Functional and molecular characterization of T cells and Natural killer (NK) cells in rainbow trout (Oncorhynchus mykiss)

Distribution of T cell subpopulations in lymphoid and mucosal organs of rainbow trout Oncorhynchus mykiss characterized by new lineage marker specific monoclonal antibodies .... MNC Mono

LUẬN VĂN TIẾN SỸ

ĐẶNG THỊ HƯƠNG

VIỆT NAM

T cells and Natural killer (NK) cells in rainbow trout (Oncorhynchus mykiss)

Inauguraldissertation zur Erlangung des akademischen Grades

doctor rerum naturalium (Dr rer nat.)

an der Mathematisch-Naturwissenschaftlichen Fakultät

der Ernst-Moritz-Arndt-Universität Greifswald

vorgelegt von Huong Dang Thi geboren am 27.05.1982

in Bac Giang, Viet Nam

Greifswald, 2015

Dekan:

1.Gutachter: Prof.Dr.Mettenleiter

2.Gutachter: Prof.Dr.Steinhagen (Hannover)

Tag der Promotion: 29.09.2015

List of figures I List of tables III Abbreviations IV Zusammenfassung VI Summary VIII

Chapter 1 General introduction 1

1.1 Aquaculture 1

1.2 Aquaculture production of rainbow trout 1

1.3 Taxonomy of rainbow trout 3

1.4 Problems in modern aquaculture production 4

1.4.1 Antibiotic treatment 5

1.4.2 Increasing survival of fish caused by probiotic treatment 6

1.4.3 Vaccination 7

1.5 Immune system of teleost 10

1.5.1 Immune organs in fish 10

1.5.2 Innate immune system in fish 12

1.5.3 Adaptive immune system 13

1.5.4 B cells and Immunoglobulins 15

1.5.5 T cells and TCR receptors in teleost 15

1.5.6 NK cells in teleost 18

1.5.7 CD56 21

1.6 Aims of the present study 24

Chapter 2 Distribution of T cell subpopulations in lymphoid and mucosal organs of rainbow trout (Oncorhynchus mykiss) characterized by new lineage marker specific monoclonal antibodies 25

2.1 Abstract 26

2.2 Introduction 27

2.3 Material and methods 29

2.3.1 Animals and organ sampling 29

2.3.2 Leukocyte preparation 29

2.3.3 Generation of monoclonal antibodies 29

2.3.4 Single and dual flow cytometry 30

2.3.5 Immunoprecitation 30

2.3.6 Separation of lymphocyte subpopulations 31

2.3.7 RT-PCR and real-time PCR 32

2.3.8 Functional assays 32

2.3.8.1 Reaction pattern of mab D11, mab D30 and mab 89 on stimulated cells 32

2.3.8.2 Kinetics of B and T cells in allogeneic stimulated trout 32

2.3.8.3 Cell mediated allogeneic cytotoxicity assay 33

2.4 Results 34

2.4.1 Mab D11, mab D30 and mab 89 display a unique staining pattern 34

2.4.2 Mab D11 and D30 recognize the same leukocyte population 39

2.4.3 The marker recognized by mab 89 is not expressed on all T cells 40

2.4.4 Naive B cells or thrombocytes are not labelled by mabs D11, D30 or 89 41

2.4.5 Immunochemical characterization of the T cell surface marker recognized by mab D11 and mab D30 42

2.4.6 Distribution of T lymphocyte subpopulations in lymphoid organs of rainbow trout42 2.4.7 CD8α - T cells are characterized by expression of CD4 mRNA as Th cells 44

2.4.8 The expression of specific transcription factors reveals the presence of Th cell subpopulations 46

2.4.9 Functional characterization of rainbow trout T cells 49

2.5 Discussion 51

Chapter 3 A multicolour flow cytometry identifying leukocyte subsets of rainbow trout (Oncorhynchus mykiss) 55

3.1 Abstract 56

3.2 Introduction 57

3.3 Material and methods 57

3.4 Results and discussion 58

Chapter 4 CD56 (NCAM1) positive leukocyte population in rainbow trout – molecular and functional characterization 62

4.1 Abstract 63

4.2 Introduction 64

4.3 Material and Methods 66

4.3.1 Fish 66

4.3.2 Leukocyte preparation and cell sorting 66

4.3.3 RNA extraction and cDNA synthesis 67

4.3.4 Cloning and sequencing of CD56 67

4.3.5 Sequence analysis 67

4.3.6 Identification of alternative splicing in trout CD56 68

4.3.7 RT-PCR analysis 68

4.3.8 Generation of monoclonal antibodies using recombinant protein 68

4.3.9 Immunofluorescence analysis of cells 69

4.3.10 NK cell cytotoxicity in xenogeneic model 69

4.4 Results 71

4.4.1 Sequence analysis and characterization of trout CD56 71

4.4.2 Phylogenetic analysis 76

4.4.3 Characterization of CD56 variability by VASE element 78

4.4.4 Characterization of CD56 variability by MSD element 80

4.4.5 In vivo expression of CD56 isoform transcripts in tissues and leukocytes 89

4.4.6 Trout CD56 expression in T and myeloid cells contrast to IgM + B cells and thrombocytes 90

4.4.7 Up-regulation of trout CD56 expression upon xenogeneic stimulation 91

4.4.8 Natural cytotoxicity assay 92

4.4.9 Characterization of anti-trout CD56 mabs 94

4.5 Discussion 97

Chapter 5 General discussion and outlook 103

5.1 Monoclonal antibody production 103

5.2 New established antibodies, new immune tools for studying T cells of fish immune system (chapter 2 of the present thesis) 106

5.3 Gene duplication 109

5.4 CD56 diversity, a typical example indicating the success of salmonids 111

5.5 Rainbow trout CD56, a marker NK cell 116

5.6 Outlook 119

Appendix 121

Appendix 1: Genbank accession numbers 121

Appendix 2: Sequence of primers 122

Appendix 3: Nucleotide sequence of a clone containing triplet “AAG” in front of M 30 exon 124

Appendix 4: The possible membrane bound trout MSD combinations of trout CD56 transcripts 125

