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

LUẬN VĂN TIẾN SỸ

ĐẶNG THỊ HƯƠNG
VIỆT NAM



Functional and molecular characterization of
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 content

Content
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


List of content
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.
2.5.

Functional characterization of rainbow trout T cells ................................................ 49

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


List of content
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.
4.4.

NK cell cytotoxicity in xenogeneic model ............................................................ 69

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 M30
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 content
List of publication and oral presentation ............................................................................. 151
Resume ..................................................................................................................................... 153
Personal Data........................................................................................................................ 153
Education .............................................................................................................................. 153
Work experience .................................................................................................................. 153

Muster der Erklärung ................................................................................................... 154


List of figures

List of figures
Figure
 1
  Global
 aquaculture
 production
 of
 rainbow
 trout
 from

 1973-­‐2012
 (FAO
 2014b)
 ______________
 2
 
Figure
 2
  World
 leading
 countries
 in
 the
 aquaculture
 production
 of
 rainbow
 trout
 in
 2012
 (FAO
 2014b)
 __
 2
 
Figure
 3
  Production
 of
 main

 fishes
 in
 Germany
 (FAO
 2014b)
 ____________________________________
 3
 
Figure
 4
  The
 evolutionary
 position
 of
 rainbow
 trout
 (Berthelot
 et
 al.
 2014)
  ________________________
 4
 
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))
 _________________________________________________________
 8
 
Figure

 6
  Receptors
 of
 CD56
 in
 NK
 cells
 (Cooper
 et
 al.
 2001b)
 ___________________________________
 22
 
Figure
 7a
  Flow
 cytometry
 pattern
 of
 mab
 D11
 with
 leukocytes
 from
 different
 lymphoid
 tissues
 ________
 34

 
Figure
 7b
  Flow
 cytometry
 pattern
 of
 mab
 D30
 with
 leukocytes
 from
 different
 lymphoid
 tissues
 ________
 35
 
Figure
 8
  Flow
 cytometry
 pattern
 of
 mab
 89
 with
 leukocytes
 from
 different

 lymphoid
 tissues
 _________
 36
 
Figure
 9
  Immunomagnetic
 sorting
 of
 leukocyte
 subpopulations
 from
 spleen
 using
 different
 mabs
 specific
 
for
 lineage
 marker
 _____________________________________________________________
 37
 
Figure
 10
  Relative
 mRNA
 expression

 of
 lineage
 marker
 molecules
 in
 immunomagentically
 enriched
 
leukocyte
 subpopulations
 _______________________________________________________
 38
 
Figure
 11
 
 Single
 and
 double
 labelling
 pattern
 of
 mab
 D11
 and
 D30
 on
 trout
 leukocytes
 from

 thymus,
 blood
 
and
 spleen
 ___________________________________________________________________
 39
 
Figure
 12
 
 Two-­‐color
 flow
 cytometry
 of
 leukocytes
 from
 thymus,
 intestine
 and
 gills
 using
 D11
 mab
 and
 89
 
mab
 ________________________________________________________________________
 40

 
Figure
 13
  Two
 color
 flow
 cytometry
 staining
 of
 spleen
 leukocytes:
 mab
 D11
 +
 mab
 anti-­‐IgM
 of
 B
 cells
 or
 
mab
 thrombocyte
 or
 intestine
 leukocytes:
 mab
 89
 +
 mab

 anti-­‐Ig
 light
 chain
 of
 B
 cells
  _______
 41
 
Figure
 14
  Immunoprecipitation
 of
 membrane
 proteins
 from
 thymus
 leukocytes
 using
 D11
 mab
 and
 D30
 
mab
 ________________________________________________________________________
 42
 
Figure
 15

  Double
 labelling
 of
 leukocytes
 with
 mab
 D11/D30
 or
 mab
 89
 and
 anti-­‐trout
 CD8α
 mab
 ______
 43
 
Figure
 16
  Double
 labelling
 profile
 of
 mab
 D11
 +
 anti-­‐trout
 CD8α
 or
 mab

 89
 +
 anti-­‐trout
 CD8α
 in
 leukocytes
 
from
 primary
 (head
 kidney,
 thymus)
 peripheral
 (blood,
 spleen)
 and
 mucosal
 (gill,
 intestine)
 
lymphatic
 organs
 ______________________________________________________________
 44
 
Figure
 17
  Fluorescence
 based
 cell

 sorting
 of
 CD8α
 and
 CD8
 T
 cells
 using
 mab
 D11
 and
 anti-­‐trout
 CD8α
  45
 
