Doctoral thesis from the Department of Immunology, the Wenner-Gren Institute, Stockholm University, Stockholm, Sweden

Phenotypic and functional studies of NK cells in neonates and during early childhood

Yvonne Sundström

Stockholm 2008

Previously published articles were reproduced with permission from the publisher. Printed by Universitetsservice AB, Stockholm 2008 Distributor: Stockholm University Library

 Yvonne Sundström, Stockholm 2008 ISBN 978-91-7155-747-6

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Till mina föräldrar

iii iv SUMMARY

During infancy, before adaptive immunity has matured, innate immunity is thought to be relatively more important. Human natural killer (NK) cells are innate immune cells involved in the control of virus-infected cells and can influence adaptive immunity mainly through cytokine production. This thesis aimed at investigating function and phenotype of NK cells in children from birth and during early childhood and to see if these features are altered in children that develop early allergy, in children latently infected by herpes viruses or born by preeclamptic mothers. In newborn mice, NK-cell function increases and expression of the different NK-cell receptors changes during the first weeks of life. In humans, similar maturation processes in early life are poorly studied. We analyzed NK-cell phenotype and function in cord blood (CB) and in the same children at the age of two and at five years. We demonstrate that the distribution of different NK-cell subpopulations and expression of NK-cell receptors changed with age. The proportion of NK cells was higher in CB compared to two years and at five years of age. The proportion of LIR-1+ NK cells was found to increase with age, while the proportion of CD94+NKG2C- (mainly NKG2A+) NK cells and the level of expression of NKG2D and NKp30 decreased with age. The induction of IFN-γ was slightly lower in CB NK cells compared to later time points. NK-cell cytokines can affect priming of naïve T helper cells and may play a role in development of allergic reactions. Therefore we evaluated CB NK cells from children that developed early allergy. Children that became allergic at the age of five years had a smaller proportion of CD56bright NK cells, which is the NK-cell subpopulation that has a high cytokine producing capacity. CB NK cells from allergic children had a tendency of an increased expression of the activating receptor NKp30 and tended to have a decreased proportion of NK-cells expressing CD94/NKG2C compared to the children that did not become allergic. Herpesvirus infection associates with a reduced risk of becoming IgE-sensitized, and NK cells are important in the control of herpes viruses. In an attempt to find a mechanism to the observed protective effect of herpesvirus latency we studied innate immunity (NK cells and monocytes) in two- year old children seropositive (SP) or seronegative (SN) for cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Herpesvirus seropositivity in early life was associated with reduced proportions of IFN- γ+ NK cells as well as lower intracellular IFN-γ levels upon in vitro stimulation (IL-15 and peptidoglycan). IFN-γ measured in cell culture supernatants and in plasma was lower in SP compared to SN subjects. In addition, SP children also tended to have a decreased proportion of proinflammatory CD14+CD16+ monocytes and lower levels of IL-6 in culture supernatants. The pregnancy disorder preeclampsia is associated with increased proinflammatory cytokine production in the mother and we wanted to study if that had an influence on CB NK cells. In CB, there was a trend towards a reduced expression of NKG2D on NK cells and a decreased NK-cell function in children born by preeclamptic mothers. We also demonstrate reduced TNF and increased IL-8 levels in CB sera from the preeclamptic group. Further, the serum levels of IL-6 in the preeclamptic mothers and their neonates were positively correlated, whereas no such association was found in the healthy group. Taken together, our results suggest that NK-cell populations are dynamic during the first years of life and start to resemble the phenotype of adults after five years of age. Early alterations in the NK- cell populations could lead to insufficient Th1 priming, with an increased risk to develop allergic disease. Early infection by common herpes viruses can influence NK-cell function and might be one important factor involved in early maturation processes of adaptive immunity. The altered NK-cell function and cytokine levels, noticed in CB from pathological pregnancies, suggest that NK cells could be influenced already in utero. These early alterations of innate immunity may affect the development of the child’s immune system, sometimes with beneficial outcome but could in some cases promote pathology.

v vi LIST OF PAPERS

This thesis is based on the following original papers, which will be referred to by their Roman numeral:

I. Yvonne Sundström, Caroline Nilsson, Gunnar Lilja, Klas Kärre, Marita Troye- Blomberg and Louise Berg. The expression of human NK cell receptors in early life. Scand J Immunol. 2007 Aug; 66(2-3):335-44.

II. Yvonne Sundström, Louise Berg, Caroline Nilsson, Gunnar Lilja, Klas Kärre and Marita Troye-Blomberg. A decreased proportion of CD56bright human natural killer cells in cord blood from children that develop early allergy. Manuscript.

III. Shanie Saghafian-Hedengren*, Yvonne Sundström*, Ebba Sohlberg, Caroline Nilsson, Marita Troye-Blomberg, Louise Berg#, and Eva Sverremark-Ekström#. Herpesvirus seropositivity in childhood associates with decreased monocyte- , induced NK-cell IFN-γ production. Under revision. J Immunol. 2008. * #Shared first and last authorship.

vii TABLE OF CONTENTS

SUMMARY...... v LIST OF PAPERS ...... vii TABLE OF CONTENTS ...... viii LIST OF ABBREVIATIONS...... xi INTRODUCTION...... 13 THE IMMUNE SYSTEM...... 13 Innate immunity...... 13 Cells and functions of innate immunity...... 13 Pattern recognition receptors...... 14 Granulocytes...... 15 Mast cells ...... 15 Monocytes/Macrophages...... 16 Dendritic cells...... 17 NK cells ...... 18 Adaptive immunity...... 18 Cells and functions of adaptive immunity...... 18 Antigen-presenting cells (APCs) and the major histocompatibility complex (MHC)...... 18 T cells...... 20 αβ T cells ...... 20 γδT cells...... 22 B cells...... 23 Cytokines...... 23 IFN-α/β...... 24 IFN-γ ...... 24 TNF ...... 24 IL-12...... 25 IL-2 and IL-15 ...... 25 IL-13 and IL-4 ...... 26 IL-10 and TGF-β...... 26 NK CELLS ...... 27 NK-cell definition and distribution ...... 27 NK cell receptors...... 28 The C-type lectin-like receptors...... 29 The Ig-like receptors...... 30 The killer Ig-like receptors (KIRs)...... 30 Leucocyte Ig-like receptor-1 (LIR-1) ...... 31 The Natural cytotoxicity receptors (NCRs)...... 31 Antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells...... 32 Chemokines and NK-cell homing...... 33 NK-cell generation...... 33 Receptor expression during development...... 34 NK-cell education...... 36 NK receptors on T cells ...... 37 NK cells bridge to adaptive immunity...... 37 NK cells and accessory cells ...... 38 HERPESVIRUSES ...... 39 ALLERGY...... 39 The allergic reaction...... 40 Type 1 and type 2 cytokines in allergy...... 40 The hygiene hypothesis ...... 41 Environmental factors in allergy development ...... 41 NK CELLS IN HEALTH AND DISEASE...... 42 NK cells in viral infections ...... 42 NK cells in allergy...... 43 THE IMMUNE SYSTEM OF NEONATES AND YOUNG CHILDREN ...... 44 PREECLAMPSIA...... 45 viii AIMS OF THE STUDY ...... 47 SUBJECTS...... 48 STUDY POPULATION (STUDY IV, PRELIMINARY DATA) ...... 48 MATERIAL AND METHODS...... 49 MATERIAL AND METHODS (STUDY IV, PRELIMINARY DATA)...... 49 RESULTS AND DISCUSSION ...... 52 PAPER I ...... 52 PAPER II...... 53 PAPER III ...... 55 STUDY IV (PRELIMINARY DATA)...... 58 Results ...... 58 Discussion...... 62 CONCLUDING REMARKS AND FUTURE PERSPECTIVES ...... 66 ACKNOWLEDGEMENTS...... 68 REFERENCES ...... 71

Tables

TABLE 1...... 14 TABLE 2...... 30 TABLE 3...... 32 TABLE 4...... 48

Figures

FIGURE 1...... 36 FIGURE 2...... 59 FIGURE 3...... 60 FIGURE 4...... 61 FIGURE 5...... 62

ix x LIST OF ABBREVIATIONS

ADCC Antibody-dependent cellular like receptor cytotoxicity LFA-1 Lymphocyte function- AEDS Atopic eczema/dermatitis associated antigen 1 syndrome LIR Ig-like receptor APC Antigen-presenting cell LPS Lipopolysaccharide BCR B-cell receptor M1 Classically activated BSA Bovine-serum albumin macrophage CB Cord blood M2 Alternatively activated CBA Cytometric Bead Array macrophage CBMC Cord blood mononuclear cell mDC Myeloid dendritic cell CD Cluster of differentiation MHC Major-histocompatibility CLMF Cytotoxic-lymphocyte complex maturation factor MIC MHC class I C chain-related CLP Common lymphoid protein progenitor NCR Natural-cytotoxicity receptor CMP Common myeloid progenitor NFκB Nuclear factor-kappa B CMV Cytomegalovirus NK Natural killer Con A Concanavalin A NKG2D NK group 2, member D CTL Cytotoxic T-lymphocyte NKP NK-cell precursor DC Dendritic cell NKT Natural-killer T cell EBV Epstein-Barr virus NOD Nucleotide-binding ELISA Enzyme-linked oligomerization domain immunosorbent assay PAMP Pathogen-associated FcγR Fragment crystallizable molecular pattern gamma receptor PBMC Peripheral blood FLK2 Fetal liver kinase 2 ligand mononuclear cell Foxp3 Forkhead box P3 pDC Plasmacytoid dendritic cell GM-CSF Granulocyte-macrophage PGN Peptidoglycan colony-stimulating factor PHA Phytohaemagglutinin HIV Human immunodeficiency PMA Phorbol 12-myristate 12- virus acetate HLA Human leukocyte antigen PML Polymorphonuclear HPC Haematopoietic precursor leukocyte cell PRR Pattern recognition receptor ICAM Intercellular adhesion SCF Stem cell factor molecule 1 SCT Syncytiotrophoblast iDC Immature denritic cell SLE Systemic lupus erythematosus IFN Interferon SPT Skin prick test Ig Immunoglobulin TCR T-cell receptor IL Interleukin TGF Transforming growth factor ILT Ig-like transcript Th T helper ITAM Immunoreceptor tyrosine- TLR Toll-like receptor based activation motif TNF Tumor necrosis factor ITIM Immunoreceptor tyrosine- Treg Regulatory T cell based inhibition motif ULBP UL-16 binding protein KIR Killer immunoglobulin- uNK Uterine natural killer cell

xi xii INTRODUCTION

THE IMMUNE SYSTEM

The immune system is able to detect the presence of harmful agents in the host, and the major goal is elimination of the threat. This task is challenging considering the enormous molecular diversity of pathogens and their high replication and mutation rates. The immune system has co-evolved with this complexity of pathogens, generating efficient strategies to fight off invaders. It is composed of several different cell types and soluble factors, which exert different protective functions against harmful invaders. The immune system is commonly divided into two parts, the innate and the adaptive. Although described as separate compartments the innate and adaptive immune systems are tightly interrelated and the combined responses most often give a highly effective protection against pathogens/infection.

Innate immunity

The innate or non-specific immune system is the first line of defense against potentially harmful agents and microorganisms. Preventing entry of pathogens will hold off infections and the first obstruction for pathogens consists of anatomic barriers such as skin and mucous membranes. Physiological conditions, such as temperature, pH and different soluble and cell- associated molecules add up the impediment. In addition, the presence of cells specialized in phagocytosis, cytotoxicity and production of microbicidal agents makes microbial survival more difficult in the host.

Cells and functions of innate immunity Cells of the innate immune system recognize and respond to pathogens by non-specific mechanisms that prevent infection by microorganisms in the host. The mechanisms include recognition of conserved molecular motifs of microbial origin that are distinguishable from molecules of the host. These motifs are collectively called pathogen-associated molecular patterns (PAMPs) and are recognized by pattern recognition receptors (PRRs). By non- specific we mean that these receptors are conserved between individuals of the same species and within the individual during ontogeny. However, the interaction between PAMPs and

13 PRRs may be referred to as specific, as not all PAMPs are recognized by all PRRs. The cells of innate immunity include granulocytes, mast cells, monocytes, macrophages, dendritic cells (DCs) and natural killer (NK) cells.

Pattern recognition receptors Among the most evaluated PRRs are the toll-like receptors (TLRs). There are 10 known functional TLRs in humans [1] (Table 1). TLR2 and TLR4 are expressed on the cell surface of mainly monocytes/macrophages, DCs and mast cells. TLR2 recognizes peptidoglycan (PGN), a type of PAMP present in the cell wall of almost all bacteria, and receptor-ligand interaction induces cellular activation and production of proinflammatory cytokines by the cell [2].

Ligands for Toll-Like Receptors- What they see on pathogens TLR Natural Ligand TLR1 (partnered with TLR2) Bacterial triacyl lipopeptides and certain proteins in parasites TLR2 (partnered with TLF6) Bacterial diacyl lipopeptides, lipoteichoic acid from Gram-positive bacteria, and zymosan from the cell wall of yeast, peptidoglycan TLR3 Double-stranded RNA from viruses (i.e., West Nile virus) TLR4 Endotoxin (lipopolysaccaride) from Gram-negative bacteria TLR5 Flagellin from mobile bacteria TLR7 Single-stranded RNA from viruses (i.e., HIV) TLR8 Single-stranded RNA from viruses (i.e., HIV) TLR9 CpG DNA from bacteria or viruses TLR10 Unknown Table 1. Immune recognition by TLRs. The ligands for each functional human TLR. Adapted from Science 2006; 312 (5771):184–187 [1]. Reprinted with permission from AAAS.

Another type of PRRs are the nucleotide-binding oligomerization domains (NODs) that mainly are located in the cytosol of monocytes, DCs and epithelial cells [3]. NOD1 and NOD2 are members of a protein family that are involved in regulation of apoptosis as well as for innate recognition of specific bacterial components [3,4]. NOD2 recognizes a peptide derived from PGN that is present in almost all bacteria, while NOD1 mainly recognizes PGN- components derived from Gram-negative bacteria and ligand recognition leads to induction of inflammatory responses through nuclear factor-kappaB (NFκB) signaling pathways [5,6].