References 128

Acknowledgement 149

About the author 151

List of publication and oral presentation 151

Resume 153

Personal Data 153

Education 153

Work experience 153

Muster der Erklärung 154

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List of tables

Abbreviations

All abbreviations used in this thesis are listed in alphabetical order

CD Cluster of Differentiation

CD56 (NCAM1) Neural adhesion molecule 1

CIS Combinatorial Immune Response

DAMPs Danger-Associated Molecular Patterns

DAP12 TYRO protein tyrosine kinase-binding protein

Facs Fluorescence Activated Cell Sorting

FNIII Fibronectin type III

GPI Glycosyl Phosphatidyl Inositol

IL-1RAcP Interleukin-1 Receptor Accessory Protein

ILT-2 Ig-Like Transcript 2

ITAM Immunoreceptor Tyrosine-based Activation Motif

ITIM Immunoreceptor Tyrosine-based Inhibitory Motif

LFA-1 Leukocyte Function-associated Antigen 1

MCSFR Macrophage Colony Stimulating Factor Receptor

MHC Major Histocompatibility Complex

MNC Mononuclear cells

NCCs Nonspecific Cytotoxic Cells

NILT Novel Immunoglobulin-Like Transcript

NITRs Novel Immune Type Receptors

PAMPs Pathogen-Associated Molecular Patterns

PRR Pattern Recognition Receptor

PSGL-1 P-selectin glycoprotein ligand 1

RAG Recombination Activating Gene

Zusammenfassung

Das Immunsystem aller Vertebraten ist verantwortlich dafür, die Homöostase des Organismus, über die Eliminierung entarteter oder gealterter Körperzellen und durch den Schutz vor Infektionen (Viren, Bakterien, Pilze, Parasiten) (Murphy et al., 2012) aufrecht zu erhalten Es ist ein komplex reguliertes Netzwerk angeborener und erworbener Immunmechanismen bei denen humorale Faktoren und zelluläre Effektoren interagieren

Diese Immunmechanismen basieren auf einer Unterscheidung zwischen “Eigen” und

“Fremd” Strukturen Pathogene oder veränderte Körperzellen werden darüber von verschiedenen Rezeptorkomplexen auf Immunzellen erkannt Muster erkennende Rezeptoren (pattern recognition receptors, PRR) binden dabei an evolutionär konservierte Strukturen auf Pathogenen (pathogen associated molecular patterns, PAMP) oder mit einer Gewebszerstörung verbundenen Zellmarker (danger associated molecular patterns, DAMP) (Takeuchi and Akira 2010) Fehlende MHC I Moleküle (major histocompatibility class I) werden von Rezeptoren auf natürlichen Killerzellen erkannt (Fischer, Koppang and Nakanishi

2013, Raulet 2006) Fremdpeptide werden in eigenen MHC I bzw MHC II Molekülen an B bzw T-zellen präsentiert und von spezifischen Rezeptorkomplexen erkannt (BCR; TCR) Diese Aktivierung führt zu einer Proliferation und Reifung der B- bzw T-Lymphozyten bis

zu Effektorstadien

Einige zelluläre Rezeptoren sind auf allen Leukozyten permanent exprimiert (z.B MHC I), andere nur in bestimmten Reifungs- und Funktionsstadien bzw auf bestimmten Leukozytenpopulationen (Monozyten, Granulozyten, NK-Zellen, Lymphozyten) (Murphy et al., 2012) Basierend spezifischen monoklonalen Antikörpern, die spezifisch solche Moleküle binden wurde für verschiedene Säuger (Mensch, Maus, Ratte, Schwein, Rind, Hund) eine System von Differenzierungsmarkern (Cluster of Differentiation Molecules, CD) auf Leukozyten etabliert (Cobbold and Metcalfe 1994, Hopkins, Ross and Dutia 1993, Haverson

et al 2001, Mason et al 2001) Mit diesen mAk ist es nicht nur möglich Entwicklungs- und Funktionsstadien einzelner Leukozytensubpopulationen zu bestimmen sondern auch die Interaktion solcher Populationen zu untersuchen

Für Knochenfisch existiert ein solches System nicht Nur eine kleine Anzahl von mAk gegen Leukozytenmarker ist bisher publiziert (Köllner et al 2004, Köllner et al 2001, Zhang

et al 2010, Ramirez-Gomez et al 2012, Wen et al 2011, DeLuca, Wilson and Warr 1983, Toda et al 2011, Toda et al 2009, Takizawa et al 2011a, Hetland et al 2010, Araki et al 2008) Die meisten von diesen mAk sind zudem strikt Spezies spezifisch

Ziel der vorliegenden Arbeit war es daher, solche mAk spezifisch für

T-Zellpopulationen der Regenbogenforelle (Oncorhynchus mykiss) zu entwickeln (Kapitel 2)

T-Lymphozyten sind durch die Expression eines T-Zell-Rezeptorkomplexes charakterisiert, der aus verschiedenen Ketten des (α α, δ β) und dem CD3 Molekül mit

α, β, γ, δ, ε und ζ) Ketten gebildet wird Zytotoxische T-Zellen binden an MHC I präsentierte Fremdpeptide über diesen TCR Komplex und dem Ko-Rezeptormolekül CD8 T-Helfer-Zellen erkennen MHC II präsentierte Fremdpeptide über den TCR Komplex und dem Ko-Rezeptor CD4

In den letzten Jahren sind die Gene, die für solche Moleküle kodieren, bei verschiedenen Fischarten kloniert und sequenziert worden Spezifische mAk gegen diese Moleküle z.B im Goldfisch halfen deren Expression auf Leukozyten in morphologischem und funktionellen Kontext zu charakterisieren Mak spezifisch für den TCR Komplex konnten bisher nicht etabliert werden Mit den in dieser Arbeit charakterisierten anti-pan-T-Zell mAk konnte die Organverteilung sowie die Aktivierung von T-Zellen in der Regenbogenforelle

erstmalig beschrieben werden (Kapitel 2) Darüber hinaus wurde eine Methode etabliert, bei

der durch Kombination verschiedener mAk spezifisch für Differenzierungsmarker die Verteilung und Reaktionskinetik von Leukozytensubpopulationen untersucht werden kann

(Kapitel 3)

Die erste Verteidigungslinie gegen Pathogene wird durch die evolutionär alten Monozyten und NK-Zellen gebildet Diese angeborenen Immunmechanismen sind hoch entwickelt in Knochenfischen Zwei Subpopulationen von NK-Zellen wurden in Fischen bisher beschrieben: natürliche zytotoxische Zellen und NK-Zellen (Shen et al 2002, Shen et

al 2003, Shen et al 2004, Fischer et al 2013) Funktionstest zur Charakterisierung von angeborenen und erworbenen zellulären Immunmechanismen sind bisher nur in wenigen Fischarten etabliert worden, ohne das jedoch spezifische mAk vorhanden sind, um diese Zellen direkt zu messen Daher wurde hier das ein NK-Zelle Marker CD56 molekular charakterisiert und die Expression auf verschiedenen Leukozytenpopulationen untersucht

(Kapitel 4)

Die hier etablierten mAk sowie die Funktionstest erlauben zukünftig eine detaillierte Untersuchung angeborener und erworbener zellulärer Immunmechnismen in der Regebogenforelle

The immune system of all vertebrates primarily is responsible to maintain the organism's homeostasis by either eliminating neoplastic or altered body cells and to protect against foreign invaders (viruses, bacteria, fungi, parasites) (Murphy 2012) It is a highly regulated network of innate and adaptive mechanisms between humoral factors and leukocytes

