bright sub-population.

These cells possess an immuno-regulatory profile. CD16 and KIR receptors are poorly expressed on their surface. CCR7 and C-type lectin receptors such as NKG2A, NKG2C or NKG2D are expressed. CCR7 allows

the CD56^bright cells to move to the secondary lymphoid organs, such as the LN or the spleen.

Here, the high affinity receptor for IL-2, IL-2Rαβγ, is found on the CD56^bright surface.

This means that this cell can proliferate with low doses of IL-2. Moreover, they secrete several cytokines such as IFN-γ, TNF-α or IL-10.

Fig. 6. Schematic view of a CD56^bright cells (20).

Next to CD56, CD16 is also an important marker for NK cells. CD16 is the low affinity FcγRIII receptor expressed by NK cells. It is an activating receptor, which binds to antibody-coated target cells and kills target cells through the antibody dependent cellular cytotoxicity (ADCC) process. ADCC consists in the coating of target cells by antibody, which will be recognized by CD16 and will induce killing activity by NK cells.

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Table 1. Summary of the differences between CD56^dim andCD56^bright NK sub-population (20).

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4. NK Cells Receptors

NK cells express a wide variety of inhibitory and activating receptors at their surface.

Fig. 7. Schematic view of the different activating (green) receptors and inhibitory (red) receptors expressed by human NK cells. In blue, are the cytokines receptors. Adapted from (21).

All these receptors can be grouped into families, and several of them have been well characterized, such as the KIR receptors families, the C-type lectins families or the natural cytotoxic receptors (NCR) families. Most of the ligands for the NK cells receptors are know, except for some activating receptor, where some ligands must still be defined. One common trait about activating ligand is their expression by cells under "stress" or infected cells (22, 23).

NK cells express also cytokines receptors, which are common through their γ-chain.

As it will be mentioned in the following chapter (see Chapter 5. NK cells development), IL-2 and IL-15 are involved in NK cell maturation and survival.

Some of these receptors are expressed in a stochastic manner and each NK cells expressed a well-defined receptor phenotype distinct from its neighbor. The function of all these receptors is to regulate NK cell response with its environment.

This response is based on the "missing-self" hypothesis, proposed by Kärre (24). This hypothesis postulates, that NK cells are activated to kill target cells, which lack self-MHC-I expression. This gives theoretically four different possibilities of NK cells

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responses (Fig. 8). First when NK cells encounter cells lacking ligand for activating receptors (Fig. 8A and 8B), no response is initiated. On the other hand, when NK cells met infected cells expressing ligands for their activating receptors, NK cells will destroyed the target cells through releasing perforin and granzymB (Fig. 8C). In fact, both type of receptors are engaged (Fig. 8D) and the signal given to the NK cells will depend on the binding strength between the receptors and its engaged ligand (25).

Fig. 8. Here is a representation of the 4 possibilities of interactions between a NK cell and a target cell. A) As no ligand is expressed by the target cell, NK cell will not receive any signals. B) Only an inhibitory receptor and its ligand are bound, this will initiate an inhibitory signal, and the target cell will not be lysed. In C), it is the opposite situation, where only the activating receptor is engaged. NK cell get activated and lyses the target cell. In D), both receptors are engaged and the outcome will depend on the amount and affinity of binding between the receptors and their ligands (25).

The most important receptors with regards to the work presented in this thesis will be described in the next chapter.

4.1. MHC-I Specific Receptors

4.1.1. KIR Receptors

KIR receptors are a family of inhibitory and activating receptors, which are expressed by NK cells and CD8⁺ T-cells. Each NK cell from an individual expresses stochastically a specific pattern of KIR receptors, and this pattern differs between

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individuals. The killer Immunoglobulin-like receptors (KIR) gene family consists of 14 genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DS1) and 2 pseudogenes (KIR2DP1 and KIR3DP1). These genes are encoded on chromosome 19 in the leukocyte receptor complex (LRC) region. The LRC gene consists of a 1Mb region, which contains several gene coding for several cells surface proteins, such as SIGLEC, LAIR or the Fcg receptor (Fig. 9). There are also genes coding for trans- membrane molecules such as DAP-10 or DAP-12. Among these genes, the KIR genes encode a region of 150bp (26, 27).

Fig. 9. Picture of the LCR region of the human chromosome 19.

In blue, the KIR gene region (28).

4.1.1.1. KIR Genes

The length of KIR genes varies between 4 to 16 Kb and comprises, 4 to 9 exons.