14 Granulocytes Polymorphonuclear leukocytes (PML), or more commonly called, granulocytes are a group of leukocytes defined by the presence of granules in their cytoplasm and a nucleus that is often lobed in segments within the cell. There are three different types of granulocytes, including neutrophils, basophils and eosinophils. Of the circulating leukocytes in humans, neutrophils are the most numerous (50-70%). These cells circulate in the blood, but upon infection they are rapidly recruited to the site of infection and are important in the subsequent progression of immune responses against the microbe. Neutrophils are very motile phagocytes, but are also involved in the inflammatory response that is induced through the release of antimicrobial molecules, stored in their granules, and also through secretion of chemokines that recruit other immune cells [7,8]. Less than 1% of the circulating leukocytes are basophils. Basophils are not phagocytic but they are suggested to be important in the early regulation of immune responses against parasitic organisms through the release of IL-4 and IL-13 [9,10]. Basophils have also been found to play a role in allergic reactions through their release of e.g. histamine [11] and cytokines [12] that contribute to allergic inflammation. Eosinophils constitute about 1-6% of all leukocytes. They are found in blood and organs like thymus, spleen and lymph nodes under normal conditions. Like those of basophils, the functions of eosinophils include combating parasitic infections [13] and eosinophils are also associated with the pathogenesis of allergic disease through the release of their granule proteins [11]. Eosinophils have also been shown to function as professional antigen presenting cells (APCs) [14].

Mast cells Like granulocytes, mast cells also have large granules in their cytoplasm and due to their similarity with basophils it has been suggested that mast cells are the tissue resident form of basophils. However, the developmental processes, function and morphology differ indicating that they are different cell types [15]. Mast cells are often mentioned in relation to pathology of allergic disease and therefore associated with harmful reactions. In a sensitized person allergen-specific immunoglobulin (Ig) E antibodies bind to the high affinity Fcε receptor I (FcεRI) on tissue-resident mast cells [16]. The subsequent exposure to the allergen that is recognized by the FcεRI-bound IgE

15 antibodies on the mast cell results in crosslinking of the antibodies, leading to degranulation of inflammatory mediators such as histamine, heparin and proteases that act on surrounding tissues. This results in an immediate phase reaction that, within minutes, leads to increased vascular permeability, mucous secretion and stimulation of sensory nerves that generates symptoms like nasal congestion, itching and sneezing. Mast cells can also be an important factor in reactions that are followed, hours later, by recruitment of cells like eosinophils, T cells and macrophages, to the area through the induction of chemokines and adhesion- molecule expression on endothelial cells. At the site, these cells become activated and the effector functions of the different cells may result in a prolonged inflammation [17,18]. However, mast cells are of great significance in other immune responses, being one key effector-cell type that protects against morbidity and pathology of infection. Mast cells are found in tissues in close proximity to blood vessels and during early responses they are involved in the induction of inflammation. For instance, upon bacterial infection mast cells are induced to release tumor necrosis factor (TNF) that is important for the recruitment of other immune cells, such as neutrophils [19]. Additionally, mast cells exhibit effector functions such as secretion of antimicrobial peptides that can kill invading bacteria [20]. By releasing proteases mast cells can disarm endogenous toxins produced to battle bacteria e.g. endothelin-1 (ET-1), [21]. Mast cells have also been shown to release effector molecules that degrade other toxins and venoms that are similar to ET-1 [22]. Another interesting feature of mast cells is the involvement in regulation of immune responses. Mast cell derived IL-10 can have immunosuppressive functions important in limiting the duration and magnitude of adaptive immune responses [23]. Taken together, these features of mast cells indicate their importance in damage control and in regulation of immune responses.

Monocytes/Macrophages Monocytes originate in the bone marrow and are released into the peripheral circulation. In human peripheral blood, 5-10% of the leukocytes are monocytes and they are morphologically and functionally heterogeneous. The initial marker for identification of monocytes was the expression of CD14. The combination of CD14 and CD16 allows the identification of two subsets of monocytes: CD14++CD16- cells and CD14+CD16+ cells. In the blood of healthy individuals the distribution of these populations is about 90% and 10%, respectively [24]. Functionally, the CD14+CD16+ cells appear to produce more proinflammatory cytokines and have higher APC activity than the CD14++CD16- monocytes

16 [25]. Monocytes are important effector cells in innate immunity and recognize microbial components through their expression of PRRs [26]. The CD14+CD16+ subset has been reported to have higher levels of TLR2 [27] and TLR4 [28]. This indicates an important role of this subset in microbial responses. After release from the bone marrow, monocytes circulate in the blood before they enter tissues were they differentiate into macrophages. Macrophages are phagocytic cells that clear the body of debris and they are potent APCs. Macrophages can be divided in subpopulations that reveal different functions depending on the mode of activation. The so-called, classically activated macrophages (M1) develop in response to IFN-γ and microbial products and are distinguished through their increased endocytic functions, ability to present antigens and ability to produce pro-inflammatory cytokines [29]. They also secrete chemokines that induce recruitment of NK cells and T cells [30]. The alternatively activated macrophages (M2) are further subdivided into three groups of cells that are activated differently and have separate functions. M2a macrophages are activated by anti-inflammatory cytokines, like IL-4 and IL- 13 and the function of M2a cells include secretion of chemokines that recruits eosinophils, basophils and T cells that promotes anti-inflammatory immune responses [30]. M2b cells are induced upon stimulation with lipopolysaccharide (LPS) or IL-1β and these cells are characterized by low IL-12 and high IL-10 production, which promote the subsequent induction of type 2 adaptive immune responses [30]. Finally, the M2c macrophages are activated by IL-10, transforming growth factor (TGF)-β or glucocorticoids (a class of steroid hormones) and their functions include down regulation of pro-inflammatory cytokines and increased scavenging activity for debris [31].

Dendritic cells DCs form another heterogeneous family of haematopoietic cells. Two main developmental pathways from hematopoeitic-progenitor cells in humans generate either myeloid DCs (mDCs) or plasmacytoid DCs (pDCs). Depending on their location, they display different morphology and function [32]. Immature DCs have high endocytic activity. Maturation of DCs is induced by sensing microbial products like PAMPs, tissue damage [33], interaction of Fc receptors or ligation to CD40L on T cells [34]. Dendritic cells process endocytosed proteins or cytosolic proteins into proteolytic peptides and load these on to major histocompatibility complex (MHC) class I and class II molecules. This process induces

17 maturation of DCs and migration to lymph nodes. In the lymph nodes, DCs present antigens to T cells and thereby initiate antigen-specific adaptive immune responses. The mDCs are distributed in peripheral tissue, secondary lymphoid organs and in the blood. Tissue resident DCs called Langerhans cells, are located in the epidermis of the skin and interstitial DCs are found in interstitial connective (non-lymphoid) tissues. The pDCs are found in peripheral blood and lymphoid tissues and respond to viruses by producing vast quantities of type 1 IFNs (IFN-α, IFN-β) [35]. It is generally considered that monocytes are precursors of DCs and human monocytes are indeed capable of differentiating into some of the DC linages in vitro [36]. It is unclear whether this occurs under physiological conditions in humans, although experiments using murine models have reported indications that sustain this hypothesis in vivo [37].

NK cells NK cells are important cells of innate immunity and are described below (starting page 27).

Adaptive immunity

The adaptive or specific immune system is composed of T- and B-cells that generate specific responses to antigens. One distinguishing feature of adaptive immunity is the rearrangement of T-cell receptor (TCR) or B-cell receptor (BCR) genes that results in a vast diversity in their respective antigen-specific receptors. This results in an enormous variability of receptors and soluble antibodies that increases the probability for recognition of specific antigens.

Cells and functions of adaptive immunity

Antigen-presenting cells (APCs) and the major histocompatibility complex (MHC) Recognition of antigens by T cells requires help/presentation from other cells. These cells are termed APCs and can be divided into two groups: the professional and the non- professional APCs. DCs, monocytes/macrophages and B cells fall into the category of professional APCs while all nucleated cells are non-professional APCs.

18 Presentation of antigen by cells is achieved through proteins encoded within the major histocompatibility complex (MHC), called MHC class I and MHC class II molecules. MHC class I can be further subdivided into MHC class Ia (or classical MHC class I) and MHC class Ib (or non-classical MHC class I). In humans the MHC complex is termed human leukocyte antigens (HLA). MHC class Ia includes HLA-A, -B and -C molecules and the MHC class Ib molecules are named HLA-E, -F and -G. MHC class II molecules include HLA-DP, HLA-DQ and HLA-DR. Nearly all nucleated cells (including professional and non-professional APCs) express MHC class I that presents endogenous antigens. After degradation of cytosolic proteins, e.g. proteins derived from the cell itself and from viruses or intracellular bacteria, into small peptides, these are transported to the cell surface as a complex with MHC class I molecules. The complex is presented to naïve CD3+CD8+ T cells in secondary lymphoid tissues, but for subsequent generation of CD3+CD8+ effector T cells, sequential signals are required. Ligation of costimulatory molecules like CD80 and CD86 on the professional APC, engaging CD28 on the T cells provides one signal that is needed for the differentiation into effector T cells. The T cell also needs signals derived from cytokines like IL-2. The activated T cells mature and migrate to sites of inflammation where they find the same antigen-MHC I complex and exert their effector functions. For CD3+CD8+ T cells these functions include killing of the target cell and/or production of cytokines that in different ways can enhance the response towards the pathogen. Professional APCs endocytose exogenous particles and proteins and process these into peptides, which are presented by MHC class II on the cell surface. Peptides in association with MHC class II are recognized by naïve CD3+CD4+ T cells and after the simultaneous interaction by costimulatory molecules the T cells become activated and are induced to mature. The functional responses by activated CD3+CD4+ T cells include production of cytokines, which regulate other parts of the immune system. The functions of MHC class Ib molecules are in large unknown and their limited polymorphism restricts the variability of peptides that can be presented. HLA-E bind sequences derived from MHC class Ia molecules and cell surface expression of HLA-E is dependent on binding of peptides provided by HLA-A, -B and –C molecules [38,39]. Due to this dependency, HLA-E is expressed by all cells expressing class Ia molecules, rendering a broad tissue distribution. Both HLA-E- and HLA-G-peptide complexes, when recognized by NK cells, either inhibit or trigger NK-cell activity [40,41]. Except for small amounts of HLA- G in some human fetal and adult tissue, HLA-G is upregulated on fetal cells (trophoblast

19 cells) in contact with maternal tissue [42] and are suggested to play a role in fetal tolerance by prevention of NK-cell mediated lysis [40,41]. Four membrane-bound forms (HLA-G1-4) and three soluble forms (HLA-G5-7) of HLA-G exist [43]. HLA-F transcripts have been detected in fetal liver, adult skin, placenta, B cells and T cells. The function of HLA-F is still elusive. The MHC class I chain related (MIC) molecules, MICA and MICB, show homology with HLA molecules but do not bind peptides needed for antigen presentation. MIC-proteins are induced on the surface of stressed cells and are recognized by cells expressing the NKG2D receptor. Some cells, like tumor cells and syncytiotrophoblast cells in the placenta, can shed soluble MIC molecules. The binding of these soluble molecules to the NKG2D receptor prevents further NKG2D dependent activation of the T- or NK-cell [44]. CD1 molecules are also related to the class I MHC molecules and present lipid antigens to T cells and NKT cells.

T cells Progenitor-T cells emanate from the bone marrow and migrate to the thymus for maturation. In the thymus, selection of developing T cells (named thymocytes) with functional TCRs takes place. The selection process authorizes the survival of T cells that recognize self-MHC molecules (positive selection), but only if the binding to MHC molecules presenting self-peptides is not too strong (negative selection). Along this maturation/selection pathway the TCR undergoes genetic rearrangement. In this process, there is recombination of multiple V, D and J genes that are randomly selected and assembled into two TCR chains, rendering a TCR with a distinctive specificity for a MHC-peptide complex. The final result of this process is a T cell with a functional TCR complex including the signaling chains of the CD3 complex. The TCR is expressed as a heterodimer, made up of a α and a β chain (αβ TCR), or a γ and a δ chain (γδ TCR).

αβ T cells

T cells with αβ TCRs (αβT cells) can be sub-divided into CD3+CD4+ T cells and CD3+CD8+ T cells and are generally termed as T helper (Th) cells and cytotoxic T- lymphocytes (CTL), respectively. Naïve CD3+CD4+ T cells can differentiate into a variety of T-cell subsets depending on the efficiency of the TCR stimulation, the magnitude of costimulation by APCs and the

20 surrounding cytokine milieu. The involvement of IL-12 induces differentiation of Th1 cells, IL-4 promotes differentiation of Th2 cells and IL-23 and IL-1β renders Th17 cells [45,46]. The Th1 response, effective against intracellular pathogens, leads to the production of cytokines like IFN-γ, IL-12 and IL-18 that mainly promotes cellular immunity and gives rise to inflammation with e.g. activation of macrophages and induction of antigen-presenting activity [47]. The Th2 response is effective against extracellular pathogens. Th2 cells produce cytokines like IL-4 that promote humoral immune responses including Ig-class switching to e.g. IgE and IgG4 by human B cells [48,49]. The characteristic of Th17 cells is the production of IL-17 [50]. All together six different homodimers exist that form the IL-17 family (IL-17A to IL-17F) of cytokines [51]. Human blood monocytes cultured in vitro and stimulated with rIL-17A, release TNF and IL-1β suggesting an important role for IL-17 in inducing and/or maintaining an inflammatory response [52]. Although the function of this Th subset is not fully clear, present data indicate a role in the immune responses against extracellular pathogens where Th1 and Th2 type of immunity are insufficient. This subset of T cells also seems to be involved in driving autoimmune diseases [53]. Another subset of T cells, called regulatory T cells, (Tregs) is important in controlling the magnitude and/or duration of immune responses and also to sustain immunological unresponsiveness to self-antigens, functions that are crucial to prevent detrimental effects to the host. The main hallmark of Tregs is expression of the transcription factor forkhead box P3 (Foxp3) and CD25, although exceptions exist (see below). Two central Foxp3 expressing populations described in the literature are referred to as natural CD4+CD25+highFoxp3+ T reg cells and peripherally induced CD4+CD25+highFoxp3+ T reg cells. The former Treg type develops from CD4+ T cells within the thymus while the latter is induced by cytokine stimulation in vitro and is thought to be induced from CD4+CD25- T cells in the periphery in vivo [54,55]. The immunosuppression mediated by these cells has been suggested to require cell-cell contact that involves cell-surface bound TGF-β1 [56]. Another type of peripherally induced Treg cells is termed Tr1 and they are induced from naïve T cells upon IL-10 stimulation. These cells do not express CD25 or Foxp3, but are defined by their production of IL-10 and TGF-β and are distinguished from Th cells by their minute production of IFN-γ and no IL-2 or IL-4 [57]. Th3 cells are antigen specific and produce TGF-β upon activation and some, but not all, appear to express Foxp3 [58].

21 Natural killer T (NKT) cells are a small heterogeneous population of CD4+, CD8+ or CD4- CD8- αβT cells that also express NK-cell receptors. Most of the αβ TCRs of human NKT cells consist of Vα24-Jα18/Vβ11 chains that recognise the MHC-like molecule CD1d. APCs constitutively express CD1d that binds lipids and glycolipids for presentation to NKT cells. The activation of NKT cells induces up-regulation of costimulatory molecules on the cell surface, which augments the antigen presenting activities by DCs and cytokine production of both Th1 and Th2 types [59]. The CD3+CD8+ T-cell effector function mainly involves induction of apoptosis of target cells but they are also capable of producing cytokines. Apoptosis can be induced through release of cytotoxic proteins or through interaction with the membrane-bound Fas ligand (FasL) on the T cells with Fas on the target cell.