The successful elimination or protection is crucially based on differentiation of self from non-self Pathogens and altered body cells are recognized by different receptor complexes on immune cells Expressed pathogen- or danger-associated molecular patterns (PAMPs or DAMPs, respectively) are bound by pattern recognition receptors (PRR) (Takeuchi and Akira 2010) Missing major histocompatibility (MHC) class I molecules or

non-self (e.g allogeneic or xenogeneic cells) MHC are recognized by natural killer cell

receptors (Fischer, Koppang and Nakanishi 2013, Raulet 2006) Foreign non-self peptides are presented through MHC class I (intracellular) or through MHC class II (extracellular) to B- cell or T cell receptor complexes

This initial activation is regulated by humoral factors or cellular interactions ligand interactions) resulting in the activation, proliferation and effector function within an immune response Some of the cellular receptors are permanently expressed on all leukocytes

(receptor-on a high level (MHC class I), whereas others (receptor-only are expressed during certain developmental or activation stages or on certain leukocyte populations (monocytes, granulocytes, NK cells, lymphocytes) (Murphy 2012, Biosciences 2010)

For different mammals (man, mouse, rat, but also swine, cattle, dog), a system of characterized leukocyte surface molecules primarily based on the recognition of these molecules by specific monoclonal antibodies (mabs) was summarized at international workshops as clusters of differentiation (CD) (Cobbold and Metcalfe 1994, Hopkins, Ross and Dutia 1993, Haverson et al 2001, Mason et al 2001) Using these mabs, it is not only possible to characterize the developmental and functional stage of different leukocyte subpopulations but also to define the interactions between these populations

For bony fish, such a system does not exist Only a limited number of mabs against leukocyte surface molecules is available and most of them are strongly specific for species (Köllner et al 2004, Köllner et al 2001, Zhang et al 2010, Ramirez-Gomez et al 2012, Wen

et al 2011, DeLuca, Wilson and Warr 1983, Toda et al 2011, Toda et al 2009, Takizawa et

al 2011a, Hetland et al 2010, Araki et al 2008)

The goal of this PhD work, therefore, was to develop monoclonal antibodies against

surface markers of rainbow trout (Oncorhynchus mykiss) T cell population (chapter 2) The

lymphocytes are characterized by the expression of a T cell receptor complex composed of TCR chains (α and β) and CD3 chains (α, β, γ, δ, ε and ζ) Cytotoxic T lymphocytes (CTLs) binds to MHC class I bound peptide on the infected host cell using their T cell receptor (TCR) and its co-receptor CD8 resulting in specific killing Th cells recognize peptides through their

T cell receptor (TCR) and their co-receptor CD4 after extracellular antigens uptake, processing and presentation via MHC class II by professional antigen presenting cells (macrophages, dendritic cells and B cells) During recent years, genes encoding MHC class I and II, TCR and their co-receptors CD8 and CD4 have been cloned in several fish species and antibodies have been developed to study protein expression in morphological and functional contexts However, mabs specific for TCR or CD3 have not been established yet Therefore, using pan-T cell marker specific mabs, the activation and kinetics of T cell subpopulation

should be investigated (chapter 2) Moreover, a flow cytometry method was established

using different lineage marker specific mabs to measure different leukocyte populations and

their involvement in immune mechanisms of trout using a single tube assay (chapter 3)

The first line of defense against altered body cells or pathogens is provided by evolutionarily ancient macrophages and natural killer (NK) cells These innate mechanisms are well developed in bony fish Two types of NK cell homologues have been described in fish: non-specific cytotoxic cells and NK-like cells (Shen et al 2002, Shen et al 2003, Shen et

al 2004, Fischer et al 2013) Functional assays for innate and adaptive lymphocyte responses have been developed in only a few fish species However, there are no tools available until now in trout to follow these cells directly in the immune response The molecular characteristics and the expression on leukocyte subpopulations of CD56 were therefore analyzed Furthermore, a mab that is specific for a molecule expressed only in NK cells but

with uncommon expression kinetics was established (chapter 4)

Overall, the established tools and methods allow a more detailed characterization of cellular immune mechanisms against intracellular pathogens in rainbow trout

1999, Marineharvest 2014, Dimitroglou et al 2011) Recently, aquaculture is considered the fastest growing animal-food producing sector, which accounted for close to half of total fish production (FAO 2014c), supplying 66,6 million tons of fish in 2012 (FAO 2014c)

The sector is currently dominated by Asia-Pacific region, with predominant role of China, which accounted for 65% of global production in 2012, while the production in Europe and North America substantially slowed Dominating position of Asia in world aquaculture also influences the composition of produced species, dominated by freshwater fish (37 million tons) such as carp, followed by crustaceans (2,5 million tons) Diadromous, molluscs and marine fishes accounted together for 1,5 million tons (Korytar 2012, FAO 2014b)

1.2 Aquaculture production of rainbow trout

Rainbow trout is native to cold water tributaries of the pacific Ocean in Asia and North America (Wikipedia 2015, Council 2000) Rainbow trout is one of the most popular and easily reared aquaculture fish because it grows very fast and is a crowding tolerant fish,

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making it well suited to captive breeding Consequently, nearly all of rainbow trout production is obtained from aquaculture (Burden 2014) Rainbow trout aquaculture production increased very fast from 197 thousand tons in 1985 to 856 thousand tons (99,77% total rainbow trout production) with value 3631 million USD in 2012, representing as one of the third biggest produced diadromous fish after Atlantic Salmon and milkfish (Fig 1) (FAO 2014b)

Figure 1 Global aquaculture production of rainbow trout from 1973-2012 (FAO 2014b)

The production of rainbow trout has grown exponentially since the 1970s, especially

in Chile and more recently in Iran and Turkey One-third of rainbow trout production comes from Europe where it is dominated by Norway, Italy, Denmark and France (Fig 2)

Figure 2 World leading countries in the aquaculture production of rainbow trout in 2012 (FAO 2014b)

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Germany belongs to top ten of main producer in Europe, producing 9394 tons of rainbow trout in the value of 39 million USD 2012 (FAO 2014a)

Figure 3 Production of main fishes in Germany (FAO 2014b)

In Germany, rainbow trout contributes around 60% of annual aquaculture production and surpass common carp and other fish species (Fig 3) (FAO 2014a)

1.3 Taxonomy of rainbow trout

The rainbow trout (Oncorhynchus mykiss) belongs to Oncorhynchus genus, family

Salmonidae that also includes Atlantic salmon Evolutionally, rainbow trout together with

Atlantic salmon belong to teleost group evolved early after the lobe and ray-finned fish branches have split (Berthelot et al 2014) One lobe-finned fish lineage evolved into tetrapods and land animals while the ray-finned fish independently led to the teleost Interestingly, vertebrates appear to have experienced two round whole genome duplications (WGD) early in their evolution and teleost fishes experienced a third round or fish-specific WGD (Meyer and Van de Peer 2005) In addition, salmon family including rainbow trout has undergone an additional and relatively recent WGD, termed Salmonid specific 4th WGD or Ss4R (Fig 4) (Berthelot et al 2014) As a consequence of duplication events, rainbow trout are polyploidy