They have been classified in 3 groups, in relation to their extra-cellular structure.

In the first group, the genes coding for KIR2D type I express two extra-cellular domains with a D1 and D2 confirmation. The second group, are the genes for KIR2D type II and express two extra-cellular domains D0 and D2. Finally, the third group represented by the KIR3D, with its 3 extra-cellular domains D0, D1 and D2.

The genes coding for KIR2D type I, which are represented by KIR2DL1, KIR2DL2, KIR2DL3 and KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 include 8 exons and one pseudoexon, which is inactive in KIR2D type I. KIR2DL1 and KIR2DL2 have a deletion in exon 7, which distinguishes them from the other KIR. The KIR types I, KIR2DL1, KIR2DL2 and KIR2DL3 are different from the KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and KIR2DS5 in their length in the cytoplasmic region, coding by the exon 9. KIR2D type II, which code for KIR2DL4 and KIR2DL5 do not have an exon 4 and the exon 1 of KIR2DL4 is longer than the one of KIR2DL5. The gene codings for KIR3D, which are represented by KIR3DL1, KIR3DL2, KIR3DL3 and KIR3DS1, include 9 exons. The

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ninth exon is coding for the cytoplasmic part of the protein, and notably KIR3DL2 is the longest of all KIR (16'256bp). KIR3DS1 has a shorter exon 8, which coded for the cytoplasmic part, compared to KIR3DL1 and KIR3DL2. KIR genes polymorphisms seem to be the biggest contributor of the KIR diversity. This allows for a tremendous variety of responses against different pathogens. Population studies of KIR genotypes showed variations in KIR gene from individual to individual. KIR are organized in a head-to-tail fashion and each KIR has a length of 10 to 16kb and 2kb separate each pair of genes (29, 30, 31, 32).

4.1.1.2. KIR Haplotype

All KIR haplotypes have the same organization; the centromeric end is delimited by KIR3DL3, and the telomeric end by KIR3DL2. In the centric part, KIR3DP1 and KIR2DL4 can be found. These 4 KIR genes constitute the framework genes, they are present on all KIR haplotypes, and between these genes, the remaining KIR gene can be located. Based on the studies of the KIR genes content within this framework, two different halpotypes appear; haplotype A and haplotype B. The main difference between these two haplotypes is the presence or not of one or more activating KIR (31, 32).

KIR haplotype A express always the same pattern, seven loci, which are KIR3DL3 (at the centromeric end), KIR2DL3, KIR2DL1, KIR2DL4, KIR3DL1, KIR2DS4 and finally KIR3DL2 at the telomeric end. This haplotype has a frequency of expression of 47 to 59% among the European population and include, only one activating KIR, KIR2DS4. By traditional typing, Hsu et al., (33) could not distinguish between the total KIR2DS4 and a variant, which has a 22 bp deletion in the D2 Ig domain. By comparing the sequence of this KIR2DS4 with a 22 bp deletion with other species, a 72% homology of amino acid appears with the Mm-KIR1D receptor, which is found in rhesus monkeys (34). The function of human KIR1D is still unknown. But the frequency of KIR1D in the European population is 78%, in comparison to KIR2DS4, which is 35%. For this reason, KIR haplotype A can be divided into 2 sub-group, the haplotype A-1D and the haplotype A-2DS4, with a frequency of 39% for A-1D and 12% for A-2DS4 (33). As the most frequent KIR haplotype A have KIR1D, which function is unknown, and we can therefore conclude that this haplotype has no

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activating KIR. But next to that, it was observed that the framework gene KIR2DL4 retains an activating function (35, 36). In contrast, haplotype B is much more variable and has one to five activating KIR. Its frequency among the European population is the same as haplotype A, however, haplotype B shows a much greater variety of subtypes (26, 32) (Fig. 10).

KIR haplotype consists of 2 parts within the central middle KIR2DL4. The centromeric half is limited by KIR3DL3 upstream and KIR3DP1, and on the other part, the telomeric half is limited by KIR3DL2 downstream and KIR2DL4. KIR2DL2 or KIR2DL3 is found in the centromeric half, although never both combined. Yet, when KIR2DL2 is present, KIR2DS2 will always be present, next to KIR3DL3. The presence of KIR2DL3 is always associated with KIR2DP1, KIR2DL1 and KIR3DP1, which defines a partial haplotype. Nevertheless, the telomeric half is characterized by the presence of KIR3DL1 or KIR3DS1, but not both. The presence of KIR3DL1 indicates a "short" telomeric end with the presence of either KIR2DS4 or KIR1D and finally KIR3LD2. In the case of KIR3DS1, a "long" telomeric end is present with KIR2DL5 paired with KIR2DS3 or KIR2DS5, then by KIR2DS1 and either KIR2DS4 or KIR1D. KIR3LD2 closes the loci (33) (Fig. 10).