+

-­‐

Figure
 18
 
 mRNA
 patterns
 of
 sorted
 CD8α
 or
 CD8α
 T

 cells
 from
 thymus,
 spleen
 and
 intestine
 _________
 45
 
+

-­‐

Figure
 19
  Expression
 patterns
 of
 transcription
 factors
 in
 CD8α
 and
 CD8α
 T
 cells
 from
 thymus
 isolated
 

+

-­‐

from
 5
 trout
 after
 fluorescence
 based
 sorting
  _______________________________________
 46
 
Figure
 20
 
 Expression
 patters
 of
 transcription
 factors
 in
 CD8α
 and
 CD8α
 T
 cells
 from
 thymus

 and
 spleen
 
+

-­‐

isolated
 from
 two
 trout
 _________________________________________________________
 47
 
Figure
 21
  Example
 of
 sorting
 trout
 thymus
 leukocytes
 into
 four
 subpopulations
 using
 mab
 D11
 and
 anti-­‐

trout
 CD8α
 ___________________________________________________________________
 47
 
Figure
 22
 
 mRNA
 pattern
 of
 sorted
 leukocyte
 subpopulation
 from
 thymus
 measured
 by
 real-­‐time
 PCR
 ___
 48
 
Figure
 23
  mRNA
 patterns
 of
 four
 leukocyte

 subpopulations
 isolated
 from
 intestine
  _________________
 49
 

I


List of figures
+
 

Figure
 24
 
 Relative
 amount
 of
 total
 versus
 CD8α T
 cells
 after
 xenogeneic
 or
 allogeneic
 stimulation

 in
 
spleen
 or
 gill
 24h
 post
 stimulation.
  _______________________________________________
 49
 
Figure
 25
  Kinetics
 of
 D11
 T
 cells
 and
 IgM
 B
 cells
 in
 spleen
 after
 allogeneic
 stimulation
  _____________
 50
 

+

+

Figure
 26
  Kinetics
 of
 D11
 T
 cells
 in
 spleen
 after
 repeated
 allogeneic
 stimulation
 ___________________
 50
 
+

Figure
 27
  Analysis
 of
 the
 cell
 composition
 in

 the
 peripheral
 blood
 leukocytes
 of
 rainbow
 trout
 using
 6 -­‐color
 
flow
 cytometry
 _______________________________________________________________
 59
 
Figure
 28
  Genomic
 trout
 CD56
 organization
 and
 three
 main
 isoforms
 in
 transcripts
 generated
 after
 

transcription
 and
 splicing
 of
 trout
 CD56
  ___________________________________________
 72
 
Figure
 29
  Deduced
 amino
 acid
 sequence
 of
 trout
 CD56
 aligned
 to
 zebrafish
 CD56
 __________________
 73
 
Figure
 30
  Evolutionary
 relationship
 between

 members
 of
 NCAM
 gene
 family
 ______________________
 77
 
Figure
 31
  Nucleotide
 sequences
 of
 trout
 CD56-­‐VASE
 gene
 a
 and
 b
 exon
 and
 intron
 boundaries
 and
 
adjacent
 splice
 junctions
 in
 trout

 genomic
 DNA
  _____________________________________
 78
 
Figure
 32
 
  Comparison
 of
 deduced
 amino
 acid
 alignment
 of
 VASE
 domain
 in
 trout
 CD56
 a
 and
 b
 genes
 with
 
those
 of
 other
 CD56

 genes
 from
 other
 vertebrates
 ___________________________________
 79
 
Figure
 33
  Expression
 patterns
 of
 transcripts
 of
 CD56
 with
 the
 VASE
 elements
 at
 the
 exon
 7/8
 junction
 in
 
different
 trout
 tissues
 __________________________________________________________

 79
 
Figure
 34
  Alternatively
 spliced
 MSD
 domain
 in
 six
 main
 trout
 CD56
 isoforms
  ______________________
 80
 
Figure
 35
  Nucleotide
 sequences
 of
 trout
 CD56-­‐MSD
 exon
 and
 intron
 boundaries
 and
 adjacent

 splice
 
junctions
 in
 trout
 genomic
 DNA
 (continue
 next
 page)
 _________________________________
 82
 
Figure
 36
  Nucleotide
 and
 deduced
 AA
 of
 trout
 MSD
 sequences
 _________________________________
 86
 