There are three main evolutionary advantages of polyploidy discussed: (1) heterosis which causes polyploidy to be more vigorous than their diploid progenitors; (2) gene redundancy which provides raw materials for mutation, diversity, innovation and the origin

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Figure 4 The evolutionary position of rainbow trout (Berthelot et al 2014)

The red stars show the position of the teleost-specific (Ts3R) and the Salmonid specific (Ss4R) WGDs The groups of species with available genomic sequence are shown in red bold type (Berthelot

et al 2014)

1.4 Problems in modern aquaculture production

Although aquaculture represents attractive alternative to the capture fishery, a number

of disadvantages are connected to the production of fish in aquaculture:

- Increased negative impact on environment,

- Increased stress to the animals, and

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Fish model for my thesis

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- Increased risk of epidemics by infectious microorganisms (The Scottish Association for Marine Science and Napier University 2002, Naylor et al 2000, Silva et al 2009).Due to the fact that fish is in modern aquacultures kept in densities more than 1000 times higher than under natural conditions, it is not surprising that this is leading to increased stress and disease susceptibility (Pulkkinen et al 2010) Subsequently, cultured trout is prone

to many disease causing organisms including bacteria, parasites, viruses and fungi which account for high economic losses (O’Neill 2006) These economical risks lead to treatments

to protect fish in aquaculture

1.4.1 Antibiotic treatment

Treatment of infected fish with antibiotics represents the first option in the fight against bacterial diseases The first fish disease treated with modern drugs such as

sulfonamides and nitrofurans was furunculosis, caused by Aeromonas salmonicida (Gutsell

and S 1948) During the 1980s, salmon farming in Norway experienced huge losses due to

bacterial diseases (mostly Vibrio spp.) and a total crash in the industry was only prevented by

the use of vast amount of antibiotics (Sommerset et al 2005)

Nowadays, antibiotics are not only used to combat bacterial disease after outbreaks, but also often used as a prophylaxis in fish feed and occasionally in bath and injections (Markestad and Grave 1997, Cabello 2006) However, the extensive use of antibiotic represents severe threat to the fish, environment and consumer A number of studies indicated that antimicrobial treatment changed the composition of bacteria in the aquatic environment surrounding aquaculture and increased number of antibiotic resistant bacteria (Miranda and Zemelman 2002b, Hektoen et al 1995, Miranda and Zemelman 2002a, Cabello 2006)

The beneficial effects of antibiotic treatment on fish survival in aquaculture is counteracted by concern that chemicals, antibiotics and pollutants can drastically affect the composition of intestinal microbiota and may lead to the elimination of individual species from the whole microbial community (Nayak and Sukanta 2010, Sugita et al 1988, Austin and Alzahrani 1988) Although the use of antibiotic may reduce the mortality during outbreaks, its negative impacts on fish and consumers health resulted in the EU moratorium

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on the banning of antibiotic growth promoters in animal feeds (European Commission 2008) which promoted search for new strategies in aquaculture

1.4.2 Increasing survival of fish caused by probiotic treatment

One current strategy aims to eliminate the chances of pathogenic bacteria to colonize and penetrate intestine by supporting proper composition of microbiota Fish intestine harbors more than 108 bacteria per gram and is dominated by the genera Acinetobacter,

Aeromonas, Flavobacterium, Lactococcus, Pseudomonas, Bacteroides, Clostridium and Fusobacterium (Austin 2002, Perez et al 2010, Nayak and Sukanta 2010) As shown by

gnotobiotic zebrafish, the role of intestine microbiota in the host development is evolutionary conserved The ‘probiotic microflora’ seems to positively influence on the organism welfare

by education of the immune system, to improve the integrity of intestinal mucosal barrier and

to play a key role in extracting and processing of nutrients (Rawls, Samuel and Gordon 2004) This positive effect is also based on the competition of microbiota with pathogens for specific receptors on mucosal surfaces and production of antimicrobial substances that restrict growth of pathogens (Bernet-Camard et al 1997, Coconnier et al 1992, Balcazar et

al 2007) Feeding with Carnobacterium augments the immune response upon challenge with Aeromonas salmonicida and Yersinia ruckeri, increasing phagocytic activity, respiratory

burst and lysozyme activity (Kim and Austin 2006) Trout fed with three freeze-dried

bacteria (Lactobacillus ramnosus, Enterococcus faecium and Bacillus subtilis) exhibits the

enhanced production of superoxide anion and activity of the alternative complement pathway (Panigrahi et al 2007) Since any negative change of the bacterial composition might have drastic effects on fish immune system, current research focuses on probiotic additives to fish feed to positively manipulate the microbial populations (Merrifield et al 2010) Importance

of microbiota and probiotic feeding was also evaluated by in vivo trials with fry or fingerlings, where the feeding with viable Carnobacteirum, Bacillus sp and Aeromonas

sorbia and even with formaldehyde inactivated Vibrio fluvialis, Aeromonas hydrophila and Carnobacterium spp reduced mortality after challenge with Aeromonas salmonicida

(Robertson et al 2000, Brunt, Newaj-Fyzul and Austin 2007, Irianto and Austin 2003) However, a routine use of probiotic treatment in aquaculture still has to overcome problems with the selection of appropriate probiotics, delivery method and assessment of dosage and duration of application (Merrifield et al 2010)

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1.4.3 Vaccination

Vaccination is still considered the most effective method in large-scale commercial fish farming to control pathogenic bacteria, as well as viruses Vaccination has been a key reason for the success of salmon cultivation In addition to salmon and trout, commercial vaccines are available for channel catfish, European seabass and seabream, Japanese amberjack and yellowtail, tilapia and Atlantic cod (Sommerset et al 2005)

Mostly empirically developed vaccines against bacterial diseases were based on inactivated bacterial pathogens The first formulation of protective vaccine was published more than 70 years ago (Duff 1942) Fish immersion vaccines based on formalin-inactivated bacterial cultures had proven to be effective against Vibriosis in the USA in the 1970s (Evelyn 1997, Sommerset et al 2005) Similar vaccines were developed in Norway against

Vibriosis diseases in salmon and efficacy of these vaccines resulted in a declined use of

antibiotics However, an immersion vaccine proved ineffective against furunculosis caused

by Aeromonas salmonicida in the early 1990s in Norway Therefore a number injection

vaccines were developed (Lillehaug, Lunder and Poppe 1992, Johnson and Amend 1984) The combination of intraperitoneal injection with a good antigen preparation and oil adjuvant resulted in dramatic decline of antibiotic treatment and in increase of produced salmon (Fig 5) The intraperitoneally delivered vaccines are still providing the best protection against a number of pathogens (Toranzo et al 2009) Recently, polyvalent vaccines protecting simultaneously against the majority of bacterial pathogens dominate the commercial market with fish vaccines