Fig. 10. Schematic view of KIR haplotype A (top) and haplotype B (bottom). In pink, the framework genes, in blue inhibitory KIR, in red activating KIR and in grey pseudogenes. Below the KIR haplotype B and shown in parentheses,

the different possibilities of KIR genes, which can form KIR haplotype B (37).

Next to these 2 KIR haplotype, the KIR genomic region possesses a high level of allelic polymorphism. It is generated by homologous recombination or by point mutation (29). It is the most variable region of the human genome, after the MHC polymorphism (38). The great advantage of this KIR allelic polymorphism is to diversify the immune response against pathogens (39). These different KIR alleles can give rise to protein variant with differential binding affinity for the MHC-I

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ligand (40). However, frameshift deletion creates premature stop codons that might generate truncated KIR proteins that are not expressed on the cell surface (41).

4.1.1.3. KIR Proteins

KIR receptors are monomeric and include 14 receptors, which can be divided into 8 inhibitory receptors (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2 and KIR3DL3) and 6 activating receptors (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 and KIR3DS1). There is a very high sequence homology between these receptors and their nomenclature is based on their structural differences.

They all possess, an extra-cellular, a trans-membrane and a cytoplasmic part. A first difference appears in the extra-cellular domain of the protein. KIR receptors have 2 or 3 Ig-like domains, and this structure is used in the KIR nomenclature. A KIR2D expresses 2 Ig-like domains, whereas a KIR3D possess 3 Ig-like domains. As said before, the extra-cellular domain is divided into D0, D1 and D2 regions. KIR2D type I, which includes KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5 expresses only the D1 and D2 Ig-like domains. KIR2D type II with KIR2DL4 and KIR2DL5 possess a D0 and D2; D1 Ig-like domain is absent. Then, KIR3D expresses the three Ig-like domains D0, D1 and D2. A second difference in the KIR structure is situated in the intra-cellular part of the protein. This cytoplasmic region can be either long or short. A long cytoplasmic tail contains two immune tyrosine-based inhibitory motifs (ITIM), which transduces an inhibitory signal to the cells. With a short cytoplasmic tail, a positive charged amino acid arginine (Arg) or lysine (Lys) is expressed in the trans-membrane domain, allowing the binding of a DNAX-activating protein of 12 KDa (DAP-12) molecule. DAP-12 has 2 immune tyrosine-based activating motifs (ITAM), and will generate an activating signal to the cells. This difference in the length in the cytoplasmic domain is also a characteristic in the KIR names. "L" represents a long cytoplasmic tail, and "S"

means a short region. The final digit indicates the number of the gene encoding a protein with this structure. As KIR exists in different allele, they were named similarly as the HLA system. After the last number, 3 digits separated by an

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asterisk indicates alleles that differ in the sequences of their encoded proteins (42, 43).

Thus KIR2DL1 for example, is a KIR with 2 Ig-like domains and a long cytoplasmic tail (Fig. 11) (26). KIR2DS1 will be its activating counterpart.

Among all of these KIR receptors, KIR2DL4 has several features, which make it special. First, it has an unusual extra-cellular D0-D2 Ig domain. Secondly, there is a positively charged amino acid (Arg) in its trans-membrane region. Thirdly, KIR2DL4 has a long cytoplasmic tail with only one ITIM motif, instead of two, and finally, KIR2DL4 mRNA is expressed in all cells and its ligand is HLA-G. With all these characteristics, KIR2DL4 shows both inhibitory and activating motifs, but remains an activating KIR (26, 35, 44, 36).

Fig. 11. Schematic view of the different KIR proteins. On the extra-cellular region, the different IgG-like domains; in red, D0; in blue D1 and in green D2. Inside the cell membrane, the red loop represents the DAP-12 molecules with the 2 ITAM motif in grey square. The 2 white squares on the long tail represent the ITIM motif.

Note the KIR2DL4 with a red loop and a white square. Adapted from (45).