γδT cells

The majority of mature T cells in peripheral blood are αβT cells, while a minority (1-10%) expresses an alternative γδ TCR heterodimer in association to CD3 (γδT cells). There are only a small number of available variable (V) γ and δ genes but the diversity of the γδ TCR repertoire is still extensive, due to e.g. N nucleotide insertions during gene rearrangement [60]. In healthy adults, the most prominent type of γδT cells in peripheral blood express the Vγ9 chain together with Vδ2, while the second most prominent type express various Vγ chains in association with Vδ1 [61]. However, the lymphocyte distribution in the intestine is different, where the majority of γδT cells are Vδ1+. The ligands for these subpopulations differ and the Vγ9/Vδ2 T cells mainly recognize phosphorylated non-peptidic molecules [62], while e.g. the intestinal Vδ1+ γδT cells recognize stress-induced MHC class I-related MICA and MICB molecules [63,64]. The recognition of antigens by γδ (Vγ9/Vδ2) T cells occurs independently of class I and class II MHC molecules or the MHC-related CD1 molecules [62]. Rather, it was recently suggested that activated γδT cells themselves can have MHC II related antigen presenting features [65]. The effector functions of γδT cells include killing of target cells through release of perforin and some γδT cells also express FasL which upon interaction with its receptor induces apoptosis of the target cell [66]. γδT cells also have the capacity to produce cytokines and are in particular potent producers of the proinflammatory cytokines IFN-γ and TNF, indicating an important role in early responses to infections [62].

22 The complexity of γδT-cell biology makes it difficult to designate them to either the innate or the adaptive part of the immune system. Since γδT cells rearrange TCR genes to produce receptor diversity and also form memory cells they could be considered as part of the adaptive immunity. Nevertheless, some subsets respond rapidly to common microbial molecules, which indicates that specific γδ TCRs may function similarly to PRRs, which are attributes of innate immunity.

B cells B cells are essential in the generation of humoral immune responses through the production of soluble antibodies. The generation of mature immunocompetent B cells takes place within the bone marrow. During several developmental stages, a random rearrangement of immunoglobulin genes generates B cells expressing IgM or IgD on the cell surface, as part of the BCR. Naïve B cells that are not self-reactive exit the bone marrow and migrate to lymphoid organs. The recognition of an antigen by the BCR on B cells with a simultaneous interaction with Th cells promotes the development germinal centers in lymph nodes. In the germinal centers the antigen-specific B cells are expanded, diversified and selected for high- affinity variants of BCRs. These form the compartment of antibody producing plasma cells and long-lived memory B cells. The quantity and the array of antibody isotypes produced by the plasma cells is in large determined by the help of cognate T cells through cell-associated signals obtained from costimulatory molecule interactions, but also through Th cell-derived cytokines. In human the Ig subclasses include: IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgE [67]. In an individual cell the class switch renders immunoglobulins with the same antigen specificity but the isotype determines the biological function.

Cytokines

Cytokines are proteins involved in cellular communication and are secreted by a variety of cells, including white blood cells, in response to different stimuli. Chemokines are a group of cytokines important for the migration and localization of cells to specific sites such as inflamed tissue. These molecules are very important in modulating both innate and adaptive immune responses. Numerous cytokines for cellular communication exist and only a few, of relevance for this thesis, will be further described.

23

IFN-α/β

IFN-α and β are included in a group of cytokines, type I interferons, which share a common receptor. An important part of host defense against viral infections is an early production of type I interferons by the infected cells [68]. The receptor for these cytokines is expressed on most cells and receptor binding results in transcription of genes encoding other cytokine and cytokine receptors [69], redistribution of immune cells [70-72], proliferation of memory CD8 T cells [73] and also help in regulating NK-cell homeostasis, activation and function [74,75]. The effect of type I interferons on NK cells include intensified NK-cell mediated cytotoxicity and indirect enhancement of proliferation through induction of IL-15 [76,77]. Type I interferons are also important regulators of NK-cell activity against tumors [75].

IFN-γ

The type II interferon, IFN-γ, is mainly produced by activated Th1 cells [78], activated type 1 CD8+ T cells [79] and NK cells after stimulation by cytokines like IL-12 and IL18 [80]. The heterodimeric IFN-γ receptor [81] is expressed on most epithelial cells, macrophages, B cells and activated T cells [82]. IFN-γ interaction with its receptor induces transcription of genes, promoting different physiological and cellular responses. Among these responses are induction of antiviral and anti-tumor effects by e.g. increased NK-cell cytotoxicity [83], activation of APCs [84,85] and promotion of Th1 differentiation [86]. NK cells are regulated in an autocrine manner by IFN-γ, resulting in enhanced secretion of the cytokine [87]. Further, IFN-γ also regulates its own receptor by down-regulating the expression on the cell surface [85] and in Th2 cells this results in cellular desensitization to IFN-γ [88]. The effect of IFN-γ on isotype regulation in B cells is inhibition of class switching to IgE [89,90].

TNF Macrophages and monocytes are major producers of the pro-inflammatory cytokine TNF, but many other cell types including NK cells, mast cells, endothelial cells, adipose tissue and fibroblasts contribute to the production. During early immune reactions against e.g. microbial

24 stimuli large amounts of TNF are released, which induce the production of other pro- inflammatory mediators that can strengthen the resistance against infection [91]. One important function of TNF is the induction of apoptotic cell death [92]. The inflammatory response mediated by TNF can also cause pathological complication associated with autoimmune disorders including rheumatoid arthritis, Crohn's disease and asthma [91].

IL-12 The pro-inflammatory cytokine IL-12 is a heterodimeric molecule composed of the two subunits p35 and p40, which together forms the biologically active IL-12p70 molecule. Macrophages, DCs, monocytes and neutrophils are major sources of IL-12 [93]. IL-12- production is induced in these cells upon microbial stimulation or after interaction of CD40 with CD40L on activated T cells in combination with IFN-γ [94]. IL-12 further induces IFN-γ production in memory Th1 cells that results in a positive feedback loop, with stabilizing effects on the Th1 polarization. IL-12 also has a self-regulatory function. In memory Th1 cells IL-12 induces the expression of IL-10 [95], which inhibits further production of IL-12 [96]. The IL-12 receptor is constitutively expressed, at low levels, on NK cells, which gives them the ability to respond rapidly to IL-12. IL-12 induces NK cells to produce IFN-γ [97], increases their cytotoxic activity and have a proliferative effect on pre-activated NK cells (and T cells) [93].

IL-2 and IL-15

The receptor for IL-2 and IL-15 consists of two shared subunits (β and γ) [98] and a cytokine-specific α-subunit [99]. Different assembly of the subunits renders different binding affinity for its ligand. For both IL-2 and IL-15 the β and γ heterodimer has intermediate affinity, while the high affinity receptor requires assembly of all three subunits (αβγ). All NK cells express the intermediate affinity IL-2 receptor, while CD56bright NK cells also constitutively express the high affinity receptor. Since the CD56bright NK cells express the IL- 2Rαβγ they respond to low doses of IL-2 in vitro by proliferation [100,101]. IL-2 is also part of induction of cytotoxicity by upregulating the perforin expression in NK cells [102]. IL-15 has been shown to be crucial for NK-cell development and is also important for NK- cell proliferation and survival [97,103-105]. NK cell-mediated cytotoxicity requires a

25 conjugate formation where cellular adhesion molecules establish binding between NK cells and target cells. One such molecule is LFA-1 on NK cells that engage ICAM-1 on target cells. IL-15 and IL-2 are important in the initiation of NK-cell mediated cytotoxicity by inducing surface expression of LFA-1 [106]. CD4+ T cells are the major source of IL-2, but also CD8+ T cells and activated DCs can produce IL-2 [107,108]. IL-15 protein is produced by many different cell types, but primarily by monocytes and DCs [109,110].

IL-13 and IL-4 Both IL-4 and IL-13 have been implicated in allergic disease. They are each capable of inducing isotype switching to IgG4 and IgE in human B cells [49,111,112] and can even have a synergistic effect on IgE synthesis [112]. Both cytokines induce proliferation of activated B cells [48,113] but have different effects on T cells. IL-4 is produced by Th2 cells and is important for further development and differentiation of Th2 cells [114] while IL-13 is not capable of inducing Th2 differentiation, since T cells do not express the IL-13 receptor [48].

IL-10 and TGF-β The biological effect of IL-10 is anti-inflammatory and immune suppressive. Several subsets of T cells, monocytes, DCs and B cells produce IL-10. Class switching in B cells can be induced by IL-10 leading to high amounts of IgG1, IgG3 and IgA [67]. IL-10 indirectly suppresses activation of NK cells through the inhibition of IL-12-production [96]. Like IL-10, TGF-β is also a cytokine with immune suppressive effects. TGF-β mediates inhibition of surface expression of NKp30 and NKG2D on the cell surface of NK cells and also reduces NK-cell cytotoxicity against immature DCs in vitro [115] NK-cell production of IFN-γ is suppressed by TGF-β [116].

26 NK CELLS

NK-cell definition and distribution

NK cells are described as large granular lymphocytes and in humans they represent approximately 15 % of circulating lymphocytes in blood [117]. Human NK cells are broadly distributed and, except from blood, are also found in small numbers in lymphoid tissues like lymph nodes, spleen and tonsils [118]. In the liver, NK cells make up a large proportion of lymphocytes [119] and during pregnancy, a predominant proportion of infiltrating leucocytes in the decidua are NK cells [120]. NK cells belong to innate immunity, as they do not need prior immunization in order to exert their activity. These cells have even been found to express mRNA for some TLRs, with relatively high expression of TLR-2, -3. -5 and -6 but very limited levels of TLR-4, -7, -8 and -9 in healthy adult blood donors [121]. Although NK cells do not appear to express TLR-7 or -8, the stimulation with related agonists results in indirect activation of NK cells through cytokines like IL-12 and IL-18 produced by i.e. myeloid cells [121]. In a recent study, surface expression of TLR-2, -3 and also TLR-4 on NK cells from cord blood (CB) was detected [122]. Even if these differences in TLR-4 expression could be due to differences in NK-cell features in CB and adult blood cells, caution should be taken in the interpretation of mRNA expression as an indication of expression of functional proteins on the cell surface. In a monocyte study, where TLR4 was evaluated, no consistency was found between mRNA levels and surface expression of TLR4 [123]. Although NK cells are generally not considered to have memory functions they have been described to respond better to haptens upon restimulation, in mice devoid of T cells and B cells. The authors suggested that this might indicate a function where NK cells can mediate long-lived, antigen-specific adaptive recall responses independent of B cells and T cells [124], but the concept is controversial and further studies are needed. Human NK cells are identified as lymphocytes that express CD56 but lack CD3 and can be further subdivided into two subsets based on the cell surface expression of CD16 and CD56. The majority of NK cells in peripheral blood expresses low levels of CD56 and high levels of CD16 and is commonly called CD56dim NK cells (CD56dimCD16+CD3- lymphocytes). The minority of NK cells in peripheral blood is the CD56bright NK-cell subpopulation, which expresses high levels of CD56 and lack CD16 (CD56brightCD16-CD3- lymphocytes) [125]. These subpopulations of NK cells are generally considered to differ in their effector functions.

27 The main effector function of the CD56dim subpopulation is cellular cytotoxicity, but they can also produce low levels of cytokines. The CD56bright NK cells are generally considered as more potent producers of cytokines and in particular IFN-γ. However, this functional dichotomy between the two main NK-cell subsets (CD56bright and CD56dim) does not always comply. The above stated differences in function between the subtypes of human NK cells appear to be valid upon stimulation with e.g. IL-12, IL-15 and IL-18 [80,126]. Following in vitro stimulation with malaria (Plasmodium falciparum) infected erythrocytes, CD56dim NK cells have a high capacity to produce IFN-γ and CD56bright NK cells show increased signs of cytotoxicity [127]. In another study, where freshly isolated peripheral blood NK cells were stimulated with MHC class I-deficient tumor cells (K562), a larger fraction of CD56dim NK cells produced IFN-γ [126]. Anfossi et al suggest a functional definition based on the outcome of the responses after exposure to target cells or cytokines where the CD56bright NK-cell subset would be referred to as “cytokine responsive” and the CD56dim NK cells as “target-cell responsive” [126]. In HIV positive individuals an expanded population of CD56-CD16+ NK cells has been described and these cells are hyporesponsive [128,129].

NK cell receptors

Through the divergent and unique repertoire of surface receptors, NK cells can recognize alterations in the expression of major histocompatibility complex (MHC) class I or class I-like molecules on target cells, respond to stress signals, molecules induced by pathogen transformation of cells and to cytokines. One of the strategies the NK cells use to recognize target cells is through detection of “missing-self”, which is dependent on MHC class I molecules [130]. A balance of negative and positive signals from cell-surface receptors determines the regulation of NK-cell activity. Cells with a normal or unaltered expression of MHC class I are protected from NK-cell-mediated killing. During e.g. virus infection, self- MHC molecules can be down regulated resulting in a lack of expression of self-molecules. The reduced interaction of inhibitory receptors with MHC class I results in a predominant positive signal that leads to activation of the NK cell with target-cell elimination as a result. NK cell can also express receptors that have non-MHC-molecule ligands [131]. Some of the receptors on NK cells will be further described below and are divided into two classes of structurally distinct receptors: C-type lectin-like receptors and Ig-like receptors.

28 The C-type lectin-like receptors The CD94 and NKG2 receptors belong to the C-type lectin receptor family and form heterodimers, with a notably high expression on CD56bright NK cells and γδ T cells [132] but also on activated CD8+ T cells and to some extent on CD4+ T cells and CD56dim NK cells [133-135]. Both inhibitory and activating functional forms of this heterodimer exist and the functional specificity is determined by which NKG2 molecule is associated to the CD94 molecule. CD94 lacks a cytoplasmic domain for signal transduction, while NKG2A and the splice variant NKG2B mediate inhibitory signals through intracellular immunoreceptor tyrosine-based inhibition motifs (ITIMs) [136]. All other NKG2 molecules that form heterodimers with CD94, including NKG2C, NKG2E and the splice variant NKG2H, mediate activating signals through DAP12 associated to immunoreceptor tyrosine-based activation motifs (ITAMs) [137,138]. The ligand for CD94/NKG2A/B/C/E/H complexes is the MHC class Ib molecule HLA-E [139,140]. NKG2D and NKG2F are also C-type lectin receptors, but they do not associate with CD94. The activating receptor NKG2D is expressed as a homodimer that signals via its associated adaptor protein DAP10 [141]. In humans, NKG2D is expressed on virtually all NK cells, CD8+αβ T cells and γδ T cells [142]. The expression levels can be altered by exposure to cytokines where e.g. the levels of NKG2D increase after exposure to IL-15 [143] and IL-12 [144]. The ligands for NKG2D are MICA and MICB, and human cytomegalovirus UL16 binding protein (ULBP) 1-4 [142,145,146]. These ligands are expressed at low levels on normal cells but are frequently expressed on tumor cells and can be induced on cells infected by viruses [147,148]. So, in the surveillance of tumors, expression of MICA and MICB by leukemic cells increases the susceptibility to cytotoxicity by NKG2D-expressing NK cells [144,149]. The interaction of NKG2D with its ligand also induces increased expression of CD25, proliferation and cytokine secretion by NK cells. [150]. NKG2F expression appears to be restricted to intracellular compartments and does not seem to be associated with CD94. NKG2F associates with the adaptor protein DAP12 and may provide activating signals by interacting with ligands present intracellularly. It is possible that NKG2F regulates cellular activation through competition for DAP12 with other receptors [151].