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Figure 5 Comparison of antibiotic use and salmon production in Norway in relation to appearance of bacterial diseases and introduction of anti-bacterial vaccines (modified from Sommerset study (Sommerset et al 2005))

Proper fish management with good hygiene and limited stress are key factors in the prophylaxis of infectious diseases and are also a necessity for the optimal effect of vaccines (Sommerset et al 2005) Today, vaccination is an integral part of most salmon farms and the use of antibiotics is very limited, at least in Northern Europe and North America

Only a few vaccines are available against viral pathogens Most of them that are polyvalent, oil-adjuvanted have to be injected and need relatively high doses to achieve protection Vaccines based on live viruses have been tested with good results in fish They provide the good protection, easy administration low costs, but safety aspects of live virus vaccines are mainly responsible for their still limited use as commercial vaccines (Sommerset

et al 2005) The first viral vaccine for fish was produced in 1982 against a carp Rhabdovirus, causing Spring Viraemia of Carp (SVC) and was based on two inactivated isolates of SVC virus emulsified in oil and administered by injection

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In salmonid farming, commercial vaccines are used for the most common viral diseases (Infectious Pancreatic Necrosis virus, Infectious Salmon Anemia Virus, Infectious Hematopoietic Necrosis Virus, Viral Haemorrhagic Septicemia Virus) but none of these anti-viral vaccines are as efficacious as the bacterial vaccines Therefore, in the past 30 years, several approaches were used to improve the efficacy of vaccines using conventional killed

or live virus, recombinant viral proteins or DNA encoding viral glycoproteins (Lepa, Siwicki and Terech-Majewska 2010, Castro et al 2014, Evensen and Leong 2013, Hølvold, Myhr and Dalmo 2014, Sommerset et al 2005) So far, one DNA vaccines have been commercialized for protection of fish against the Rhabdovirus caused disease Infectious Hematopoetic Necrosis in Atlantic salmon (Castro et al 2014) The vaccine plasmids encoded the viral glyko(G)protein and post intramuscular injection of purified plasmid DNA,

a nonspecific antiviral immune response is initially generated followed later by a specific immune response The high efficacy of the experimental DNA vaccines against fish Rhabdoviruses including the Viral Hemorrhagic Septicaemia Virus, warrants testing of a similar vaccination strategy against other infections in fish (Gersdorff Jorgensen et al 2012)

Commercial vaccines against parasites are not yet available and only chemical or medical treatments are performed parasitic infections, but research for development of

effective vaccines against Ichthyophthirius multifiliis is ongoing (Gersdorff Jorgensen et al

2012) In general, fish possess both humoral and cell mediated defense mechanisms against many parasites and there are many reports on immunity/increased resistance among fish surviving natural parasite infections (Alvarez-Pellitero 2008, Sommerset et al 2005) Therefore, there are no principal biologic limitations hindering vaccine development against

at least some parasitic diseases in fish Experimental parasite vaccines based on whole pathogens have been reported and live vaccines appear to be superior to killed vaccines However, due to the impossibility of cultivating the parasite for large-scale production a recombinant vaccine is required for vaccination under commercial aquaculture conditions

However, most of the vaccines against viral pathogens are not as effective as those once against bacterial diseases Furthermore, still most of the vaccines are delivered by injection which causes additional handling stress to the fish and is also cost intensive The alternative oral vaccination does not induce effectively a protective, long lasting humoral immunity under ‘field conditions’ for most viral or bacterial pathogens

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Hence, the key for successful vaccines requires the understanding of the immune system that is poorly characterized in fish

1.5 Immune system of teleost

The immune system of all vertebrates is primarily responsible to maintain the organism's homeostasis sharing an essential immune structure characterized by: (i) highly conserved innate functions, (ii) consistent development of a combined immune system, and (iii) multilateral communication between components of the innate and adaptive immunity

The immune defense against foreign invaders is based on layered defense mechanisms First, the physical barriers prevent pathogens from entering the organism (Kum and Sekkin 2011, Tort, Balasch and Mackenzie 2003) If a pathogen can overcome these barriers, the innate immune system provides an immediate response based on the recognition

of pathogen associated molecular pattern (Litman, Cannon and Dishaw 2005) If pathogens successfully evade the innate response the second, anticipatory adaptive immune system provides protection This system recognizes specific epitopes by clonally proliferated immune cells that recognize the antigens by specific receptor complexes Adaptive immune functions are activated by the innate response that provides the necessary time for producing enough effectors (Mayer 2015) Adaptive immunity arose early in vertebrate evolution, between the divergence of cyclostomes (lampreys) and cartilaginous fish (sharks) around 450 million years ago in an evolutionary time span estimated to be less than 20 million years (Marchalonis and Schluter 1998, Flajnik and Kasahara 2010) The bony fish are the first vertebrate family which immune system comprises of fully developed innate and adaptive immune functions Specifically, the innate immune mechanisms are here developed to its broadest variety

1.5.1 Immune organs in fish

Fish represents to the most successful and diverse group of vertebrates One reason might be that the immune system of fish is not only conditioned by the particular environment, but also by their poikilothermic physiology

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The immune organs found in fish are homologous to those of the mammalian but lymph nodes are missing and the bone marrow is part of a combined organ with both hematopoietic and renal function the kidney (Kum and Sekkin 2011, Tort et al 2003, Press and Evensen 1999) The entire kidney contains immune cells In the anterior part (head kidney) has mainly hematopoietic functions (Meseguer, lópez-Ruiz and García-Ayala 1995, Zapata et al 2006, Deivasigamani) andcontains developing B- cells and antibody-secreting cells (ASC) (Bromage et al 2004, Zwollo et al 2005, Zwollo et al 2008, Zwollo, Mott and Barr 2010) Moreover, it seems to be immune organ responsible for phagocytosis from circulation (Danneving et al 1994), antigen processing (Brattgjerd and Evensen 1996, Kaattari and Irwin 1985) and B cell immune memory through melano-macrophage centres Furthermore, it is the major site of antibody production (Whyte 2007) In trout, the head kidney or pronephros is formed by two Y arms, which spreads underneath the gills In fish, the head kidney is a well innervated organ and is served as an endocrine organ with key regulatory functions and the central organ for immune–endocrine interactions and neuroimmunoendocrine connections The posterior kidney possesses both renal and immune tissues (Zapata and Cooper 1990, Zwollo et al 2005, Zwollo et al 2008)

The thymus also is used as a primary lymphoid for T cell generation, education and maturation It is a paired organ situated near the opercular cavity in teleost (Bowden, Cook and Rombout 2005) Interestingly, the composition of leukocytes and the involution depend more on seasonal variations than on age T lymphocytes are the major cell type found in the thymus The organization of thymus is less complex than in mammals