KIR are expressed in a stochastic way, which means that each NK cells express one or more KIR at their surface. It should be noted that significant fraction of NK cells do not express any KIR (46).

4.1.1.4. KIR and HLA Interaction

The role of the KIR receptors is to control NK cell response with its environment.

NK cell recognizes cells expressing self-MHC-I, and kill cells, which lack or down- regulate expression of self-MHC-I expression. NK cell reactivity will depend on which type of KIR is bound to its ligand. An inhibitory KIR will give an inhibitory signal to the NK cell, whereas an activating KIR will stimulate the NK cell, and

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perforins and granzyms will be secreted in order to kill the target cell. Some studies have suggested that activating KIR have a very weak specificity for MHC-I, while other conducted studies did not show this interaction (47). One of the main ligand for inhibitory KIR is HLA-C, but a difference in the amino acid at position 80, results in two groups, HLA-C1 and HLA-C2. HLA-C1 has an asparagines at position 80 (Asn80), and recognize KIR2DL2 and KIR2DL3, whereas, KIR2DL1 recognized by HLA-C2, has a lysine at position 80 (Lys80) (48).

Behind these 2 ligands, there is an evolutionary process (32). An HLA-C locus was first observed in orangutans (49). This HLA-C, called Popy-C, shows only an Asn80, and no HLA-C with Lys80 were present in orangutans. Then, when orangutan and chimpanzee/human predecessors separated, a mutation of Asn80 to Lys80 in HLA-C1 emerged and produced HLA-C2 (50). Binding measurements show that KIR2DL1 and HLA-C2 are stronger and more specific compared to KIR2DL2/DL3 and HLA-C1, which binding is weaker and less specific. Therefore, most probably, in the evolutionary process, the weak KIR2DL2/2DL3-HLA-C1 interaction appeared first, and then the stronger KIR2DL1-HLA-C2 evolved. In humans, both interactions are present. HLA-C2 did not eliminate HLA-C1, in all probability for the reason that, both interactions are complementary.

For HLA-B, which is the ligand for KIR3DL1, there is also a sequence dimorphism at the C-terminal, which gives 2 groups HLA-Bw4 and HLA-Bw6 (51). The binding strength depends on the amino acid sequence at position 80. HLA-Bw4 with an isoleucine residue at position 80 (Ile80) is the strongest inhibitor (52), whereas, HLA-Bw6 with threonine residue at position 80 (Thr80) is weak. Next to the knowing ligand, it has been shown that KIR3DL2 bind to HLA-A3/-A11. Surprisingly, HLA-A3/-A11 was reported to be expressed by Epstein-Barr virus (EBV) transformed B-cells and direct binding of HLA-A3 to KIR3DL2 is peptide specific. Hansasuta et al.

(53), used tetramer HLA-A3 with several antigenic peptides, and demonstrated that only tetramers, which refold with a peptide from EBV, bind to KIR3DL2. The last knowing KIR ligand involved KIR2DL4, which is mainly expressed by trophoblasts cells, and bind to HLA-G (54).

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4.1.1.5. KIR and Diseases

This binding strength between KIR and MHC-I can influence susceptibility to diseases. KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1 bind to their ligand HLA-C and HLA-B with varying strength. As KIR2DL3 and HLA-C1 have a weak binding compared to KIR2DL1 and HLA-C2, KIR2DL1 and HLA-C2/C2 homozygote has a stronger inhibition effect compared to KIR2DL3 and HLA-C1/C1 homozygote. On the other hand, KIR haplotype can also play a role; KIR haplotype A/A homozygote has less activation in comparison to KIR haplotype B/B homozygote, which will have between 1 and 5 activating KIR. Autologous KIR-HLA interaction appears in infectious or autoimmune diseases, whereas in pregnancy, maternal KIR will interact with fetal MHC-I, and in transplantation, donor KIR will interact with recipient MHC-I molecules (55, 37).

In pregnancy, which is in a way, a successful allograft transplantation, KIR-MHC-I interaction plays an important role in preeclampsia. Preeclampsia is a serious complication in pregnancy, where the fetus receives too little blood, which as a consequence leads to maternal and fetal mortality. Hiby et al. (56), show that when maternal KIR receptors, with a KIR haplotype A homozygote (no activating KIR), are in the presence of a fetus, which possess a HLA-C2/C2 homozygote ligand, there is an increasing risk of preeclampsia. The authors explain that, as KIR2DL1/HLA-C2 is a strong association, there is too much inhibition and trophoblasts are not able to move into the uterine arteries. They also show, no association with preeclampsia in individual, which have KIR haplotype A homozygote and HLA-C1 ligand. Finally, with maternal activating KIR (haplotype A/B or B/B) and HLA-C2, the strong inhibition of KIR2DL1/HLA-C2 is balanced by the activating receptors.