29

Receptor (alternative Ligands Function Signaling Refs name) (alternative name) CD94/NKG2A HLA-E Inhibitory ITIM [136,139,140] (CD159a) CD94/NKG2C HLA-E Activating DAP12 [137,139,140] (CD159c) NKG2D MICA, MICB, ULBPs Activating DAP10 [141,142,152] (CD314) NKG2F Unknown Activating DAP12 [151] CD161 LLT-1 (osteoclast Inhibitory ITIM [153,154] (NKR-P1A) inhibitory lectin, CLEC2D) NKp80 AICL (CLEC2B) Activating Unclear [155-157] (KLRF1, CLEC5C) Tyrosine-based motifs (untypical ITIMs) suggested, with undefined adaptor proteins Table 2. General summary of some C-type lectin-like receptors on human NK cells.

The Ig-like receptors

The killer Ig-like receptors (KIRs) The KIRs are expressed on NK cells (preferably CD56dim NK cells) and on a minor subpopulation of both CD8+αβ and γδT cells [158,159]. The ligands for KIRs are MHC class I molecules (including HLA-A, -B, -C and -G) [160]. KIR interaction with MHC class I molecules results in inhibitory or activating signals. The inhibitory KIRs have a long cytoplasmic tail containing ITIMs that mediate negative signals. The activating KIRs have short cytoplasmic tails and a positively charged amino acid in the transmembrane domain. Association of the adaptor protein DAP12 to the charged amino acid in the transmembrane domain allows for activating signals through ITAMs of DAP12 [161,162]. To date, 16 different KIR genes have been described and the genetic setup differs between individuals. The KIR genes are highly polymorphic and different NK-cell clones express a random combination of the present KIR genes, rendering a great diversity of the KIR-receptor repertoire between individuals and when comparing NK cells within one individual [160,163]. Although the gene expression is highly variable, there is some degree of organization. Two main groups, the A haplotype and the B haplotype, have been characterized with different KIR gene content. The A haplotype only exhibits one gene for an activating KIR and several inhibitory KIRs, while the B haplotype is more diverse in gene

30 content and contain several activating KIR genes [163]. In nearly all haplotypes, so-called framework genes are present, including the three KIR genes 3DL3, 2DL4 and 3DL2 [160].

Leucocyte Ig-like receptor-1 (LIR-1) Another type of Ig-like receptor is LIR-1; also called Ig-like transcript (ILT)-2 or CD85j. LIR-1 is expressed on NK cells and T cells but also on myeloid cells like B cells, monocytes and DCs [164]. LIR-1 recognizes MHC class I molecules on surrounding cells. Among MHC molecules, LIR-1 binds to HLA-A, -B, -C and HLA-G [164]. HLA-G has the highest affinity while UL18, a virally encoded MHC-like molecule, has by far the highest affinity for LIR-1 [165,166]. The function of the receptor is inhibitory and binding results in negative signaling mediated by four intracellular ITIMs [167].

The Natural cytotoxicity receptors (NCRs) The NCRs include NKp30, NKp46 and NKp44, of which the two first mentioned are constitutively and selectively expressed on all peripheral NK cells. NKp44 is upregulated on IL-2 activated NK cells and is also expressed on some γδT cells [168,169]. Natural cytotoxicity receptors (NCRs) are Ig-like receptors that trigger lysis of tumors and virus- infected cells upon interaction with ligands on the target cells. Both NKp44 and NKp46 can bind viral hemagglutinins, supporting the role of NCRs in recognition of virus-infected cells [170,171]. HIV infected cells have been reported to express a ligand (still undefined) for NKp44 [172]. In herpes simplex virus-infected cells, expression of a viral gene product is recognized by NCRs leading to increased susceptibility to NK-cell mediated lysis [173]. The NK-cell mediated cytotoxicity has been reported to be inhibited by interaction between the tegument protein, pp65, from human cytomegalovirus (CMV) and NKp30 [174] perhaps demonstrating an escape mechanism by the virus. NKp30 has also been showed to be involved in the NK-cell induced maturation of DCs and in killing of iDCs [175,176].

31

Receptor Ligands Function Signaling Refs (alternative (alternative name) name) KIR2DL1 HLA-C2 Inhibitory ITIM [160] (CD158b) KIR2DL2 HLA-C1 Inhibitory ITIM [160] (CD158b1) KIR2DL3 HLA-C1 Inhibitory ITIM [160] (CD158b2)

KIR2DL4 HLA-G Activating/inhibitory ITIM/FcεRI-γ [160,177,178] (CD158d) KIR2DL5 Unknown Inhibitory ITIM [160] (CD158f) KIR3DL1 HLA-Bw4 Inhibitory ITIM [160] (CD158e1) KIR3DL2 HLA-A3, A11 Inhibitory ITIM [160] (CD158k) KIR2DS1 HLA-C2 Activating DAP12 [160,177] (CD158h) KIR2DS2 Unknown Activating DAP12 [160,177] (CD158j) KIR2DS3 Unknown Activating [160] KIR2DS4 HLA-Cw4 Activating DAP12 [160,177] (CD158i) KIR2DS5 Unknown Activating [160] KIR3DS1 HLA-Bw4? Activating DAP12 [160,177] (CD158e2) KIR3DL3 Unknown Unknown [160] LIR-1 HLA-class I Inhibitory ITIM [165,167] (CD85j, ILT2) LAIR-1 Collagen Inhibitory ITIM [179] (CD305) CD244 CD48 Activating/Inhibitory ITSM, SAP [180,181] (2B4)

NKp30 Unknown Activating FcεR1-γ [181] (CD337) NKp44 Unknown Activating DAP12 [181,182] (CD336)

NKp46 Unknown Activating FcεR1-γ [181] (CD335) Table 3. General summary of some immunoglobulin (Ig)-like receptors on human NK cells.

Antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells

The CD16 receptor is expressed on almost all CD56dim NK cells. Another name for CD16 is FcγRIIIA that is the low affinity receptor for IgG. Ligation of CD16 to IgG bound to specific antigen on the target cells, IgG-immune complexes or anti-CD16 monoclonal antibodies (mAb) induces cytolysis through ADCC [183] and also increased expression of the IL-2 receptor and production of several cytokines [184]. Crosslinking of CD16 has also been

32 shown to induce apoptosis in NK cells [185]. The surface expression of CD16 can be altered by cytokines. CD16+ NK cells convert to CD16- NK cells in the presence of TGF-β [186]. After in vitro culture with IL-2 and IL-12 the expression of CD16 is decreased and can be re- expressed to some extent by IL-15 [187]. A polymorphism in the extracellular domain 2 of CD16 has been reported to affect ligand binding by NK cells and may play a role in human autoimmune disease [188]. It was recently described in a mouse model that NK cells were activated by IgE through CD16 and responded by both production of IFN-γ and ADCC [189].

Chemokines and NK-cell homing

Chemokines and chemokine receptors are important for the circulation of immune cells to different organs and tissues. For NK cells to be able to mediate their cytolytic and regulatory functions, they must be recruited to the site where their effector functions should be applied. The CD56bright and CD56dim NK-cell subsets express different chemokine receptors, suggesting that they home to different sites in vivo. CD56bright NK cells express high levels of e.g. CCR7, involved in homing of immune cells to secondary lymphoid organs [190], and also the adhesion molecule L-selectin (CD62L) important for the interaction with endothelial cells [191]. CX3CR1 and the IL-8 receptor (CXCR1) are expressed on CD56dim NK cells but not on CD56bright NK cells [115]. Thus, the CD56dim NK cell subset migrates in response to IL-8 (chemokine CXCL8) [192] which is released during early responses to infection. Several chemokines have been shown to be involved in the regulation of NK-cell mediated cytolysis by promoting cytotoxic granular release [193].

NK-cell generation

NK cells, along with other lymphocytes, originate from CD34+ haematopoietic precursor cells (HPCs) present in the bone marrow of adults. During fetal life NK cells are generated from precursors in the liver in humans [194], and in the thymus in mice [195]. The bone marrow is considered to be the primary site of NK-cell generation in adults because it provides the required interaction with stromal cells and composes a rich source of cytokines and growth factors. The importance of the bone marrow for generation of NK cells is shown by the loss of NK cells upon bone marrow depletion [196]. However, NK cells at different developmental stages have been found in other organs of the body, including liver, spleen, lymph nodes and in blood [192,197-200]. Organs like thymus and spleen are not essential in

33 the generation of NK cells, since normal numbers of functional NK cells are present, in humans and mice, even though they lack functional organs of this type [192]. This does not preclude that NK cells can develop at these sites. It is unclear whether NK-cell precursors (NKPs) found at these sites are derived from precursors migrating from the bone marrow or if these sites can actually support NK-cell differentiation from local HPC. The ontogeny of the CD56bright and CD56dim NK cells is unclear. It has been suggested that CD56bright NK cells are precursors for the CD56dim NK cells [201,202]. The CD56bright subpopulation of human NK cells is preferentially found in secondary lymphoid tissues and at sites of inflammation [203-205]. These cells might develop within lymphoid tissue as CD34+ HPC isolated from lymph nodes can be induced to differentiate in vitro to CD56bright NK cells in the presence of IL-2 or IL-15 or after culture with activated T cells alone [198]. NK cells have been reported to reside in close proximity to T-cell rich regions in lymph nodes in vivo [198,206]. This could support the theory of a direct interaction between NK cells and T cells at the site of T-cell activation and thus a possibility of NK cells to affect this process. Data concerning the life span of mature NK cells in vivo is diverse, perhaps due to different methods of transfusion and tracking of the cells. Splenic mature NK cells, labelled with tracking dyes and transferred to normal adult mice, have been estimated to have a half- life of 10 days [207] up to almost 3 weeks [105]. In humans, transfused allogenic NK cells in patients with cancer have been detected for up to 3 days in blood [208]. In another study, human blood NK cells in healthy individuals were suggested to have a turnover rate of approximately 2 weeks [209].

Receptor expression during development

The general consensus is that HPC develop into either common lymphoid progenitors (CLP), (giving rise to B, T and NK cells) or common myeloid progenitors (CMP), (giving rise to monocytes, granulocytes, erythrocytes and mast cells). The understanding of human NK- cell development is incomplete. To begin with, it is uncertain exactly what factor/s that determines the commitment of CLPs to the NK-cell linage. Although, in vitro studies have shown that cytokines like stem cell factor (SCF), fetal liver kinase 2 ligand (FLK2) and IL-7 can promote the development of NKPs, they are not essential for NK-cell commitment. However, once the CD34+CD45RA+CD117+ CLPs acquire expression of the interleukin-2 receptor-β (IL-2Rβ) they are denoted NKPs and have the potential to develop into mature NK cells. The further development has been described to take place in different stages where

34 CD161 expression is evident very early on immature NK cells. The immature NK-cell stage is followed by a sequential process of maturation that seems to be initiated with the acquisition of CD56 together with features of cytotoxicity [192]. The maturation stages proceed as NK cells start to express receptors that are specific for self-MHC molecules, such as CD94/NKG2A or KIRs, which bind to MHC class I molecules. The CD94/NKG2A heterodimeric receptor complex is more dominantly expressed compared to the KIRs in humans and most of the analogous Ly-49 inhibitory receptors in mice [210,211] during early stages of development (Figure 1). The KIR receptor repertoire on NK cells is mainly determined by gene variations and polymorphisms, as described above. It has, however, been suggested that MHC class I might have some influence on the KIR expression on NK cells where many optional MHC class I ligands is associated to fewer cognate KIRs. This inverse relationship also extends to the frequency of NK cells expression NKG2A, being inversely correlated to the number of KIR:MHC class I pairs [212]. In general, developing NK cells express inhibitory receptors before the activating, as if to assure that they are able to be inhibited by self molecules before they gain the capacity to be activated [213]. Notably, a subpopulation of human NK cells without inhibitory receptors has been found and the tolerance to self seems to be achieved through hyporesponsiveness by these NK cells [126]. Upon in vitro stimulation with IL-15 and stromal cells, these anergic NK cells start to express inhibitory receptors and acquire effector functions [214].

35

Figure 1. Phenotypic and functional features of developing NK cells. Several NK-cell related molecular markers are expressed in a sequential manner during NK cell development. NKPs do not express specific NK-cell markers but give rise to NK cells. Immature NK cells express CD161 together with receptors required for growth and survival. In the periphery, NK cells can become activated after e.g. detection of missing or altered self-MHC class I molecules or by cytokines. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology 2007; 7 (9) 703-714 [200].

NK-cell education

To prevent autoreactivity, NK cells need to discriminate aberrant cells from normal cells and this is achieved through a combination of activating and inhibitory receptors. Although the process is not well understood, host MHC class I molecules are believed to be part of the development of NK-cell reactivity and self-tolerance. An educational model called “licensing” has been suggested by Yokoyama et al. [215]. In this model NK cells are induced to mature only after successful engagement of inhibitory ligands. Another model named “the disarming theory” that have been proposed by Raulet et al suggests that, NK cells that fail to receive inhibition become anergic [216].

36

NK receptors on T cells

NK-cell receptors were first described on NK cells, hence the name. However, other immune cells like T cells, B cells and monocytes also express some of these receptors [217]. When NK receptors are expressed on T cells they can function in a costimulatory or coinhibitory fashion [218]. On human naïve CD8+ T cells, NKG2D serves as a costimulatory receptor for the TCR resulting in priming to a type 1 phenotype [219]. CD8+ T cells with specificity for CMV show increased cytokine and cytolytic responses upon NKG2D ligation [148]. Inhibitory KIRs are expressed mainly on CD8+ αβT cells [158]. CD8+ T cells expressing KIRs are not found in CB [220] but memory KIR+CD8+ T cells have been reported to accumulate with increasing age [221]. The expression of KIRs by T cells is associated with resistance of effector-cell- mediated cell death [222]. Of the receptors present in the LIR family, only LIR-1 has been described on T cells. LIR-1 appears to be primarily expressed on effector T cells that are KIR negative [223] whereas the KIR expression appears to be more restricted to memory T cells [224]. The co-ligation of TCRs and inhibitory KIRs or LIRs can reduce or prevent T-cell effector functions including both cytokine production and cytotoxicity [158,220,225-227]. Although the mechanism is unkown, CD8+ T cells have been reported to be able to mediate MHC-independent lysis of CMV infected cells through UL18 and LIR-1 interaction, suggesting alterations in the function of LIR-1 [228].