The spleen in fish is the major secondary immune organ with high amounts IgM+mature B cells (Zwollo et al 2005, Zwollo et al 2008, Bromage et al 2004) It works as a

“filter” for trapping and clearance of blood-borne antigens and immune complexes in splenic ellipsoids (Uribe et al 2011, Tort et al 2003) Here, the antigen is presented and then initiate the adaptive immune response (Whyte 2007) Blood filtration and erythrocytic destruction are performed by the melano-macrophage centres (by the accumulation of macrophages associated to ellipsoid capillaries) These centres may retain antigens as immune complexes for long periods Much like the mammalian immune system, teleost (fish) erythrocytes, neutrophils and granulocytes are believed to be present in the spleen

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The gut-associated lymphoid tissue (GALT) of teleost is less developed than in mammals and contains lymphocytes, different types of granulocytes and eosinophil granular considered T cells Lymphoid cells present in the lamina propria are mainly B lymphocytes (Zapata et al 2006) In bony fish, prethymic T-like cells occur in the gut similar to extra-thymic origin of some T cells in mammals (Rombout et al 2005, Rocha 2007)

In teleost fish, all immune cell types as in higher vertebrates are already present like neutrophils, monocytes, thrombocytes, plasma cells, B cells, T cells, natural killer cells and eosinophils (Whyte 2007, Uribe et al 2011) Certain leukocytes also act as Ag-presenting or dendritic-like cells (Alvarez-Pellitero 2008, Whyte 2007, Uribe et al 2011) However, fish leukocytes are still poorly characterized due to the lack of specific tools

1.5.2 Innate immune system in fish

The innate response is the first line of defense protecting an organism from foreign invaders in non-specific way Most of the innate immune components are as old as multicellular animals themselves Cells of the innate system recognize and respond to pathogens using pattern recognition receptors (PRR) The memory of innate immune recognition is based on an evolutionary experience of vertebrate ancestors encountering similar pathogens (Boehm 2012) The innate immune function also regulates the acquired immune response and homeostasis The innate immune system includes physical and chemical barrier, humoral and cellular components

In fish, the first physical and chemical barrier is composed of live cells and produced mucus containing a variety of antimicrobial substances which blocks attachment and penetration of microbes (Kum and Sekkin 2011, Tort et al 2003, Deivasigamani) If a microbe accesses to the tissues of the fish, it is met with a ray of humoral and cellular defenses Various studies in teleost show a huge variety of humoral molecules (antimicrobial peptides, various lytic enzymes, agglutinins and precipitins cytokines, components of complement pathways), display an effective defense against microbial pathogens (Alejo and Tafalla 2011, Secombes et al 2001, Reyes-Cerpa et al 2013, Robertsen 2006, Zou and Secombes 2011, Holland and Lambris 2002, Boshra, Li and Sunyer 2006) These humoral molecules play roles in growth, differentiation and activation of immune cells (Alejo and Tafalla 2011, Secombes et al 2001, Reyes-Cerpa et al 2013) They also promote the

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responses against the pathogens (Robertsen 2006, Zou and Secombes 2011); or help

antibodies and phagocytic cells to clear pathogens from an organ through complement components (Holland and Lambris 2002, Boshra et al 2006)

Fish innate immune cells consist of monocytes, macrophages, non-specific cytotoxic cells, thrombocytes and granulocytes, which have main features as in higher vertebrates (Kum and Sekkin 2011, Magnadottir 2006) Monocytes, macrophages and granulocytes (or neutrophils) play roles in phagocytosis, phagocyte activation, cytokine production, intracellular killing, antigen presenting and T- lymphocytes stimulation (Kum and Sekkin 2011) while non-specific cytotoxic cells are involved in recognition and lysis of target cells (Evans and Jaso-Friedmann 1992)

Briefly, innate system of teleost possesses the main features as higher vertebrate but components of innate system in teleost are more complex and more diverse than in mammals (Grinde 1989, Saurabh and Sahoo 2008, Nikoskelainen, Lehtinen and Lilius 2002, Sunyer and Tort 1995, Zarkadis et al 2001, Sunyer et al 1996, Boshra et al 2005)

1.5.3 Adaptive immune system

If a pathogen escapes the innate defense mechanism, the adaptive immune system is activated (Kum and Sekkin 2011) This dichotomy of immune defense is thought to have arisen in the first jawed vertebrates about 450 million years ago It is composed of highly specialized lymphocytes recognizing pathogens by the generation of a diverse repertoire of antigen receptors of the immunoglobulin family Adaptive immune has, therefore, a clonal specificity to individual antigenic epitopes After first contact to an antigenic epitope, a subsequent activation and clonal expansion of cells carrying the appropriate antigen-specific receptors is induced The adaptive response is regulated by a network of humoral factors and cellular interactions to eliminate or prevent recurrent infections by generating memory cells Adaptive immunity is biologically costly, time-consuming and exists only in vertebrates excluding the most primitive jawless fish The appearance of this adaptive immunity about

450 million years ago and its relatively short expansion to the complex system seem already

in teleost was leading to the so-called “big bang”-theory (Litman, Rast and Fugmann 2010)

Th1, Th2 and Th17 cytokines Yes Yes

Spleen, thymus and bone marrow Spleen and thymus but no

true bone marrow Yes Mucosa-associated lymphoid tissue Yes Yes

Germinal centers and lymph nodes Yes Yes

Teleost displays all genetic elements essential for a combinatorial immune response (CIS): Ag-recognizing lymphocytes, immunoglobulins (Abs and Ig-family TCR), MHC products, and recombination-activating (RAG) 1 and 2 genes (Litman et al 2010) It includes

B and T lymphocytes, which recognize foreign non-self peptides presented through MHC class I (intracellular) or through MHC class II (extracellular) by their antigen receptor complexes (Fischer et al 2013, Zhu et al 2014b, Rombout, Yang and Kiron 2014) During recent years, genes encoding MHC class I and II, TCR and its co-receptors CD8 and CD4 have been cloned in several fish species Both B and T lymphocytes have diverse re-arranging and adapting antigen receptors and can survive as long-lived memory cells (Sunyer

2013, Litman et al 2010) The overall shape of the specific immune elements and the recombination mechanisms (causing of diversity in TCR molecules and Igs) are similar in fish and mammals (Castro et al 2011, Sunyer 2013) Furthermore, the repertoire of regulation cytokines is as variable as in mammals (Papewalis et al 2008, Zou and Secombes

2011, Alejo and Tafalla 2011, Kono and Korenaga 2013, Reyes-Cerpa et al 2013, Wang et

al 2011c)

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1.5.4 B cells and Immunoglobulins

