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 General introduction 1.1 Aquaculture 1.2 Aquaculture production of rainbow trout 1.3 Taxonomy of rainbow trout 1.4 Problems in modern aquaculture production 1.4.1 Antibiotic treatment 1.4.2 Increasing survival of fish caused by probiotic treatment 1.4.3 Vaccination 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 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 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 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 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 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 References Miranda, C D & R Zemelman (2002a) Antimicrobial multiresistance in bacteria isolated from freshwater Chilean salmon farms Science of the Total Environment, 293, 207-218 - (2002b) Bacterial resistance to oxytetracycline in Chilean salmon farming Aquaculture, 212, 31-47 Mizuno, T., M Kawasaki, M Nakahira, H Kagamiyama, Y Kikuchi, H Okamoto, K Mori & Y Yoshihara (2001) Molecular diversity in zebrafish NCAM family: three members with different VASE usage and distinct localization Mol Cell Neurosci, 18, 119-30 Modrek, B & C J Lee (2003) Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss Nature Genetics, 34, 177-180 Modrek, B., A Resch, C Grasso & C Lee (2001) Genome-wide detection of alternative splicing in expressed sequences of human genes Nucleic Acids Res, 29, 2850-9 Montaldo, E., G Del Zotto, M Della Chiesa, M C Mingari, A Moretta, A De Maria & L Moretta (2013) Human NK cell receptors/markers: a tool to analyze NK cell development, subsets and function Cytometry A, 83, 702-13 Monte, M M., T Wang, M M Costa, N O Harun & C J Secombes (2012) Cloning and expression analysis of two ROR-gamma homologues (ROR-gammaa1 and ROR-gammaa2) in rainbow trout Oncorhynchus mykiss Fish Shellfish Immunol, 33, 365-74 Monte, M M., T Wang, J W Holland, J Zou & C J Secombes (2013) Cloning and characterization of rainbow trout interleukin-17A/F2 (IL-17A/F2) and IL-17 receptor A: expression during infection and bioactivity of recombinant IL-17A/F2 Infect Immun, 81, 340-53 Moore, L J., J M Dijkstra, E O Koppang & I Hordvik (2009) CD4 homologues in Atlantic salmon Fish Shellfish Immunol, 26, 10-8 Moore, L J., T Somamoto, K K Lie, J M Dijkstra & I Hordvik (2005) Characterisation of salmon and trout CD8alpha and CD8beta Mol Immunol, 42, 1225-34 Morales, H D., L Abramowitz, J Gertz, J Sowa, A Vogel & J Robert (2010) Innate immune responses and permissiveness to ranavirus infection of peritoneal leukocytes in the frog Xenopus laevis J Virol, 84, 4912-22 Moulana, M., J Evenhuis, M Albertino, U Godwin, E I Kountikov, T B Stuge, M Wilson, E Bengten, N W Miller & T J McConnell (2008) Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies Vet Immunol Immunopathol, 126, 120-30 Mudge, J M., A Frankish, J Fernandez-Banet, T Alioto, T Derrien, C Howald, A Reymond, R Guigo, T Hubbard & J Harrow (2011) The origins, evolution, and functional potential of alternative splicing in vertebrates Mol Biol Evol, 28, 2949-59 Murphy, K (2012) Janeway's_Immunobiology Garland Science Nakanishi, T., K Aoyagi, C Xia, J M Dijkstra & M Ototake (1999) Specific cell-mediated immunity in fish Vet Immunol Immunopathol, 72, 101-9 Nakanishi, T., U Fischer, J M Dijkstra, S Hasegawa, T Somamoto, N Okamoto & M Ototake (2002) Cytotoxic T cell function in fish Dev Comp Immunol, 26, 131-9 140 References Nakanishi, T., H Toda, Y Shibasaki & T Somamoto (2011) Cytotoxic T cells in teleost fish Dev Comp Immunol, 35, 1317-23 Nayak & K Sukanta (2010) Role of gastrointestinal microbiota in fish Aquaculture Research, 41, 15531573 Naylor, R L., R J Goldburg, J H Primavera, N Kautsky, M C Beveridge, J Clay, C Folke, J Lubchenco, H Mooney & M Troell (2000) Effect of aquaculture on world fish supplies Nature, 405, 1017-24 Nelson, R W., P A Bates & U Rutishauser (1995) Protein determinants for specific polysialylation of the neural cell adhesion molecule J Biol Chem, 270, 17171-9 Nemunaitis, J (1993) Macrophage function activating cytokines: potential clinical application Crit Rev Oncol Hematol, 14, 153-71 Nielsen, N., N Odum, B Urso, L L Lanier & P Spee (2012) Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A PLoS One, 7, e31959 Niethammer, P., M Delling, V Sytnyk, A Dityatev, K Fukami & M Schachner (2002) Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis J Cell Biol, 157, 52132 Nikoskelainen, S., J Lehtinen & E M Lilius (2002) Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement Dev Comp Immunol, 26, 797-804 O’Neill, B (2006) Seafood Watch Seafood Report: Farmed Rainbow Trout Monterey Bay Aquarium, 126 Ohtani, M., N Hayashi, K Hashimoto, T Nakanishi & J M Dijkstra (2008) Comprehensive clarification of two paralogous interleukin 4/13 loci in teleost fish Immunogenetics, 60, 383-97 Okada, K & K Asai (2008) Expansion of signaling genes for adaptive immune system evolution in early vertebrates BMC Genomics, 9, 218 Ostergaard, A E., S A Martin, T Wang, R J Stet & C J Secombes (2009) Rainbow trout (Oncorhynchus mykiss) possess multiple novel immunoglobulin-like transcripts containing either an ITAM or ITIMs Dev Comp Immunol, 33, 525-32 Panigrahi, A., V Kiron, S Satoh, I Hirono, T Kobayashi, H Sugita, J Puangkaew & T Aoki (2007) Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding Dev Comp Immunol, 31, 372-82 Papanastasiou, A D & I K Zarkadis (2005) Gene duplication of the seventh component of complement in rainbow trout Immunogenetics, 57, 703-8 Papewalis, C., B Jacobs, M Wuttke, E Ullrich, T Baehring, R Fenk, H S Willenberg, S Schinner, M Cohnen, J Seissler, K Zacharowski, W A Scherbaum & M Schott (2008) IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo J Immunol, 180, 1462-70 141 References Partula, S., A de Guerra, J S Fellah & J Charlemagne (1994) Characterization of cDNA of T-cell receptor beta chain in rainbow trout C R Acad Sci III, 317, 765-770 - (1995) Structure and diversity of the T cell antigen receptor beta-chain in a teleost fish J Immunol, 155, 699-706 - (1996) Structure and diversity of the TCR alpha-chain in a teleost fish J Immunol, 157, 207-12 Paul, W E (2004) Fundamental Immunology Immunology and Cell Biology, 82, 453 Perez, T., J L Balcazar, I Ruiz-Zarzuela, N Halaihel, D Vendrell, I de Blas & J L Muzquiz (2010) Host-microbiota interactions within the fish intestinal ecosystem Mucosal Immunol, 3, 355-60 Poli, A., T Michel, M Theresine, E Andres, F Hentges & J Zimmer (2009) CD56bright natural killer (NK) cells: an important NK cell subset Immunology, 126, 458-65 Pollscheit, J., N Glaubitz, H Haller, R Horstkorte & K Bork (2012) Phosphorylation of serine 774 of the neural cell adhesion molecule is necessary for cyclic adenosine monophosphate response element binding protein activation and neurite outgrowth J Neurosci Res, 90, 1577-82 Polo-Parada, L., F Plattner, C Bose & L T Landmesser (2005) NCAM 180 acting via a conserved Cterminal domain and MLCK is essential for effective transmission with repetitive stimulation Neuron, 46, 917-31 Press, C M & O Evensen (1999) The morphology of the immune system in teleost fishes Fish & Shellfish Immunology, 9, 309-318 Pulkkinen, K., L R Suomalainen, A F Read, D Ebert, P Rintamaki & E T Valtonen (2010) Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland Proc Biol Sci, 277, 593-600 Ramirez-Gomez, F., W Greene, K Rego, J D Hansen, G Costa, P Kataria & E S Bromage (2012) Discovery and characterization of secretory IgD in rainbow trout: secretory IgD is produced