NK cells bridge to adaptive immunity

The interaction between NK cells and several cell types can have immuno-modulatory effects on the outcome of the adaptive immunity. Lymph node resident CD56bright NK cells can, through cell contact together with GM-CSF and CD154, trigger CD14+ monocytes to differentiate into DCs with a Th1-promoting activity [229]. CD56bright NK cells have been reported to produce IFN-γ in response to IL-12 derived from LPS activated DCs [230], which might be important for the priming of adaptive immune cells, as IL-12 exposed NK cells have been shown to favor the selection of mature DCs that are associated to Th1-cell priming [231]. In a murine model it was shown, in vivo, that upon antigen stimulation in lymph nodes, NK cells are recruited and there they provide an early source of IFN-γ needed for Th1 priming of T cells [232].

37 NK cells have been shown to affect antibody responses by B cells. NK cell knock-out mice can develop a Th1 response and produce IFN-γ but are deficient in their antigen-specific IgG2a antibody response [233]. In humans, NK cells are able to induce B-cell differentiation into antibody-producing cells and to isotype switching to IgG and IgA. This function seems to be regulated through CD40-CD40 ligand interactions [234]. Human IFN-γ producing NK cells have also been shown to inhibit in vitro IgE production [90].

NK cells and accessory cells

Many pathogens are recognized by accessory cells like DCs, monocytes and macrophages that activate NK cells through cytokines and/or by signals that are contact dependent [235]. The effects on NK-cell activity by accessory cell derived cytokines are variable. IL-12 induces IFN-γ production by NK cells [97], increases the cytotoxic activity and have a proliferative effect [93]. IL-15 is important in the initiation of NK-cell mediated cytotoxicity by inducing surface expression of the adhesion molecule LFA-1 [106] but is also crucial for NK-cell development and induction of NK-cell proliferation and survival [97,103-105]. Cytokines delivered to NK cells through synaptic formation is an efficient route of activation [236]. NK cells also affects accessory cells, suggesting a reciprocal regulation, which might depend on the nature of the cellular stimuli. In in vitro cultures with LPS, NK cells can enhance DC maturation and IL-12 production [237] and the maturation of DCs can be mediated by NK-cell derived TNF and IFN-γ [176]. Ligation of the NKp30 receptor on NK cells has been suggested to be involved in both the killing of iDCs and in the induction of IFN-γ production by NK cells [175]. DC-derived transforming growth factor beta 1 (TGF-β1) can inhibit the expression of NKp30, and thus, decrease the NK-cell mediated DC regulation [115]. A reciprocal regulation between macrophages and NK cells has also been reported. Macrophages can induce NK-cell proliferation and functions, while NK cells control macrophage activity through elimination of overstimualted macrophages [238]. The importance of accessory cell and NK cell interaction has been demonstrated in relation to different diseases like malaria [239], rheumatoid arthritis [229] and respiratory allergic disorders [240].

38 HERPESVIRUSES

The family of herpesviruses (Herpesviridae) includes viruses of the α-, β- and γ-subfamily. CMV belongs to the β-herpesviruses, which all have a rather restricted host range and a replicating cycle of days resulting in enlarged cells. Epstein-Barr virus (EBV) is included in the family of γ-herpesviruses that replicate in lymphoblastoid cells. Both CMV and EBV are widespread pathogens with a majority of the population living in industrial countries being seropositive and the prevalence of seropositivity is even higher in populations with low socioeconomic status. Primary infection usually occurs during early childhood. The infection is persistent throughout life and asymptomatic in healthy people. However, if primary EBV infection is acquired later in life it can cause infectious mononucleosis that is associated with e.g. fever, sore throat and fatigue. Breast milk and saliva is the most common route of transmission of CMV and for EBV through saliva. Upon primary infection the virus is amplified in a permissive cell type and during the asymptomatic latent phase of infection the virus is sustained in B cells for EBV and in cells of the myeloid linage for CMV [241,242]. Herpesvirus infections are in large controlled by T-cell immunity [243] but also B cells and NK cells are important in combating the virus. During early phases of EBV infection in vitro, tonsillar NK cells stimulated by EBV-activated DCs produce vast amounts of IFN-γ that limits the EBV-induced B-cell proliferation. For an in vivo scenario, the authors suggest that the innate immune response by NK cells and DCs would limit primary EBV infection until the specific immunity has been recruited [244]. The expression of MHC class I molecules on B cells during EBV latency protects against NK-cell mediated attack. However, during lytic EBV infection, the surface expression of MHC class I molecules is reduced, which increases the risk of NK-cell mediated lysis [245]. CMV in particular has evolved several escape strategies to evade immune responses, including a whole set of genes responsible for downmodulation of MHC class I expression and genes responsible for evading NK-cell recognition [246].

ALLERGY

Allergy is caused by a hypersensitivity reaction to a common antigen (i.e. allergen) and is mediated by allergen-specific IgE antibodies or allergen-specific lymphocytes causing symptoms from mucosal membranes. An IgE-sensitized person has allergen-specific IgE antibodies, but do not display allergic symptoms. The definition of an allergic person is

39 presence of allergen-specific IgE antibodies and allergic symptoms. Atopy is referred to as a personal and/or familial tendency to become IgE sensitized [247].

The allergic reaction

For some unknown reasons, some individuals start to produce IgE antibodies against antigens (called allergens) that normally are harmless. Initially, allergens are taken up by APCs in e.g. the respiratory tract. In the lymph node the allergen is presented to naive T cells in a peptide-MHC class II complex. Several other types of immune cells are present (mast cells, NK cells and T cells) that can influence the priming of the naïve T cell. IL-4 promotes the naïve T cell towards the Th2 type that produces IL-4 and IL-13. The interaction of these Th2 cells with B cells, under the influence of IL-4 and IL- 13 in addition to ligation of co-stimulatory molecules, induces immunoglobulin class-switch recombination to the IgE class. The produced IgE antibodies are distributed systemically through the blood. The allergen-specific IgE antibodies bind to the FcεRI on tissue-resident mast cells [16]. This is referred to as the sensitization phase and is not symptomatic. The symptoms may appear upon a second exposure to the allergen. Allergen-binding, crosslinks the FcεRI-bound IgE antibodies on the mast cell, which results in degranulation of inflammatory mediators. This immediate phase reaction leads to the recruitment of other cells, including eosinophils, T cells and macrophages, to the area. At the site, these cells become activated and the result in a prolonged state of inflammation. This late phase reaction can be evident around 6 hours after allergen exposure [17,18]. The symptoms, caused by IgE-mediated allergic reactions, vary depending on location and severity. Allergic eczema/dermatitis syndrome (AEDS) is caused by reactions in the skin, asthma in the bronchial respiratory tract and rhino-conjunctivitis in the upper respiratory tract and eyes. In systemic anaphylaxis all the sites are involved simultaneously. If severe enough it can cause massive vasodilation and anaphylactic shock and be life threatening.

Type 1 and type 2 cytokines in allergy

T-cell responses have traditionally been classified based on two effector scenarios, Th1 and Th2. The Th1 cell and Th2 cell nomenclature was originally suggested by Tokuhisa et al. [248] and further distinguished in their cytokine profiles in mice by Coffman et al. [249]. Since then it has for long been a general consideration that the function of the immune system

40 is dependent on a Th1/Th2 balance in healthy individuals. This balance is reciprocally regulated where Th1 responses downmodulate Th2 responses and vice versa. Typical Th1 inducing cytokines are IL-12 and IFN-γ, while Th2 inducing type 2 cytokines include IL-4, IL-5 and IL-13. In view of the classical Th1/Th2 paradigm, a Th2 skewed immune response has been considered to be involved in the risk of early development of allergic disease. Neonates, with an increased risk of developing allergic disease (i.e. having allergic parents) have a Th2 skewed type of response [250]. In line with this, lower numbers of IL-12- and IFN-γ- producing cells in CB is associated with IgE sensitization [251]. However, there are some contradictory results revealing a mixed Th1/Th2 type of response after allergen stimulation in young atopic children [252,253]. Also other immune cells, like DCs [254] and NK cells [90,255] have been described as polarized towards type 1 and type 2 cytokine production, probably affecting the fate of naïve helper T-cell activation in lymphoid tissue.

The hygiene hypothesis

The reason for the increased incidence of allergic diseases during the last decades is not fully understood but several possible theories have been proposed. Although there appear to be a genetic association [256] this does not explain the rapidly increased occurrence. Another plausible explanation includes environmental factors. Through the observations by Strachan that more siblings decreased the risk of developing hay fever [257], the theory of the ”hygiene hypothesis” came forth. The “hygiene hypothesis” states that due to changes in the exposure pattern to microbial components (both infectious and non-infectious) there are insufficient qualities and/or quantities of these factors, leading to an altered development of the immune system with a increased tendency to develop allergic diseases.

Environmental factors in allergy development Several studies support the hygiene hypothesis. Exposures, during early childhood, to an environment with higher infection rate seem to have a protective effect against allergy development. In addition to the protective effect of increasing number of siblings [257,258] it has been documented that day care attendance in early life is associated with a reduced risk of developing allergic disease [259,260]. Growing up on a farm is associated to an environment

41 rich in microbial components. LPS or endotoxins are components of the cell wall of Gram- negative bacteria that are highly prevalent in households of farmers [261]. The level of endotoxin has been shown to be inversely related to the occurrence of allergic disease [262,263]. Other bacterial components, like N-acetyl-muramic acid (a component of PGN) have been inversely associated with asthma [264] and the high levels of bacterial DNA found in farm barns [265] may also be part of the protective effect. In line with this, children growing up in farm families have a reduced risk of developing allergic disease [266-270]. In families with part-time farming, the protection is not as strong as in families with full-time farming [269], suggesting that dosage is important and that exposure should be constant in order to reach optimal effect. However, the current scientific evidence also reports on opposing effects by infections on the development of allergic disease. Helminth and mycobacteria have a protective effect but depend on at what age the infection occurs, the route and the dose of infection. Infection by viruses has been reported to be associated to both protection and increased risk to develop allergic disease [271-273]. It is possible that a decreased exposure to e.g. Th2-inducing helminths or a delayed infection during early life to Th1-inducing viruses alters the balance of immune responses to allergens [274].

NK CELLS IN HEALTH AND DISEASE

Implication of NK cells in health and disease include many areas of research. For the topics involved in this thesis, the focus will be on the role of NK cells in virus infections (mainly EBV and CMV) and in allergic disease.

NK cells in viral infections

NK cells are important in the defense against viral infections. In humans this is supported by data from cases of natural infections where NK-cell deficiencies increase susceptibility, mainly to herpesviruses. Genetic mutations can cause NK-cell defects. Patients with Chediak-Higashi syndrome suffer of immunological deviations, such as deficient NK-cell cytotoxicity and ADCC. As a result these patients have chronic active EBV infection [275]. X-linked lymphoproliferative syndrome is associated with severe immune deficiencies. NK-cell cytotoxicity is often markedly reduced and patients are commonly affected by fatal infectious mononucleosis,

42 caused by EBV, or/and lymphoreticular malignancies [276]. The Griscelli syndrome is caused by another gene mutation and defects in NK-cell cytotoxicity leading to increased susceptibility to EBV [277]. Gene mutations that cause hemophagocytic lymphohistiocytosis affect the perforin protein, NK-cell activity or/and the percentages of NK cells. For some patients this disease is associated with EBV infection [278]. A complete lack of NK cells has been reported, resulting in increased susceptibility to several herpesviruses and bacterial infections with fatal outcomes [279,280]. The importance of NK cells for antiviral responses is further highlighted by a case study where a young girl (3 months old) was able to recover from CMV disease without treatment, although she virtually lacked T cells. During recovery, the viral load correlated with the number of NK cells and NK-cell derived cytokines [281].

NK cells in allergy

During infection and/or exposure to non-pathogenic microbes, cells of innate immunity are probably activated early, leading to initiation of adaptive immune responses. The functions of NK cells include, lysis of target cells and also regulation of immune responses by the release of cytokines, like IFN-γ, TNF, GM-CSF, IL-4, IL-5, IL-10 and IL-13 that can affect immune responses by other cells in different ways. Several reports suggest a role for NK cells in allergic disease. It has been shown that atopic asthmatic patients have a higher ratio of IL4-producing NK cells than healthy individuals [282]. Aberrant NK-cell frequencies and functions have also been observed in patients with atopic dermatitis, where circulating NK cells were decreased and had a defective ability to produce TNF and IFN-γ, but not IL-4. These effects were suggested to be due to apoptosis of NK cells, induced after cell contact with activated monocytes [283]. In a study by Buentke et al. NK cells were shown to interact with DCs in skin lesions of atopic dermatitis patients [284]. It has further been shown that peripheral mononuclear cells from atopic patients have a reduced capacity to produce IFN-γ in response to IL-12 [285]. Although this was not related to NK cells specifically, another study show reduced numbers of IL-12 producing monocytes and IFN-γ producing NK cells in adult allergic patients with rhinoconjunctivtis [286]. In a recent study allergic adult individuals were reported to have a reduced proportion of CD56bright NK cells and decreased production of IFN-γ in response to DCs in vitro [240]. These data might suggest an aberrant feedback loop of IFN-γ and IL-12 between NK cells and monocytes/DCs in allergic individuals.

43 In resemblance to the T-cell subsets Th1 or Th2, NK cells have also been shown to be able to polarize in vitro into two subsets with different cytokine profiles. NK cells with type 1 responses produce IFN-γ and IL-10 while type 2 NK cells mainly produce IL-5 and IL-13 [90,287]. Freshly isolated human NK cells, from both adults and neonates, contain a distinct subpopulation of immature IL-13+ NK cells that are induced to proliferate to IL-4 in vitro [288]. In cell cultures with IL-4 and IL-13, mature IFN-γ+ NK cells are prevented to accumulate [288] and IFN-γ in turn prevents the accumulation of IL-13+ NK cells, suggesting a negative feedback loop between these two subsets of NK cells [289]. This could be of importance in an allergy context, where the presence of IL-4 and IL-13 may drive the progression of IL-13+ NK cells and prevent type 1 responses through inhibition of IFN-γ and aggravate atopic reactions or reciprocally, in the presence of proinflammatory cytokines suppress type 2 responses.