B lymphocytes are characterized by the expression of surface immunoglobulins creating with co-receptor molecules the B cell antigen receptor Teleost B lymphocytes primarily express immunoglobulin µ heavy chain (IgM) that is quite similar to the mammalian mechanism (Sunyer 2013) In Atlantic salmon, two different isoforms are characterized by their recognition using anti-IgM mabs whereas in trout just one isoform is found in trout (Hedfors et al 2012, Kamil et al 2013) Two further heavy chain isotypes δ (IgD) and τ/ζ (IgT/IgZ) have been identified in fish IgD is a cell membrane Ig presumably acting as an antigen receptor (Hansen, Landis and Phillips 2005, Ramirez-Gomez et al 2012, Tian et al 2009, Zhu et al 2014a, Hordvik et al 1999, Zimmerman et al 2011) IgT/IgZ was found in zebrafish, carp and trout appeared to be a unique heavy chain isotype restricted to bony fish (Hansen et al 2005)

Fish B cells display Ig H-chain rearrangement and allelic exclusion (Fillatreau et al 2013) Germline VH and VL elements, as well as the joining (J) segments are highly conserved among distinct vertebrate species (Flajnik and Kasahara 2010) The number of VH families varies among different teleost species (Golub and Charlemagne 1998) Fish IgM molecules are usually tetrameric and not limited to the serum Antibodies are also found in the fish epithelial mucus, and these IgM molecules may be of local origin rather than derived from the serum, which suggested that fish immune system could be viewed as consisting of systemic and mucosal part (Rombout et al 2014, Lorenzen 1993, Castro et al 2013a) Besides, some monomeric and dimeric forms of IgM are also reported (Choudhury and Prasad 2011)

The major transcription factors Ikaros, E2A, early B cell factor-1 (EBF1), paired

box-5 (Paxbox-5), B lymphocyte-induced protein-1 (Blimp1), and X-box binding protein-1 (XbpI) have been characterized at least to some extent in teleost, too (Zwollo 2011)

1.5.5 T cells and TCR receptors in teleost

T cells play a central role in cell mediated immunity (Murphy 2012) Through specific TCR receptors, nạve T cells recognize a pathogen presented nạve via MHC class I

Ig domains comparing to one mammalian CD4 molecule with 4 Ig domains (Moore et al 2009) TCR gene expression is restricted to IgM- and IgT-negative lymphocytes in teleost fish (Zhang et al 2010, Blohm, Siegl and Köllner 2003) and confirmed in T cell lines (Wilson et al 1998) All four elasmobranch TCR genes are co-expressed by thymus and spleen derived lymphocytes in a manner that follows the mammalian paradigms

The presence and functions of T cell subsets also are supported by the presence and responses against bacteria and virus of interleukin and cytokines such as IL-12, IL-4, IL-17, IL-10, IL-22 and IFNγ (Monte et al 2013, Costa et al 2012, Uribe et al 2011, Fischer et al 2013) Despite the characterization of numerous genes associated with T cell functions and signaling identified in fish, the functional correlation between presumed T cell associated genes and T cell activity in adaptive immunity in fish is poorly understood So far, most studies on T cell functions are based on characteristics of transcriptional sequences A few studies about T cell functions involving interactions and responses of T cells in teleost immune system have been described

Cytotoxic T lymphocytes (CTLs) bind to MHC class I bound peptide on the infected host cell using their T cell receptor (TCR) and its co-receptor CD8 resulting in specific killing In fish, cytotoxic functions of CD8+T cells have been described (Nakanishi et al

2011, Somamoto, Nakanishi and Nakao 2013, Somamoto, Nakanishi and Okamoto 2002, Nakanishi et al 2002) Specific cell mediated cytotoxicity as a function of sIgM-lymphocytes

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expressing TCR and CD8 which kill allogeneic cells and virus- infected cells are described for crusian carp and trout (Fischer et al 2006, Nakanishi et al 1999, Somamoto, Nakanishi

success of CD8α antibody production in some teleost, it is demonstrated that CD8α+ T cells

Somamoto, Koppang and Fischer 2014b, Lakshmikanth, Karre and Sreerama 2011) In rainbow trout and carp, CD8α+ T cells express CTL effectors molecules such as perforin, granualysin, granzymes and IFNγ which can kill microbes by perforin pathway (Takizawa et

al 2011a, Somamoto et al 2002, Somamoto et al 2000, Somamoto et al 2013, Nakanishi et

al 2002, Nakanishi et al 1999, Raulet 2006) Carp CD8+ T cells are anti-viral specific cytotoxic effector cells when the purified CD8α+ cells killed CHNV-infected cells (Somamoto et al 2013, Somamoto et al 2000, Somamoto et al 2002, Castro et al 2011) CD8+ T cells also are the effectors in graft rejection shown by the infiltration of CD8+ T cells into graft site (Nakanishi et al 2011)

Th cells recognize peptides through their T cell receptor (TCR) and its co-receptor CD4 after extracellular antigens uptake, processing and presentation via MHC class II by professional antigen presenting cells (macrophages, dendritic cells and B cells)

CD4+ T cells that recognize pathogen peptides presented on MHC class II, have functions in immune regulation, immune pathogenesis and host defense CD4+ T cells assist

in the maturation of B cells into plasma and memory B cells and activation of CD8+ T cells and macrophage Activated CD4+ T cells also secrete many different cytokines that regulate and propagate the immune responses including antibody production from B cells after recognizing pathogen on APCs

Recently, the function of Th cells has been investigated using anti-CD4antisera in

carp and Japanese pufferfish Takifugu rubripes (Fugu) (Toda et al 2011, Wen et al 2011,

Kono and Korenaga 2013) Isolated CD4+ T cells were characterized by the expression of cytokines corresponding to Th1, Th2, Th17 during stimulation (Kono and Korenaga 2013) Using an anti-CD4 mab in carp, the functions of carp CD4+ T cells were examined In vitro

proliferation of CD4+ carp T cells after sensitization with co-administered ovalbumin or by

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allogeneic combination of mixed leukocyte culture suggests that CD4+ carp T cells proliferate specifically in presence of antigen on antigen presenting cells (Toda et al 2011) Furthermore, in another study, it was proved that CD4+ carp T cells induced a secondary

antibody response in vivo (Somamoto et al 2014a) The characterization of T-bet, Gata3,

RORγT, Foxp3 and BCL-6 transcription factors indicated that already Th1, Th2, Th17, Treg (regulatory T cells) and Tfh (follicular T cells) subsets might already exist in bony fish (Wang et al 2010b, Monte et al 2013, Wang et al 2010a, Monte et al 2012, Ohtani et al

2008, Zou and Secombes 2011) For instance, the up-regulation of T-bet expression in

Tetracapsuloides bryosalmonae infection model indicates that Th1 cells are involved in

protection against this parasite (Wang et al 2010a) Furthermore, the higher expression of