through a novel splicing mechanism J Immunol, 188, 1341-9 Randelli, E., G Scapigliati & F Buonocore (2011) CD3gamma/delta in sea bass (Dicentrarchus labrax): Molecular characterization and expression analysis Results Immunol, 1, 31-5 Rao, Y., X F Wu, P Yip, J Gariepy & C H Siu (1993) Structural characterization of a homophilic binding site in the neural cell adhesion molecule J Biol Chem, 268, 20630-8 Raulet, D H (2006) Missing self recognition and self tolerance of natural killer (NK) cells Semin Immunol, 18, 145-50 Rawls, J F., B S Samuel & J I Gordon (2004) Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota Proc Natl Acad Sci U S A, 101, 4596-601 Rees, M., A J A.Butler, I G Brons, M C Negus, J N Skepper & P J Friend (2005) Evidence of macrophage receptors capable of direct recognition of xenogeneic epitopes without opsonization Xenotransplantation, 12, 13-9 Reyes, A A., S V Schulte, S Small & R Akeson (1993) Distinct NCAM splicing events are differentially regulated during rat brain development Brain Res Mol Brain Res, 17, 201-11 142 References Reyes-Cerpa, S., K Maisey, F Reyes-López, D Toro-Ascuy, A M Sandino & M Imarai (2013) fish cytokines and immune response Richard Garand, R., E Emanuelle Homolle, B Bernard Husson, E Emilienne Kuhlein, G L Calvez, Danielle., C Arnoulet, F B Brignole-Baudouin, B Drénou, D Duchayne, A Falkenrodt, J Feuillard, M Jacob, F Valensi, M Maynadié, R L Gressin, L Chaperot, Sainty, S Sotto, F Trimoreau & M Béné (2002) Clinical and biologic features of CD4+CD56+ malignancies The American Society of Hematology, 99, 1556-1563 Robertsen, B (2006) The interferon system of teleost fish Fish Shellfish Immunol, 20, 172-91 Robertson, P A W., C O'Dowd, C Burrells, P Williams & B Austin (2000) Use of Carnobacterium sp as a probiotic for Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum) Aquaculture, 185, 235-243 Rocha, B (2007) The extrathymic T-cell differentiation in the murine gut Immunological Reviews 166177 Roederer, M (2004) Cytometry: new developments 4th Edition Methods in cell biology, 95 Rombout, J H., G Yang & V Kiron (2014) Adaptive immune responses at mucosal surfaces of teleost fish Fish Shellfish Immunol, 40, 634-43 Rombout, J H W M., H B T Huttenhuis, S Picchietti & G Scapigliati (2005) Phylogeny and ontogeny of fish leucocytes Fish & Shellfish Immunology 19, 441-455 Roothans, D., E Smits, E Lion, J Tel & S Anguille (2013) CD56 marks human dendritic cell subsets with cytotoxic potential Oncoimmunology, 2, e23037 Saffell, J L., F S Walsh & P Doherty (1994) Expression of Ncam Containing Vase in Neurons Can Account for a Developmental Loss in Their Neurite Outgrowth Response to Ncam in a Cellular Substratum Journal of Cell Biology, 125, 427-436 Salgado-Miranda, C., E Loza-Rubio, R.-A E & G García-Espinosa (2013) Viral vaccines for bony fish: past, present and future Expert Rev Vaccines, 12, 567-78 Sato, A., W E Mayer, P Overath & J Klein (2003) Genes encoding putative natural killer cell C-type lectin receptors in teleostean fishes Proc Natl Acad Sci U S A, 100, 7779-84 Sato, Y & M Nishida (2010) Teleost fish with specific genome duplication as unique models of vertebrate evolution Environmental Biology of Fishes, 88, 169-188 Saurabh, S & P K Sahoo (2008) Lysozyme: an important defence molecule of fish innate immune system Aquaculture Research, 39, 223-239 Secombes, C J., T Wang, S Hong, S Peddie, M Crampe, K J Laing, C Cunningham & J Zou (2001) Cytokines and innate immunity of fish Dev Comp Immunol, 25, 713-23 Seliger, C., B Schaerer, M Kohn, H Pendl, S Weigend, B Kaspers & S Hartle (2012) A rapid highprecision flow cytometry based technique for total white blood cell counting in chickens Vet Immunol Immunopathol, 145, 86-99 143 References Shen, L., T B