Human immunodeficiency virus (HIV) is a viral infection causing immunosuppression and progression of acquired immunodeficiency syndrome (AIDS) over a variable period of time. Several publications (57, 58) show an interaction between KIR3DL1/3DS1 and HLA-Bw4. They demonstrated that patients NK cells, which contain KIR3DS1 and its ligand HLA-Bw4-80Ile, show a slower progression of the disease, compared to patients with no KIR3DS1 or no HLA-Bw4. This is interesting;

beside the role of the T-cells that were largely investigated in HIV this KIR-HLA interaction demonstrates an interestingly anti-viral role of the NK cells.

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In autoimmune diseases, KIR receptors can also be important. In patients with rheumatoid arthritis (RA), expression of KIR2DS2 is much higher compared to healthy individuals (83% vs. 47%). Moreover, this KIR2DS2 is expressed by a CD4⁺/CD28^- T-cells sub-population. In absence of the corresponding inhibitory receptors, this sub-population can be directly activated through KIR2DS2 receptors, instead of the T-cell receptor (TCR), and become cytotoxic (55, 59).

Still, one of the most interesting roles of the KIR-MHC-I interaction appears in allogeneic BM transplantation (see Chapter 7. NK cells and Transplantation).

4.1.2. Ly49 Receptors

As much as KIR receptors are specific to humans, their counter part in mice is Ly49.

As this work is focused on human KIR, mouse Ly49 receptors could not be relevant, but several similarities exist between these two receptors, and a specific characteristic appears with activating Ly49, which makes it interesting.

Initial studies on mice identified genes on chromosome 6 coding for Ly49 receptors (60). Whereas KIR receptors are Ig-related proteins, Ly49 are C-type lectin-related proteins. Ly49 encoding for activating and inhibitory receptors and their ligands are also MHC-I. Ly49 intra-cellular structure has some similarities with KIR receptors.

Two ITIM motifs in the cytplasmic part on the inhibitory receptor, and activating Ly49 recruit DAP-12 molecule with 2 ITAM motifs (31). The extra-cellular part differs from the KIR. Ly49 is attached to the cell membrane through a α-helix of 70 amino acids, on top of which are 2 C-type lectin-like domains, which can bind to its ligand, MHC-I (61). Notice that on human chromosome 12, a Ly49 pseudogene is present in the natural killer complex (NKC) region; this could suggest that human KIR probably evolved from an ancestral common Ly49 (62). Some mice strains, such as C57BL/6 have a Cmv-1 promoter, which makes them resistant to mouse CMV infection, which allows the expression of the activating Ly49H receptor. Mouse CMV encodes an MHC-like protein, m157 that is specific for the activating Ly49H receptor. With this binding, mouse NK cells increase their cytotoxicity and their IFN-γ secretion in order to kill mouse CMV-infected cells (63). As human KIR and

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mouse Ly49 shared several similarities, the human m157 counter part has as not yet been found.

4.1.3. C-type Lectins Receptors

The receptors, belonging to the C-type lectins family, are represented by the NKG2 family. The genes coding for these receptors and the CD94 are located on chromosome 12 (31). These receptors show much less allelic variations comparing to the KIR receptors; they formed a heterodimeric receptor with CD94. Four transcripts has been characterized as, NKG2A, NKG2C, NKG2E and NKG2F (64). A fifth member exists as NKG2D, it is a homodimeric receptor and as its ligands are different from the other NKG2 family, NKG2D receptor will be discussed in Chapter 4.2.1.

NK cells and CTL express NKG2A/CD94. It is the only inhibitory receptor, and it has a long cytoplasmic tail, which expresses an ITIM motif (as do the KIR receptors). All the other CD94/NKG2 are activating receptors, with a short cytoplasmic tail. As the activating KIR, NKG2C has a positive charge amino acid in the trans-membrane region, which allows it to bind to DAP-12 and transduces an activating signal to the cell. In all these signal pathways, CD94 is not involved (22). The ligand for CD94/NKG2 family is the non-classical HLA-E. HLA-E is identical to MHC-I, but has a limited polymorphism and its expression is much weaker on the cell surface.