Figure
 37
  Nucleotide
 and

 deduced
 AA
 sequence
 of
 isoforms
 containing
 M65,
 M15’M58,
 intron281a
 _______
 87
 
Figure
 38
  In
 vivo
 expression
 of
 MSD
 domain
 in
 different
 trout
 tissues
 by
 RT-­‐PCR
  ___________________
 88
 
Figure

 39
  In
 vivo
 expression
 of
 six
 different
 trout
 CD56
 isoform
 transcripts
 in
 different
 tissues
 by
 RT-­‐PCR
  89
 
Figure
 40
  In
 vivo
 expression
 of
 six
 different
 trout
 CD56
 isoforms
 in

 leukocytes
 at
 mRNA
 level
 by
 RT-­‐PCR
 _
 90
 
Figure
 41
  Expression
 of
 mRNA
 transcripts
 in
 leukocyte
 subpopulation
 ____________________________
 90
 
Figure
 42
  mRNA
 expression
 analysis
 of
 trout
 CD56
 and

 effector
 molecules
 after
 xenogeneic
 injection
  __
 92
 
Figure
 43
 
 Natural
 cytotoxicity
 of
 trout
 spleen
 leukocytes
 against
 P815
 cells
 at
 different
 effector
 to
 target
 
cell
 ratio
  ____________________________________________________________________
 93

 
Figure
 44
  Natural
 cytotoxicity
 of
 trout
 spleen
 leukocyte
 subpopulations
 against
 P815
 cells
 at
 different
 
effector
 to
 target
 cell
 ratio
 ______________________________________________________
 93
 
Figure
 45
  Immunofluorescense
 staining
 of
 transfected

 3T3
 cells
 using
 anti-­‐trout
 CD56
 mab
  __________
 94
 
Figure
 46
  Immunofluorescense
 staining
 of
 trout
 brain
 cells
 using
 anti-­‐trout
 CD56
 mab
 ______________
 94
 
Figure
 47
  Immunofluorescense
 staining
 of
 trout

 leukocytes
 using
 anti-­‐trout
 CD56
 mab
 ______________
 95
 
Figure
 48
  Flow
 cytometry
 of
 leukocytes
 from
 different
 lymphatic
 tissues
 staining
 with
 anti-­‐trout
 CD56
 mab

  ___________________________________________________________________________
 96
 
Figure
 49
  Two-­‐color

 flow
 cytometry
 staining
 trout
 leukocytes
 using
 anti-­‐trout
 CD56
 and
 other
 anti-­‐trout
 
mabs
 _______________________________________________________________________
 96
 
Figure
 50
  Evolution
 of
 trout
 CD56
 genes
 __________________________________________________
 110
 
Figure
 51
  Predicted
 proteomic

 diversity
 of
 trout
 CD56
  _______________________________________
 115
 

II


List of tables

List of tables
Table
 1
 

Fundamental
 features
 of
 adaptive
 immune
 systems
 of
 teleost
 fish
 and
 mammals
 (Sunyer

 2013)
  14
 

Table
 2
 
  Antigens
 expressed
 differentially
 by
 resting
 human
 NK
 cell
 subsets
 (Cooper
 et
 al.
 2001a)
  _____
 23
 
Table
 3
 
  Percentage
 of
 labelled
 leukocytes

 from
 different
 organs
 taken
 from
 10
 healthy
 unstimulated
 
trout.
 ________________________________________________________________________
 36
 
Table
 4
 

The
 specificity
 of
 monoclonal
 antibodies
 together
 with
 selected
 fluorochrome
 used
 in
 current
 

study
 is
 listed
 in
 table
 below
 ______________________________________________________
 58
 

Table
 5
 
  The
 identity
 of
 trout
 CD56
 with
 CD56
 from
 other
 animals
 at
 amino
 acid
 level
 _______________
 71
 

Table
 6
 
  Summary
 of
 MSD
 sequences
 ______________________________________________________
 84
 
Table
 7
 
  Antibodies
 against
 teleost
 surface
 molecules
 ________________________________________
 103
 
Table
 8
 
  CD
 markers
 pattern
 of
 vertebrate
 leukocyte

 subsets
 __________________________________
 104
 

III


Abbreviations

Abbreviations
All abbreviations used in this thesis are listed in alphabetical order.