THE IMMUNE SYSTEM OF NEONATES AND YOUNG CHILDREN

The ability of neonates to mount an immune response is insufficient in comparison to older individuals. However, the process of birth is followed by an age-dependent maturation of the immune system. The distribution and function of mononuclear cells in blood differs between newborns and adults. Very low numbers of memory CD4+CD45RO+ T cells are present in CB [290] but the proportion of the CD4+ [291,292] and CD8+ [293] T-cell memory pool increase with age. CB cells proliferate less than cells from adults in response to in vitro stimulation with low doses of the T-cell mitogens concanavalin A (Con A) and phytohaemagglutinin (PHA), but reach equivalent proliferation levels as adults with higher concentration [294]. The fractions of DCs and monocytes from CB appear to be similar to adults, but the function of cord-blood DCs as accessory cells for T-cell responses to mitogens is insufficient, perhaps due to lower expression of molecules like ICAM-1, MHC class I and II on DCs, needed for antigen presentation [294]. The highest proportion of NK cells, in relation to lymphocytes, is found in CB followed by a decline during early childhood years and then an increase again in adults, although the levels do not reach that of newborns [295]. NK-cell cytotoxicity is low in CB but is improved quickly during infancy [296]. Cytokine responses by newborns are considered to be skewed towards Th2. The concentration of IFN-γ in serum from CB is low compared to adults, but increases during the first days of life, while IL-2 and IL-4 can be found at higher concentration in CB than in

44 adults [297]. This early cytokine profile is also detected in T cells where, relative to adults, smaller proportions of both CD4+ and CD8+ CB T cells are positive for IFN-γ but not IL-2 after cell culture with phorbol myristate acetate (PMA) and ionomycin [290]. CB NK cells are, however, readily activated by cytokines. In cell cultures with IL-12 and IL-18 the activating marker CD69 is more effectively upregulated on NK cells from CB as compared to adults. This activation is mirrored in the release of IFN-γ by these cells that by far exceed that of adult NK cells [298]. The production of IL-12p70 and IFN-α by CB cells is impaired in neonates [299], which may be part in the initial inefficiency to mount Th1 responses. The mode of delivery affects the frequency of lymphocyte populations and cytokine production. In full term neonates, NK cells are increased in children that are vaginally delivered (VD) as compared to children born by elective Cesarean section (ECS) [300]. CB cells from children born by VD produce more IFN-γ and IL-12 in response to mitogens [301].

PREECLAMPSIA

Preeclampsia is a disorder seen in the later part of pregnancy with symptoms like proteinuria, hypertension and oedema. If the condition is severe enough the outcome can be lethal for both mother and child. Approximately 3-5% of all pregnancies are diagnosed with preeclampsia. The cause for this pregnancy disorder is unknown but the disease is related to the placenta since the symptoms cease upon removal of placenta and fetus [302]. Further, the development of the placenta during preeclamptic pregnancies is deficient as seen by altered placental morphology [303]. This can be a result of the poor penetration by extravillious trophoblasts into the endometrium and the establishment of fewer placental arteries resulting in poor utero-placental blood circulation [120]. Healthy pregnancies are associated with a mild inflammation in the circulation, which is further enhanced in preeclampsia [304]. Cytokines like IL-12p40 and IL-15 are increased in the circulation of preeclamptic women. Alterations of the cytokine balance are also local in the placenta where IL-12p70 is practically absent in preeclampsia while it is abundant in the placenta of healthy pregnancies [303]. A subset of NK cells is found in the human uterus and is referred to as uterine NK (uNK) cells. During early phases of placentation these cells are thought to be involved in the regulation of trophoblast invasion in the decidua through NK- receptor and trophoblast-ligand interactions and cytokine production [120]. One mechanism that have been suggested in the regulation of placenta development is the interaction of maternal KIRs on the uNK cells and fetal expression of MHC class I molecules on

45 trophoblasts cells. This statement is based on the finding that mothers with the A haplotype for KIRs, bearing a fetus with a HLA-C2 allotype are at increased risk of preeclampsia [305]. Further, higher numbers of CD56+ cells in the deciduas of preeclamptic women [303] and a larger proportion of NK cells in CB of neonates from preeclamptic mothers compared to the newborns from healthy mothers [306,307], has been reported.

46 AIMS OF THE STUDY

During infancy, before adaptive immunity is matured, innate immunity is thought to be rather important. Human natural killer (NK) cells are innate immune cells involved in the control of virus-infected cells and can influence adaptive immunity mainly through cytokine production. The overall aim of this study was to evaluate function and phenotype of NK cells in children from birth and during early childhood years and if these features are altered in children that develop early allergy, are latently infected by herpes viruses or are born by preeclamptic mothers. In more detail, we wanted to evaluate:

Paper I: The general understanding of the maturation kinetics of NK-cell immune functions after birth is modest. Comparisons are mainly done between CB cells and cells from adult donors, while data on blood mononuclear cell from birth and young children are scarce. In paper I we wanted to examine the receptor expression and IFN-γ production by NK cells in CB and in the same children at the age of 2 years and at 5 years.

Paper II: NK cells are important during early life. Here, we wanted to evaluate early features of NK cells that could be of importance for immune responses later in life. In paper II, the aim was to elucidate if NK cells were altered regarding phenotype and/or function at birth in individuals that developed allergic disease at 5 years of age.

Paper III: Herpes-virus infection associates with a reduced risk of becoming IgE-sensitized, and NK cells are important in the control of herpes viruses. In paper III we aimed at analyzing the functional capacity and interaction of innate immune cells (NK cells and monocytes) in healthy 2-year-old children that were either seropositive for EBV, EBV and CMV or seronegative for both viruses.

Study IV: Preeclampsia is associated with escalated inflammation in the circulation of the pregnant woman. In study IV, we wanted to evaluate if the pro-inflammatory immunological profile of the mother had an influence on the immunological profile in their neonates in regard to the functional and phenotypic features of CB NK cells from healthy and preeclamptic pregnancies.

47 SUBJECTS

The three first studies utilize samples from the same longitudinal and prospective study cohort that was initiated to investigate the parental influence on the immunology of the child. The cohort has previously been described by Nilsson C. [308]. The subjects included in the respective studies are described in the individual papers I-III, while the study population for the preliminary Study IV is described below.

Study population (Study IV, preliminary data)

Eighteen children from healthy mothers and 19 children from preeclamptic mothers were included in the study. Preeclampsia was divided into moderate (n=11, >0.3 g proteinuria, systolic/diastolic blood pressure >140/90) and severe preeclampsia (n=8, >3 g proteinuria, systolic/diastolic blood pressure >160/110). The study included 14 mothers delivered by vaginal delivery (VD) (n=7 healthy and n=7 preeclamptic mothers) and 23 mothers delivered by elective caesarean section (ECS) (n=11 healthy and n=12 preeclamptic mothers) (see Table 4). CB was collected at time of delivery at the delivery unit, Karolinska University Hospital and all women gave their informed consent to participate in the study. The Ethics Committee of Karolinska Institute, Stockholm, Sweden, approved the study.

Group Cases Maternal age Mode of Gestational age Weight (children) at delivery delivery

(years) (VD/ECS)* (weeks) (g) Neonates from 18 33 (24-38) 7/11 39 (37-43) 3540 (2810-4900) healthy mothers Neonates from 19 33 (22-44) 7/12 38 (31-41) 3270 (1158-4125) preeclamptic mothers Mild cases of 11 33 (27-44) 5/6 40 (37-41) 3295 (2510-4096) preeclampsia Severe cases of 8 33 (22-41) 2/6 37 (31-40) 2580 (1158-4125) preeclampsia *VD=vaginal delivery; ECS=elective caesarean section

Table 4. Demographic data of all neonates born from healthy and preeclamptic mothers included in the study. Median (range) values are shown in the table.

48 MATERIAL AND METHODS

The methods used for the individual papers I-III are summarized in short below and described in detail in the respective papers. For the preliminary Study IV the material and methods are described in more detail here.

• Separation of CBMCs and PBMCs (I-III) • Clinical evaluation (I-III) • Phadiatop/Cap-FEIATM (I-III) • Skin prick test (SPT) (I-III) • In vitro activation of blood cells (I-III) • ELISA (III) • Cytometric Bead Array (CBA) (II) • Flow cytometry (I-III) • Intracellular staining for analysis by flow cytometry (I and III) • Statistical analyses (Paper I-III)

Material and methods (Study IV, preliminary data)

Collection and isolation of cord blood mononuclear cells (CBMCs) CBMC samples were prepared within 24 hours after collection. For preparation of serum, CB was centrifuged at 1500 rpm for 10 minutes and the serum samples were stored in -20°C until use. Cells were isolated using Ficoll-PaqueTMPLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and stored in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, L-glutamine (2mmol/L), penicillin G sodium (100 units/ml) and streptomycin sulphate (100µg/ml) (complete tissue culture medium, CTCM) containing 10% Dimethyl sulphoxide (DMSO). Freezing was performed gradually at 1°C/min to -70°C in a freezing container (Nalgene Cryo 1°C, Nalge Co, Rochester, NY, USA) and thereafter stored in - 135°C until use.

In vitro activation of cells All CBMCs were thawed quickly and then washed twice in CTCM. CBMCs included in

49 the phenotypic study were aliquoted in 0.5x106 cells/well and incubated for 20 minutes in 37°C prior to surface staining. For stimulation experiments, CBMCs at a concentration of 1x106 cells/mL were cultured in CTCM alone or in the presence of IL-15 (20 ng/mL) and peptidoglycan (PGN) (1 µg/ml) in 37°C for 24 hours.

Flow cytometry To prevent non-specific binding of antibodies to FcR, the cells were incubated in 1% BSA and 0,02% NaN3 in PBS (wash buffer) supplemented with 5% human serum (blocking buffer) prior to staining with specific antibodies. APC-conjugated anti-CD56, Per-Cp- conjugated anti-CD3 and PE-conjugated anti-CD16 monoclonal antibodies were used to identify NK cells (all from BD Biosciences, San Diego, CA). NK-cell receptor expression was studied by the following antibodies: Anti-CD69, anti-NKG2D (both PE-conjugated from BD Biosciences), PE-conjugated anti-NKp30 and anti-NKp46 (both from Beckman Coulter, Immunotech, Marseille, France), un-conjugated anti-NKG2A, NKG2C (RnD Systems, Minneapolis, MN, USA) detected with PE-conjugated goat anti-mouse (DakoCytomation). As negative controls, IgG1 (DakoCytomation, Glostrup, Denmark) and IgG2a (RnD Systems) were used. To study intracellular cytokine production from NK cells, antibodies for IFN-γ (FITC-conjugated from BD Biosciences) and IL-4 (PE-conjugated from BD Biosciences) were used. Flow cytometry was performed on a FACSCalibur (BD Biosciences, Mountain View, CA, USA) and analyses were made using Cellquest Pro software (BD, version 5.2.1).

Cytometric Bead Array IL-12p70, TNF, IL-1β, IL-6, IL-8 and IL-10 in sera from children and mothers were measured with Cytometric Bead Array (CBA) (BD Biociences, San Diego, CA, USA) according to the manufacturer’s instructions. Calibration of the flow cytometer was performed using BD FACSCompTM and BD CaliBRITETM Beads and analysis was using BD CBA Software (all from BD Biociences, San Diego, CA, USA). The detection limits for the cytokines were, IL-12p70 (1.9 pg/ml), TNF (3.7 pg/ml), IL-1β (7.2 pg/ml), IL-6 (2.5 pg/ml), IL-8 (3.6 pg/ml) and IL-10 (3.3 pg/ml).

IFN-γ Enzyme-Linked Immuno Sorbent Assay (ELISA) IFN-γ production in culture supernatants, after 24 hours of stimulation with IL-15 and PGN, alone or in combination, was studied with ELISA. Ninety-six well plates (Costar 3690,

50 Corning Inc., Corning, NY, USA) were coated with anti-IFN-γ mAbs (2µg/ml, Mab 1-DIK, from Mabtech, Stockholm, Sweden) and incubated ON at 4°C. Prior to adding the samples in duplicates blocking with 0.5 % bovine serum albumin (BSA) in PBS was performed. Biotin labelled mAb (1µg/ml, 7-B6-1-Biotin, Mabtech, Stockholm, Sweden) and streptavidin- alkaline phosphatase (Mabtech, Stockholm, Sweden) were added followed by the substrate (SIGMA-Aldrich, Stockholm, Sweden). All incubations were performed for 1 h in 37°C, except when adding the substrate, when plates were incubated in room temperature. Standard curve was used with calculated dilutions of recombinant IFN-γ (NIBSC, Hertfordshire, UK). Detection limit was 3.9 pg/mL and optical densities were measured at 405 nm.

Statistics Data is shown as median (range) in the results section. The non-parametric Mann-Whitney U test was used to evaluate differences between the healthy and preeclamptic children. Correlations between mother and child were calculated with Spearman Correlation test. Statistical analyses were performed with Statistica 7.1 (STATISTICA Statsoft Inc., Tulsa, USA). Statistical significance was assumed when p<0.05.