Moreover and surprisingly, HLA-E peptide binding groove is filled with a peptide derived from the leader segment of other classical HLA-I (65). Binding studies show that CD94/NKG2A-HLA-E is stronger compared to CD94/NKG2C-HLA-E (66).

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4.2. MHC-I Non-Specific Receptors

4.2.1. NKG2D

Unlike to NKG2A and NKG2C, NKG2D is a homodimeric receptor, encoded by chromosome 12. NKG2D is constitutively expressed by NK cells and by CTL. As there is 70% of homology between NKG2A and NKG2C, only 28% of homology exists between NKG2A/C and NKG2D (67). NKG2D is an activating receptor and instead of recruiting a DAP-12 molecule to activate NK cells, it requires a DAP-10 molecule, which has nearly the same role as DAP-12 (68). DAP-12 activates the Syk family tyrosine kinase pathway, which will trigger the activation cascade, whereas DAP-10 triggers the PI-3 kinase pathway (22). There are 2 main ligand families for NKG2D, the MHC-I chain related protein A or B (MIC-A/-B) (69) and the UL-16 binding protein (ULBP) -1/-2/-3 (70). These molecules are not part of the MHC-I family.

What characterizes these molecules is that their expression is correlated with viral infection and is induced under cellular stress. MIC-A/-B and ULBPs are well expressed by CMV-infected cells.

4.2.2. Natural Cytotoxic Receptors

NCR are a specific monomeric receptor family expressed by NK cells; they are not expressed by T-cells. They belong to the immunoglobulin family, but have little homology with human immunoglobulin receptors. There are three receptors, NKp30, NKp44 and NKp46; all three of them are activating receptors and infected cells express their ligands. NKp30 binds to several ligands, such as pp65, a CMV tegument protein (71), BAT3, which is released from tumor cells (72), or B7-H6 another tumor cells protein (73). NKp44 and NKp46 seems to bind to viral hemagglutinins (23). The extra-cellular domain has 2 Ig-C2 for NKp46, and a single Ig-like domain of type V for NKp44 and NKp30. The trans-membrane domain contains positive amino acid and each receptor binds to specific adaptor molecules, containing ITAM motifs. NKp30 binds to a dimmer CD3ζ, and NKp44 to DAP-12 molecules. NKp46 binds to FcRIγ-CD3ζ, an adaptor molecule. As NKp30 and

(39)

NKp46 are constitutively expressed, NKp44 is only expressed in presence of IL-2 (74, 75).

(40)

5. NK Cells Development

In adults, the main site of NK cells generation is the BM. Similar to B- and T-lymphocytes; NK cells develop from a common hematopoietic progenitor that resides in the CD34⁺ cell compartment. BM contains a rich environment of cytokines and growth factors, which can help NK cell development. When mice are treated with radiations, which will destroy the BM, NK cell development is more affected than other haematopoietic lineages. This shows that neither the thymus, nor the spleen, is important for NK cells development (76). Additionally, this was observed either with patients affected with an absence of thymus (DiGeorge Syndrome) (77), or which were thymectomized (78). In all these situations, NK cell number and functionality were found to be normal.

Compared with the understanding of T- and B-cells development, knowledge of NK cell maturation is still at an early stage. Nonetheless, a model incorporating three stages is emerging. The first stage involved NK cell precursors derived from a CD34⁺ HS cells. The second phase, concern immature NK cells, which will express some of the NK cells specific markers. Finally, immature NK cells differentiate further through a process called education to become fully activated NK cells and self-tolerant.

5.1. Stage I: NK Cells Precursors

Two cell lineages are derived from HS cells, the myeloid progenitors (79) and the lymphoid progenitors (80). The first ones will generate erythrocytes, but also monocytes or neutrophiles. The latter, will give rise to the T-, B or NK cells.

Unfortunately, there is no proof of a pure NK cell precursor.

Most of the evidences were found in vitro, by generating NK cells from HS cells with soluble factors. Starting with CD34⁺ HS cells and stroma-cell contact, NK cells precursors arise and could be driven toward NK cells in presence of IL-2 or IL-15.

Several clues show that IL-15 is far more important in the maturation of NK cells than IL-2. IL-2 is produced mainly by activated T-cells and T-cells deficient patients have normal NK cells (77). IL-2 deficiency in humans is associated with a small

The role of natural killer cells in the immune response against CMV infection in immunosuppressed patients

Thesis

Reference