RORγT in vaccinated versus unvaccinated fish during Yersina ruckeri challenge

demonstrates not only the presence of teleost Th17 but also roles of RORγT in protection against extracellular bacteria as in higher vertebrates (Monte et al 2012) Based on the analysis of interleukin - 4/13A and Gata3 expression in thymus and gills, Th2 helper cell responses were suggested (Takizawa et al 2011b)

Although the functions of T cells in teleost have been suggested, the development, differentiation and functions of T cells as well as T cell subsets are still less investigated To study their functions, T cells have to be identified and separated by specific antibodies recognizing differentiation and activation markers on T cells that still are not available in rainbow trout

1.5.6 NK cells in teleost

Originally, NK cells are defined as cytotoxic lymphocytes, which are active in innate immune responses against viruses and tumors, by their cytotoxicity Furthermore, NK cells also participate in the complex network of cell-to-cell interaction that leads to the development of adaptive immune responses (Vivier et al 2008, Marcenaro et al 2011) NK cells are able to destroy rapidly lacking MHC class I cells without prior sensitization Interestingly, recent findings show that NK cells also can develop long-lived and highly specific memory to a variety of antigens, which are a crucial feature of adaptive responses (Biosciences 2010, Sommerset et al 2005, Sun and Lanier 2009) Therefore, NK cells are

an important marker of NK cells

Functionally, NK cells possess dual activating and inhibitory receptors which are involved in the regulation of different NK cells functions (Shen et al 2004) Activating receptors can trigger the NK cell to kill their target bearing low levels of MHC class I by the release of cytokines such as IFNγ and cytotoxic granules such as perforin, granzyme and granualysin To activate NK cells, the tyrosine residues in the ITAM bearing DAP12

NK cells and finally lead to the release of cytotoxic granules for killing tumor cells or infected cells In contrast to activation receptors, inhibitory receptors act to prevent NK cells

2004) Upon engagement of ligands to inhibitory receptors containing ITIM motif, the tyrosine in ITIM is phosphorylated Subsequently, it can bind intracellular phosphatases, which remove phosphates from tyrosine residues on other intracellular signaling molecules (Lightner et al 2008) These molecules antagonize the kinase of activation signals (Lightner

et al 2008) Therefore, presence or absence of particular signaling motifs in their cytoplasmic domain defines whether activating or inhibitory functions of NK receptors Overall, the balance of activating and inhibitory receptors on NK cells clarifies whether NK cell is activated or inhibited by a target cell (Lightner et al 2008, Du Pasquier et al 2004) In structure, both NK receptor groups fall into two large families: killer cell immunoglobulin-like receptors such as KIR-2D, KIR-3D, which contain two, or three Ig domains and killer C-type lectin-like receptors such as Ly49 receptor (Yoder et al 2010, Stet et al 2005, Kock and Fischer 2008)

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In contrast to much work on NK cells in mammals, the knowledge of fish NK cells is

exhibited significant cytotoxic activity against crucian carp hematopoietic necrosis

cytotoxic roles by perforin mediated pathway (Somamoto et al 2013)

At present, there are two fish subsets that behave like NK cells: nonspecific cytotoxic cells (NCCs) and NK like cell lines (Nakanishi et al 2002, Fischer et al 2013, Evans and Jaso-Friedmann 1992) For many years, NCCs identified in catfish and rainbow trout are used as the ‘‘evolutionary precursor to mammalian NK cells’’ or the ‘‘lower vertebrate homologue of NK cells’’ by their ability to recognize and kill transformed human cell lines

or mammalian tumor target cells spontaneously (Evans and Jaso-Friedmann 1992, Bruce et

al 2002) Moreover, NCCs also express multiple granzymes and other components of cytotoxic granules as well as TNFα, suggesting the ability of NCCs to kill virus-infected cells by granzyme mediated apoptosis pathway (Boyton and Openshaw 2002) However, functional data of NCCs is still missing and shows significant differences to mammalian NK cells Firstly, functional studies of enriched NCC typically utilized a mixture of cell types

NK cells Thirdly, NCCs are morphologically small and agranular; more closely resembling

have been reported based on size and granularity Finally, NCCs are distinct to NK-like cell lines developed from catfish peripheral blood leukocytes (Yoder 2004, Evans and Jaso-Friedmann 1992)

NK like cell lines in catfish are identified as NK cells because they do not express Igµ, TCR α, β and γ , meaning that they are neither B nor T cells Additionally, they also lack the markers that define neutrophils, macrophages or NCCs (Shen et al 2004) Functionally, they can kill the stimulating allogeneic cells, unrelated allogeneic targets without prior sensitization and also enable to kill virus-infected cells (Yoder 2004, Nakanishi

et al 2002, Shen et al 2003) However, to answer whether NCCs and NK like cells belong to fish NK cells as well as to study more detail about cytotoxicity of teleost, it is necessary to separate subsets presenting NK cells by specific antibodies against fish NK receptors

non-rearranging receptors with a similar molecular structure and signaling to that of mammalian KIRs and involve in recognizing self-determinants in lower vertebrates (Cannon et al 2008) According to the structure, NITRs and NILTs enable to play both activating and inhibitory response because they contain either or both ITIM and ITAM motifs bearing DAP molecules which have functions in activating NK response in mammals (Cannon et al 2008, Stet et al

2005, Wei et al 2007, Yoder et al 2007) Recent findings have been shown that DAP12 and

mucosal lymphocytes (Takizawa et al 2011a) This result suggests the presence of NKT cells

in non-mucosal lymphocytes and NK cells in mucosal lymphocytes

Taken together, genes used as NK cell markers for distinguishing to other lymphocytes (B and T cells) such as CD56 and CD16 have not been reported in fish so far

1.5.7 CD56

Because of important roles of NK cells in the immune system, investigation of NK cells is necessary NK receptor information and antibodies against NK receptors are used to analyze NK cells development, subsets and functions (Montaldo et al 2013) As described above, although NK cells have numerous receptors but none of them are specific unique receptors for NK cells However, in mammals CD56 is expressed in more than 90% NK

can also be induced on cytotoxic T cells via TCR or cytokine stimulation, and CD56+ T cells were found to be more auto-aggressive as effectors than their CD56 counterparts (Ahn et al 2005)

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Figure 6 Receptors of CD56 in NK cells (Cooper et al 2001b)

A) CD56 bright NK cell; B) CD56 dim NK cell

In accordance to expression of CD56, NK cells are divided in CD56bright NK mainly responsible for NK cell cytokine production in response to monokines and CD56dim playing a key role in exocytosis mediated cytotoxicity (Fig 6) (Cooper et al 2001c, Chan et al 2007, Poli et al 2009) Regarding to phenotype, CD56dim NK cells in blood express strongly CD16, low CD56, low NKp46, present KIR and ILT2 but very low or not CD94/NKG2A and not CD117 in contrast to CD56bright as described in Table 2