51 RESULTS AND DISCUSSION

Paper I

The overall understanding of the maturation kinetics of NK-cell immune functions after birth is modest. Comparisons are mainly done between CB cells and cells from adult donors, while data on immune characteristics of NK cells from birth and during early childhood years are limited. In Paper I we examined the receptor expression and IFN-γ production by NK cells in CB and in the same children at the age of 2 years and at 5 years. In this study, we demonstrate alterations in the distribution and the phenotype of NK cells with increasing age. The proportion of NK cells was higher in CB compared to later time points. In line with our data, a higher fraction of NK cells in neonates compared to older age groups has been reported [295,300]. Further, the mode of delivery has been shown to influence the frequency of lymphocyte subsets in CB with a larger proportion of NK cells in VD full term neonates compared to babies delivered by ECS [300]. All subjects included in this study were VD. Human labour and delivery are characterised by an inflammatory response with cytokines [309] that directly or indirectly could influence NK cells. Taken together, our results and others indicate that the increased proportion of NK cells at birth may be a consequence of the systemic inflammatory response induced by the process of delivery. Further, the gradual decrease of peripheral blood NK cells, with age, might indicate that the frequency drops after birth due to for example retention in secondary lymphoid tissues or at sites of infection. We also found that the CD56bright NK-cell population comprised similar proportions in CB and at five years of age, but increased marginally at two years of age. An increase of CD56bright NK cells in peripheral blood has been reported in individuals that are healthy but infected by Mycobacterium tuberculosis [310], as well as during chronic systemic inflammation [311]. This indicates that ongoing immune activation can result in an altered distribution of NK cells in the periphery. The marginal increase of CD56bright NK cells at two years of age may thus be explained by increased exposure to infectious agents during this age. Concurrent with that theory, many of the children included in this study attended day care centres, which could be a source of increased infection load. Further, the in vitro induction of IFN-γ production was slightly lower in CB NK cells compared to later time points. Although CB NK cells have been reported to have low

52 cytotoxicity [296] they are readily activated by cytokines in vitro and respond by producing vast amounts of IFN-γ [298]. Our results regarding intracellular IFN-γ in CB NK cells are in concordance with findings by others where similar levels of IFN-γ was induced in NK cells from CB and adults after in vitro culture with PMA and ionomycin [312]. Taken together, our data would agree with reports showing that the capacity of NK cells to respond to stimuli by producing cytokines is not impaired in healthy neonates. Rather, the lower levels of IFN-γ in CB NK cells, compared to later time points, may be due to other factors such as impaired IL- 12p70 production by accessory cells [299], which could result in lower activation of NK cells. The receptor expression on NK cells changed with increasing age. The proportion of LIR- 1+ NK cells was found to increase with age. To our knowledge this is the first report about expression of LIR-1 on NK cells in CB or during the early years of childhood. In adult peripheral blood approximately 30% of the NK cells express LIR-1 [223]. Cytokines, like IL- 2 or IL-15, can induce expression of LIR-1 on NK cells in vitro [313]. Thus, the increased proportion of LIR-1+ NK cells in our study may be explained by an altered cytokine milieu with increasing age. The proportion of CD94+NKG2C- (NKG2A+) NK cells and the level of expression of NKG2D and NKp30 decreased with age. The changes in proportion of CD94+NKG2C-, reflecting CD94+NKG2A+ NK cells, is in line with data showing acquisition of inhibiting receptors (CD94/NKG2A) in early NK-cell development, in both mice and humans [314,315]. The inhibitory properties appear to be acquired in an orderly pattern so that NK cells first can be inhibited, preventing autoreactivity. The expression of the activating receptors NKG2D and NKp30 can be influenced by cytokines [144,316-318] and the increased expression on NK cells in CB may be an effect of the pro-inflammatory cytokine profile induced by labour at delivery [309]. The reason why CB NK cells express more of the activating receptors compared to later in life, and why inhibitory receptor expression rather increase with age is not clear. One hypothesis is that NK cells and innate immunity are relatively more important in early life when adaptive immunity is immature. The first years of life involve an increased adaptation to a broad variety of external and internal stimuli that may influence the NK-cell receptor expression and function.

Paper II

The immune responses mediated by innate cells helps to determine the outcome of the subsequent acquired immune responses, which could be important in the context of allergy

53 development. In Paper II, we wanted to elucidate if NK cells were altered regarding phenotype and/or function at birth in individuals that later developed allergic disease. In this study, we show alterations in the NK-cell subpopulation composition and NK-cell receptor expression in CBMCs from newborns that became allergic at the age of 5. No variations were detected in the T-cell compartment. Children that became allergic had a smaller proportion of CD56bright NK cells compared to the children that did not become allergic. Several reports show alterations in the proportion of CD56bright NK cells in peripheral blood in pathological conditions. During treatment with IL-2R blockade in uveitis [319] and multiple sclerosis [320] and also during IFN-β therapy of multiple sclerosis [321] the proportion of CD56bright NK cells is increased. Furthermore, in primary HIV infection, the proportion of CD56bright NK cells is reduced while it is expanded in chronic HIV infection [322]. In a recent study, patients with the autoimmune inflammatory disease, systemic lupus erythematosus (SLE) were reported to have increased proportions of CD56bright NK cells, independent of treatment [311]. In the context of SLE being a disease promoted to some extent by increased production of type 1 interferons and allergies generally being considered to be driven by type 2 cytokines, it is interesting that the proportions of CD56bright NK cells are increased in SLE [311] and decreased in children that are developing early allergy but also in adults with manifested allergic symptoms [240]. Activated CD56bright NK cells are capable of producing IFN-γ that have multiple effects on the immune system, including activation of APCs [84,85], Th1 differentiation [86] and inhibition of class switching to IgE [89,90]. The CD56bright NK-cell subset and DCs are located in close proximity to T cells in human lymph nodes [97], providing prerequisites for NK-cell effects on T-cell priming. The local cytokine milieu might be of importance for the contributing effects of NK cells on DC induced polarization of naïve T cells [323]. In a murine model, injection of mature DCs induced recruitment of NK cells to lymph nodes and NK-cell derived IFN-γ was necessary for Th1 priming [232]. In conjunction with these reports, it is tempting to speculate that a decreased proportion of CD56bright NK cells could possibly reduce Th1 priming, favouring Th2 priming. In line with this, it was recently reported that also adult allergic individuals have a reduced CD56bright NK-cell subpopulation in the periphery and that their NK-cell IFN-γ-production in response to stimulation by DCs was reduced [240]. The group of children developing early allergy also tended to have an increased expression of the activating receptor NKp30. Cytokines derived from accessory cells like DCs are

54 important for activation of NK cells [324] and NK cells have been reported to regulate DC by means of the NKp30 mediated killing of immature dendritic cells (iDCs) and maturation of DCs in vitro [175,176]. Both NK cells and DCs are recruited early to sites of inflammation, which, besides from lymphoid tissue and priming of T cells as discussed above, could be another possible place for interaction between the cells types. If the NKp30-mediated regulation of DCs occurs in vivo, the reduced proportion of CD56bright NK cells in combination with the increased expression of NKp30 on NK cells could result in an altered regulation of early immune responses to infections. An altered ability by NK cells to stimulate DC maturation with subsequent Th1 responses, in pre-allergic individuals, may be one factor involved in the development of allergy. Further, the proportion of NK cells expressing CD94/NKG2C, in children that developed early allergy tended to be decreased while both groups had equally large proportions of NKG2A expressing NK cells. Although the difference is not big, the allergic group, with lower NKG2C but normal NKG2A, may be more inhibited than NK cells in the control group. An early alteration in the activating capacity of NK cell could perhaps contribute to the reduced Th1 priming discussed above. Whether the altered receptor expression have an effect on NK-cell function remains to be evaluated. However, upon in vitro stimulation with IL-2 and IL-12, CB cells from the two groups of children produced similar levels of cytokines (IFN-γ, IL-10 or TNF). Since IL-2 and IL-12 are cytokines that readily activates NK cells, these results could be indicative of equal activation of NK cells to cytokines between the groups. Our results imply that an early imbalance in NK-cell subpopulations may correlate with the development of allergy even before the onset of disease. However, if NK cells are involved in the process of disease development the mechanism still remains to be evaluated.

Paper III

Children seropositive (SP) for certain herpesviruses have been suggested to be less likely to become IgE-sensitized than seronegative (SN) children [271]. Recent findings in mice with herpesvirus latency, suggest resistance to infection with bacterial pathogens through prolonged production of the antiviral cytokine IFN-γ and systemic activation of macrophages [325]. In Paper III, we wanted to evaluate the functional capacities of innate immunity (NK cells and monocytes) in healthy young children that were either SP for EBV, EBV and CMV or SN for both. We hypothesized that herpesvirus latency would be associated with a higher

55 activation capacity of innate immune responses, which could be involved in Th1 priming and perhaps explain the reduced risk of becoming IgE-sensitized, observed by Nilsson et al [271]. In this study we demonstrate that 2-year old children SP for EBV have reduced proportions of IFN-γ+ NK cells as well as lower intracellular IFN-γ levels upon in vitro stimulation with IL-15 and PGN, (known to promote NK-cell survival/proliferation [97,326] and monocyte activation [327], respectively). This reduction was even more pronounced in children who were SP for both EBV and CMV. Since data on intracellular NK-cell IFN-γ and levels of IFN-γ detected in cell culture supernatants correlated, kinetic differences is unlikely the reason for the difference observed between the groups. We rather argue for a difference in NK-cell and/or monocyte reactivity between the groups of children. Concurrent with our in vitro data, plasma IFN-γ levels in SP children were significantly lower than in their SN counterparts, again with the lowest levels in CMV co-infection. Both CMV and EBV evade the immune system by interfering with the surface expression of MHC class I on the cell surface [245,328]. The disturbed balance of inhibitory and activating ligands may lead to direct activation of NK cells, which upon interaction with accessory cells could induce subsequent activation of adaptive immunity. Thus, our data could be a direct cause of viral infection, by interaction between infected cells and e.g. NK cells, or could be indirectly associated to infection with herpesvirus. The fact that some children are SN may reflect a very potent innate reaction towards viral infections, which theoretically could block productive viral infections and thus preclude adaptive immune responses. Indeed, we have observed an overall decreased cytokine production by PHA- stimulated PBMCs from SN children (Nilsson et al., unpublished), which could indicate that adaptive immunity, including T cells, is less activated. On the other hand, if herpes virus latency confers some kind of suppression of NK-cell immunity, it might be beneficial to the host for the development of adaptive immunity during early childhood years. SP children also tended to have a decreased proportion of the CD14+CD16+ monocytes subpopulation, which are more likely to differentiate into DCs upon stimulation [329]. In line with this finding there was a tendency towards lower levels of IL-6 in culture supernatants from SP subjects upon stimulation with IL-15 and PGN, indicating less proinflammatory monocyte function. Concurrent with the discussion of NK cells above, also alterations in monocytes subpopulations and function could be either directly due to viral infections, or a more indirect consequence thereof. The activation of accessory-cell function could be modified by herpesvirus infections. It has been shown that infection of monocyte-derived

56 DCs with CMV [330] and herpes simplex virus 1 [331] inhibits their maturation process as well as their release of pro-inflammatory cytokines. We did however, not observe any statistically significant differences between the groups in monocytes-related cytokine levels (culture supernatant concentrations of IL-6 and IL-10). It is possible that the quantification of IL-10 could have been affected by EBV and CMV encoded homologues for human IL-10 [332], although it is unlikely since the frequency of CMV infected monocytes from seropositive donors is very low. Although, the levels of IL-12p70 in cell culture supernatants were below the detection limit of our assay, low levels of IL-12p70 may be produced by monocytes in the cell culture, and delivered to NK cells through synaptic formation [236]. Altered IL-12 levels/delivery could possibly contribute to the differences in NK-cell IFN-γ responses between the groups. The fact that the NK-cell IFN-γ production in vitro and plasma levels of IFN-γ correlated, could imply that NK-cell derived IFN-γ was the major source of IFN-γ in plasma. T cells are also important in controlling viral infections [242], but here their contribution to the plasma IFN-γ is unclear. However, since the SP subjects had lower IFN-γ in their plasma, and we have observed an overall increased cytokine production by PHA-stimulated PBMCs from SP children (Nilsson et al. unpublished), this strongly argues against T cells as the major contributors to the differences in plasma IFN-γ between the groups. At first sight, the present data may seem difficult to reconcile with our previous findings of a high cytokine production in SP children (Nilsson et al. unpublished). However, in the study by Nilsson et al., PBMCs were activated in vitro with PHA, which mainly induces T- cell derived cytokines. Thus, the two studies indicate that seropositivity for EBV and CMV is associated with a lower NK-cell but higher T-cell cytokine response. This is interesting in relation to allergy development, since exposure to pathogenic and non-pathogenic agents during childhood has a strong influence on the progressive shift from the allergy-related Th2- polarization towards a Th1-Th2 balanced type of immunity [333]. NK-cell derived IFN-γ regulates Th1 development which is essential for long-term control of many pathogens [116] and it might also be important in preventing the development of aberrant immune reactions. Taken together, herpesvirus infections could be one of the contributing factors needed for maturation of adaptive immune responses. A very potent innate immunity may delay these infections and thereby also influence the development of allergies.

57 Study IV (preliminary data)

Results

No alterations of NK-cell proportions and the expression of NK-cell receptors in children from preeclamptic mothers We analysed the NK-cell populations in neonates born by preeclamptic and healthy mothers. The proportion of all NK cells and the proportion of CD56bright NK cells did not differ between the groups. Further, there were no differences in receptor expression of NKG2A and NKG2C with regard to the proportion of NK cells expressing the respective receptor [NKG2A; 66% (40-72) in healthy and 63% (51-66) in preeclampsia, NKG2C; 15% (13-78) in healthy and 19% (17-23) in preeclampsia] or in the level of expression on NK cells in either of the subsets (data not shown). In the few cases were there was enough cells, NK cells were further phenotyped for the activating receptors CD69, NKp30 and NKp46 (preeclamptic n=5 and healthy n=3 neonates). No significant differences were seen when comparing either proportion or expression levels of the receptors on NK cells (data not shown). Although no statistically significant difference could be demonstrated, there was a tendency towards a lower expression of NKG2D on NK cells in preeclamptic children [MFI 60 (25-105)] when compared to healthy children [MFI 101 (99-142)] (p= 0.4, Figure 2). Another study, performed in our group, with new material, that has continued to evaluate the NKG2D expression in preeclampsia, supports these findings (Ebba Sohlberg, personal communication). To study if the mode of delivery could influence NK-cell proportion and receptor expression, neonates were divided into VD (n=3) and ECS groups (n=5). An increased number of NK cells were detected in VD (p=0.03). However, there was a statistically significant difference in gestational age (p=0.012) where pregnancies that ended by ECS were shorter, which may influence the distribution among lymphocytes.

58

Figure 2. Median fluorescence intensity (MFI) of NKG2D on CB human NK cells from healthy pregnancies (n=3) and from preeclamptic pregnancies (n=5).

Low capacity of NK cells to produce IFN-γ and IL-4 in both healthy and preeclamptic neonates The ability of NK cells to produce IFN-γ and IL-4 in response to in vitro stimulation (IL- 15 and PGN) was measured by intracellular staining. There was very low spontaneous and induced production of IFN-γ by NK cells in both the groups [0.3% (0.0-0.5) in the preeclamptic group and 0.4% (0.0-0.8) in the healthy group]. We confirmed this poor production of IFN-γ by ELISA, run on culture supernatants and in most cases the levels were close to the detection limit of the assay (3.9 pg/ml). Both groups did have NK cells positive for IL-4 although no differences in proportion of IL-4+ NK cells [5.8% (1.7-14.2) in the preeclamptic group and 6.4% (3.1-11.6) in the healthy group] or expression level [MFI 43.5 (26.5-63.7) in the preeclamptic group and MFI 51.1 (22.3-75.6) in the healthy group] were detected. To evaluate if the cellular response to stimulation differed at an individual level we compared the stimulation indexes of the groups. The index is obtained by dividing the value for stimulated cells with the value for unstimulated cells, for each individual. An index of 1 means no cellular response. A value below 1 is a downregulation, while a value above 1 is an upregulation of the response by NK cells to IL-15 and PGN. The two groups tended to differ in their response to in vitro stimulation of IL-15 and PGN where the proportion of IL-4+ NK cells in neonates from healthy pregnancies decreased but were unchanged in the preeclamptic group (p=0.14, Figure 3). No differences between the groups were detected in the regulation,

59 measured as MFI of IL-4+ NK cells.

Figure 3. Stimulation index (ratio of IL-15 and PGN stimulated/unstimulated from cell cultures) of the proportion of IL-4+ NK cells of all lymphocytes.