IV

Ab

Antibody

AIS

Adaptive Immune System

APC

Antigen Presenting Cell

AS

Alternative Splicing

CCR7

CC-chemokine receptor 7

CD

Cluster of Differentiation

CD56 (NCAM1)

Neural adhesion molecule 1

CIS

Combinatorial Immune Response

CTL

Cytotoxic T Lymphocyte

CXCR

CX-chemokine receptor

Cys

Cysteine

DAMPs

Danger-Associated Molecular Patterns

DAP12

TYRO protein tyrosine kinase-binding protein

Facs

Fluorescence Activated Cell Sorting

FcγR

Fc gamma receptor

FNIII

Fibronectin type III

GPI

Glycosyl Phosphatidyl Inositol

IC

Intracellular

ICOS

Inducible Costimulator

IFN

Interferon

IFNγ

Interferon gamma

Ig

Immunoglobulin

IL

Interleukin

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

KIR

Killer Ig-Like Receptor

LFA-1

Leukocyte Function-associated Antigen 1

mab

Monoclonal antibody

MCSFR

Macrophage Colony Stimulating Factor Receptor

MHC

Major Histocompatibility Complex


Abbreviations
MNC

Mononuclear cells

MSD

Muscle Specific Domain

NCCs

Nonspecific Cytotoxic Cells

NILT

Novel Immunoglobulin-Like Transcript

NITRs

Novel Immune Type Receptors

NK

Natural Killer

NKT

Natural Killer T cell

ORF

Open Reading Frame

PAMPs

Pathogen-Associated Molecular Patterns

PBS

Phosphate Buffered Saline

PHA

Phytohaemagglutinin

PLCγ

Phospholipase C gamma

PRR

Pattern Recognition Receptor

PSGL-1

P-selectin glycoprotein ligand 1

RAG

Recombination Activating Gene

SP

Signal Peptide

SVC

Spring Viraemia of Carp

Tc

T cytotoxic cells

TCR

T Cell Receptor

Th

T helper

Th1, Th2, Th17, Treg, Tfl

Subsets of Th: Th1, Th2, Th17, regulatory T, follicular T

TM

Transmembrane

TNF

Tumor Necrosis Factor

WGD

Whole Genome Duplication

V


Zusammenfassung

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.

VI

Zusammenfassung
Ziel der vorliegenden Arbeit war es daher, solche mAk spezifisch für TZellpopulationen 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-HelferZellen erkennen MHC II präsentierte Fremdpeptide über den TCR Komplex und dem KoRezeptor 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.

VII


Summary

Summary
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 Bcell or T cell receptor complexes.
This initial activation is regulated by humoral factors or cellular interactions (receptorligand interactions) resulting in the activation, proliferation and effector function within an
immune response. Some of the cellular receptors are permanently expressed on all leukocytes
on a high level (MHC class I), whereas others 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
VIII


Summary
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.

IX



Chapter 1. General introduction

Chapter 1.
1.1.

General introduction

Aquaculture
The importance of fish for human nutrition in the light of environmental protection is

mentioned in 2014 by the general Director of the Food and Agricultural Organization José
Graziano da Silva: “In a world where more than 800 million continue to suffer from chronic
malnourishment and where the global population is expected to grow by another 2 billion to

reach 9.6 billion people by 2050 – with a concentration in coastal urban areas – we must
meet the huge challenge of feeding our planet while safeguarding its natural resources for
future generations” (FAO 2014c). This fact “(...) highlights the significant role that fisheries
and aquaculture play in eliminating hunger, promoting health and reducing poverty. Never
before have people consumed so much fish or depended so greatly on the sector for their
well-being. Fish is extremely nutritious – considered a vital source of protein and essential
nutrients, especially for many poor members of our global community. Fisheries and
aquaculture are a source not just of health but also of wealth.” (Lloyd, Horrocks and Yeo
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,
1


Chapter 1. General introduction

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).
900
800
700

Thousand tons

600
500
400
300
200
100

10
20
11
20
12

09

20

06

20

03

20

00

20

97

20

94

19

91

19

88

19

85

19

82

19

79

19

76

19

19

19

73

0

Year

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)

2


Chapter 1. General introduction
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).

Tons

!

Year

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
3


Chapter 1. General introduction
of novel gene functions; (3) Asexual reproduction enabling polyploids to reproduce in the
absence of sexual mates (Comai 2005). The genomic complexity of particular rainbow trout
or teleost suggests that it might be the reason for their evolutionary success and astounding
biological diversity (Meyer and Van de Peer 2005).

Fish model for my thesis

!
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

4


Chapter 1. General introduction
- 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
5


Chapter 1. General introduction
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).
6


Chapter 1. General introduction

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

7