Altered levels of pro-inflammatory cytokines in sera from neonates born by preeclamptic mothers The levels of the cytokines IL-12p70, TNF, IL-1β, IL-6, IL-8 and IL-10, in sera from neonates born by preeclamptic and healthy mothers were evaluated. In sera from preeclamptic neonates there were significantly decreased levels of IL-12p70 (p=0.003, Figure 4a) and TNF (p=0.001, Figure 4b) compared to neonates from healthy mothers, while the levels of IL-8 were increased (p=0.047, Figure 4c). No differences in the levels of IL-1β, IL-6, and IL-10 between the two groups of newborn children were found.

60

Figure 4. Cytokine levels of a) IL-12, b) TNF and c) IL-8 in CB sera from healthy (n=16) and preeclamptic (n=10) mothers.

Labour can cause elevated levels of pro-inflammatory cytokines (IL-12p70, TNF and IL-8) in neonates [309]. To evaluate effects of labour all children, healthy and sick, were divided into VD and ECS. There were statistically significant differences between the two groups with regard to the levels of IL-6 (p=0.02) and IL-8 (p=0.004) where the VD group had higher levels of both cytokines. There was a significant difference in gestational age, being higher in the VD group (p=0.0006, median VD = 40 weeks, median ECS = 38 weeks). To exclude labour as a confounding factor, only healthy and preeclamptic neonates delivered by ECS were evaluated next. Levels of TNF were still significantly lower (p=0.03, Figure 5a) and IL-8 was higher (p=0.03, Figure 5b) in the preeclamptic group. The preeclamptic group had a significantly lower gestational age compared to the healthy group (p=0.02) (healthy group, median=38 weeks and preeclamptic group, median=37 weeks). Only one neonate in the ECS group was not delivered by full term.

61

Figure 5. Cytokine levels of a) TNF and b) IL-8 in CB sera from healthy (n=11) and preeclamptic (n=4) mothers delivered by ECS.

Positive correlation of serum IL-6 levels between mother and neonate in preeclampsia The morphology of both moderate and severe cases of preeclamptic placentae is altered, with disturbed layers of syncytiotrophoblasts and malformed villi [303]. To evaluate if an altered placental barrier in preeclampsia was associated to abnormal function of maternal- foetal communication, we studied the correlation of cytokines between the children and their mothers (neonates from healthy mothers n=8, neonates from preeclamptic mothers n=8). There was a significant correlation between IL-6 levels in the preeclamptic mothers and their neonates (r= 0.83 p=0.042), but not in the healthy group.

Discussion

In this study we wanted to investigate whether the escalated inflammation in the circulation of preeclamptic women [334-337] and altered placental morphology [303] had an influence on the immunological profile in the neonates. The following parameters were evaluated: NK-cell proportions, NK-cell receptor expression, NK-cell IFN-γ and IL-4 production and serum levels of pro- and anti-inflammatory cytokines. Maternal preeclampsia had no effect on CB NK-cell proportions and NK-cell-subset distribution, which is in contrast to what have been reported before [306,307]. Children born to preeclamptic mothers are more often delivered by ECS [307] and the mode of delivery

62 [300] as well as gestational age [338] can influence NK-cell numbers. In our study we found increased proportions of NK cells in CB from children delivered by VD, which is in line with the study by Samelson et al [300]. Preeclampsia did not influence the proportion of NK cells expressing NKG2A, NKG2C, CD69, NKp30 and NKp46 or the receptor levels on NK cells. To note is the large proportion of human cord-blood NK cells expressing NKG2A compared to data obtained from analyis of adult blood donors, reported in Paper I and a recent report [339]. The high prevalence of NKG2A+ NK cells in CB could possibly explain their low cytotoxicity [296,339], as NKG2A is an inhibitory receptor with an ubiquitously expressed ligand, i.e. HLA-E. Except for inhibition of NK-cell activation, co-engagement of NKG2A can suppress CD69-initiated cytotoxicity [340]. The proportion of cord-blood NK cells expressing NKG2C is also concurrent with our previous findings (Paper I). Also this receptor is more prevalent in children than healthy adults although increased proportions of NKG2C+ NK cells are evident in adult individuals seropositive for CMV [341]. Preliminary data showed a trend towards reduced levels of NKG2D on NK cells from children with preeclamptic mothers. NKG2D expression can be influenced by cytokines [143,316,342] where e.g. NK cells treated with IL- 15 and IL-12 upregulate the expression of NKG2D [143,144], and T-cell expression of NKG2D is upregulated by TNF [343]. The reduced NKG2D expression on NK cells from neonates born from preeclamptic mothers could depend on the lower IL-12 and TNF levels found in their cord sera. Further, pregnancy has also been reported to influence NKG2D expression. The NKG2D receptor can be downregulated on PBMCs, in the mother, by soluble MICA and MICB (sMICs) derived from the syncytiotrophoblasts in the placenta [344]. Although we did not substantiate the presence of these molecules in the mothers or their children we found a strong correlation between IL-6 measured in sera from preeclamptic, but not healthy, mothers and their neonates indicative of a transfer of IL-6 over the maternal- foetal interface. If sMICs would be transferred to the foetal side by leakage or active transfer, it could possibly influence the NKG2D levels in the preeclamptic children. Although we did not detect any differences between the groups regarding the other receptors that were evaluated, the reduced levels of the activating receptor NKG2D on NK cells from children born by preeclamptic mothers may indicate a population of NK cells that are less activated and/or less capable of cytolysis or cytokine production. The proportions of CB NK cells positive for IFN-γ and IL-4 following in vitro stimulation was very low, as was the levels of released IFN-γ in culture supernatant with no detectable differences between groups. The low levels of released IFN-γ is in line with earlier reports

63 [345], but our results regarding intracellular IFN-γ in CB NK cells do not agree with other findings [312,346], which could be due to the different stimulations used in vitro. When evaluating the stimulation index, there was a tendency towards a downregulation of IL-4 producing NK cells in children with healthy mothers. In an attempt to explain this finding, we looked into serum levels of IL-12p70 and TNF. According to the literature, the immune system of the neonate is skewed towards a type 2- profile while the type 1-response is ameliorated during the first days of life [290,297]. This may be due to an impaired TLR-induced APC production of pro-inflammatory cytokines seen in neonates [347,348] and defects in intracellular events have been explained for the low IL- 12p70 and TNF production [349]. However, the production of cytokines in APCs found in early life might depend on the chosen stimuli [299,345,349]. In our study, we found that CB serum levels of IL-12p70 and TNF in foetal sera from healthy pregnancies are similar to adult levels, while children born from preeclamptic mothers had significantly lower levels of these cytokines. Thus, the downregulation of IL-4+ NK cells possibly reflects a dampened type 2- response driven by a more pronounced pro-inflammatory response induced by APCs in healthy neonates that do not function equally well in neonates born by preeclamptic mothers. Although the mode of delivery can influence levels of TNF in CB, with higher levels in VD than in unlaboured ECS [301,309], this did not explain the differences between the groups observed here. IL-8 levels were significantly increased in sera from neonates of preeclamptic pregnancies. IL-8 production can be influenced by labour and therefore we subdivided all children into mode of delivery. Not surprisingly, IL-6 and IL-8 were elevated in VD children. These cytokines are produced in higher levels during labour [309]. IL-6 induces oxytocin, an important hormone believed to induce labour work [350]. IL-8 can also be further escalated during stressful deliveries like acute ECS and VD with assistance and in preterm ECS associated with infections [351,352] and it is possible that preeclamptic pregnancies could be associated with a higher stress during delivery. To investigate if the increased IL-8 levels in preeclamptic cord sera were influenced by mode of delivery, we further studied neonates from the two groups delivered by ECS, which still showed increased IL-8 in the preeclamptic group. The higher IL-8 levels in sera from neonates with preeclamptic mothers could be due to an overall higher load of maternal stress or to an infection in preeclamptic pregnancies. Taken together, our results suggest that NK cells in neonates born by preeclamptic mothers are phenotypically and functionally altered compared to neonates from healthy controls. The cytokine levels in sera were also altered indicating that preeclampsia has an effect on the

64 immune system of the child. However, the results are preliminary and need to be confirmed before any firm conclusions can be drawn.

65 CONCLUDING REMARKS AND FUTURE PERSPECTIVES

During early childhood years a continuous exposure to pathogens and harmless microbial products modulates the immune system and an altered and/or delayed exposure might affect the immune responses later in life. With age being an important factor for the outcome of both innate and adaptive immune responses, the mechanisms responsible for disease development might origin from early immunological variations. This work has focused on evaluating NK cells at birth and during early childhood years. According to the results from study I, the NK-cell populations in peripheral blood, defined by their phenotype, are continuously changing with increasing age in healthy children. This was paralleled by an increasing capacity to produce IFN-γ. These results indicate that environmental factors modulate the immune system. The innate and adaptive immune systems are tightly interrelated and disturbances of the innate responses could affect the outcome of the adaptive responses. In study II, we found that children that developed early allergy had a decreased proportion of CD56bright NK cells and a slightly altered receptor expression by NK cells at birth. We speculate that these early alterations of NK cells could have an influence on adaptive immunity and subsequently on development of allergic disease. The reason for these alterations at birth is unknown. However, from the preliminary results of study IV, we show data that suggest that preeclampsia can have an influence on the innate immunity of the child. These data indicate a maternal effect on the immune status of the child. A main finding from study III is that innate immunity seems to be affected by herpesvirus seropositivity in children at two years of age. This is exhibited by lower production of cytokines by innate cells that may be one factor that can influence the maturation of the adaptive immunity. CB NK cells have been reported to be inefficient in their cytotoxic capacity [296]. For future evaluations it could be of interest to study their functional capacities, including cytotoxicity and cytokine production during early childhood years. Infection by herpesvirus, before the age of 2, associates with decreased function of innate immunity. The future plan is to evaluate if this effect is sustained at 5 years of age. Since herpesvirus seropositivity has been suggested to decrease the risk of becoming IgE-sensitized [271], it could be of interest to evaluate features of adaptive immunity. In study II we found a difference in NK cell proportions between allergic and non-allergic children, present already in CB. In a pilot study we found that this difference did not remain at 2 and 5 years (not shown). A recent study shows that allergic adults have a reduced proportion of CD56bright NK cells in the periphery

66 [240]. Thus, it could be of interest to follow the children from study II to see if the observed alteration at birth reappears at an older age. For the last study, preliminary data associating preeclampsia with CB NK-cell phenotype and function and with CB serum levels of monokines are intriguing and it would be interesting to evaluate if these associations can be validated in a larger cohort of neonates.

67 ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to everyone that in one way or another has contributed to this work and especially:

My supervisor Marita Troye-Blomberg for your support and dedication to this project.

My co-supervisor Louise Berg. You are the best supervisor a student could ever wish for! You are always willing to take the time to discuss questions and I am ever so grateful for your dedication, patients and encouragement. Your enthusiasm is contagious! 

The seniors at the department of immunology: Klavs Berzins, Carmen Fernandez, Eva Severinson and Eva Sverremark-Ekström for your support.

Thanks to my co-workers at Sachs’ Children’s Hospital, Södersjukhuset; Caroline Nilsson, Gunnar Lilja, Monica Nordlund and Anna Stina Ander.

Also great thanks to Klas Kärre at MTC, Karolinska Institutet for your valuable comments on manuscripts.

Thanks to all students at the Department of Immunology that make the working environment so warm and friendly! John Arko-Mensah who realized that my blood sugar was low before I did and sent me away for lunch when we were teaching together! Nancy Awah, Nora Bachmayer, Halima Balogun, Jacqueline Calla, Olga Daniela Chuquima, Salah Farouk. Pablo Giusti for your refreshing, happy attitude! Ulrika Holmlund, Nnaemeka Iriemenam. Elisabeth Israelsson for the extra support during the last few months. Maria Johansson, Valentina Mangano, Amre Nasr, Jubayer Rahman, Shanie Saghafian-Hedengren, Ylva Sjögren, Olivia Simone, Ebba Sohlberg, Fernando Sosa, Stefania Verani.

Former students at the department: Anna Tjärnlund, Camilla Rydström, Ariane Rodriguez, Manijeh Vafa Homann, Nina-Maria Vasconcelos, Qazi Khaleda Rahman,

68 Anna-Karin Sigfrinius (Larsson), Jacob Minang, Khosro Masjedi. Petra Amodruz, my former room mate for our discussions and mutual encouragement.

Special thanx to Nora Bachmayer for the occasional brakes during the days to get some “finkaffe” and for always making me laugh. Anna Tjärnlund, my fellow “norrlänning”, for your encouraging support and the inspiration you supply by just being the person that you are. Camilla Rydström the best creator of “chokladpraliner”! It is always great to see you when you come around for a visit. Ylva Sjögren, the greatest traveling companion. Shanie Saghafian-Hedengren for brightening up the working days with your lively personality.

Special thanx to Margareta “Maggan” Hagstedt for putting up with all the questions! You are truly an asset for the department and a nice companion during coffee brakes! Ann Sjölund for the help with practical things and the plommon-deliveries! Yummy! Thanks to Gelana Yadeta for all the help. You always have a positive attitude! Anna-Lena Jarva for all your fun parties and quizzes!

My deepest and warmest thanks to my friends. I treasure our friendship and you mean the world to me!

Kamilla Östberg, my very dear friend from the childhood years in Gnarp. We have a special bond that keeps us close even if we are apart.

My “gymnasiepolare” Jenny Elwell, Jenny “Väris” Värhammar, Fredrica Jonsson and Mathilda Hermansson. I am very privileged to have you as my friends! Jenny E, we have to go out dancing soon! Seriously! And “Väris”, thank you for taking my to the gym on a more regular basis than I would have been able to achieve without your company! Especially during this last year…

Jenny Wolin, that shares my interest in creative activities! Anna Lövgren, the master of mixing drinks!

My dear “biologkompisar” Lina Enell, Åsa Andersson, Ida von Schéele, Anna Davidsson, Julia Borg and Agnes Karlson. It always makes my happy to see you! What should we “leka” next? 

69

During later years my family have been extended to include Bosse (who sadly passed away recently), Berit, Ulf, Richard, Anneli and Henrik. Thank you for embracing me into your family and making me feel as a part of it! Thanks also to Lars-Erik and Barbro, Margot and Harry.

Most importantly I would like to express my warmest gratitude to my family; my sister Susanne, my brother Niklas, my mother Siv and my father Rolf! Your support and love throughout my life is invaluable. Mamma och pappa, jag är lyckligt lottad som har er till föräldrar! You are the best! My sincere thanks also to Sara for you support and understanding of this process, Emil for directing the focus towards the really important things in life, Stig for your kindness and Barbro for all our nice chats and wonderful times together.

Fredrik, min älskling! For always focusing on the positive things in life. Without your love and support I would not have been able to do this.

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