Stuge, E Bengten, M Wilson, V G Chinchar, J P Naftel, J M Bernanke, L W Clem & N W Miller (2004) Identification and characterization of clonal NK-like cells from channel catfish (Ictalurus punctatus) Developmental and Comparative Immunology, 28, 139–152 Shen, L., T B Stuge, J P Evenhuisa, E Bengten, M Wilson, V G Chinchara, L W Clema & N W Millera (2003) Channel catfish NK-like cells are armed with IgM via a putative FcµR Developmental and Comparative Immunology, 27, 699-714 Shen, L., T B Stuge, H Zhou, M Khayat, K S Barker, S M Quiniou, M Wilson, E Bengten, V G Chinchar, L W Clem & N W Miller (2002) Channel catfish cytotoxic cells: a mini-review Dev Comp Immunol, 26, 141-9 Silva, S S D., T T T Nguyen, G M Turchini, U S Amarasinghe & N W Abery (2009) Alien Species in Aquaculture and Biodiversity: A Paradox in Food Production AMBIO: A Journal of the Human Environment, 38, 24-28 Somamoto, T., M Kondo, T Nakanishi & M Nakao (2014a) Helper function of CD4(+) lymphocytes in antiviral immunity in ginbuna crucian carp, Carassius auratus langsdorfii Dev Comp Immunol, 44, 111-5 Somamoto, T., E O Koppang & U Fischer (2014b) Antiviral functions of CD8(+) cytotoxic T cells in teleost fish Dev Comp Immunol, 43, 197-204 Somamoto, T., T Nakanishi & M Nakao (2013) Identification of anti-viral cytotoxic effector cells in the ginbuna crucian carp, Carassius auratus langsdorfii Dev Comp Immunol, 39, 370-7 Somamoto, T., T Nakanishi & N Okamoto (2000) Specific cell-mediated cytotoxicity against a virusinfected syngeneic cell line in isogeneic ginbuna crucian carp Dev Comp Immunol, 24, 633-40 - (2002) Role of specific cell-mediated cytotoxicity in protecting fish from viral infections Virology, 297, 120-7 Somamoto, T., Y Yoshiura, T Nakanishi & M Ototake (2005) Molecular cloning and characterization of two types of CD8alpha from ginbuna crucian carp, Carassius auratus langsdorfii Dev Comp Immunol, 29, 693-702 Sommerset, B Krossøy, E Biering & P Frost (2005) Vaccines for fish in aquaculture Expert Rev Vaccines, 4, 89-101 Stet, R J M., T Hermsen, A H Westphal, J Jukes, M Engelsma, L B M Verburg-van Kemenade, J Dortmans, J Aveiro & H F J Savelkoul (2005) Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44 Immunogenetics, 57, 77-89 Storset, A K., I O Slettedal, J L Williams, A Law & E Dissen (2003) Natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs Eur J Immunol, 33, 980-90 Sugita, H., M Fukumoto, H Koyama & Y Deguchi (1988) Changes in the Fecal Microflora of Goldfish Carassius-Auratus with the Oral-Administration of Oxytetracycline Nippon Suisan Gakkaishi, 54, 2181-2187 144 References Sun, J C & L L Lanier (2009) Natural killer cells remember: an evolutionary bridge between innate and adaptive immunity? Eur J Immunol, 39, 2059-64 Sunyer, J O (2013) Fishing for mammalian paradigms in the teleost immune system Nat Immunol, 14, 320-6 Sunyer, J O & L Tort (1995) Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway Vet Immunol Immunopathol, 45, 333-45 Sunyer, J O., I K Zarkadis, A Sahu & J D Lambris (1996) Multiple forms of complement C3 in trout that differ in binding to complement activators Proc Natl Acad Sci U S A, 93, 8546-8551 Takashi Aoki, T., T Takano, M D Santos, H Kondo & I Hirono (2008) Molecular Innate Immunity in Teleost Fish: Review and Future Perspectives fisheries for global welfare and Environment, 5th World Fisheries Congress Takeuchi, O & S Akira (2010) Pattern recognition receptors and inflammation Cell, 140, 805-20 Takizawa, F., K Araki, M Ohtani, H Toda, Y Saito, V S Lampe, J M Dijkstra, M Ototake, T Moritomo, T Nakanishi & U Fischer (2014) Transcription analysis of two Eomesodermin genes in lymphocyte subsets of two teleost species Fish Shellfish Immunol, 36, 215-22 Takizawa, F., J M Dijkstra, P Kotterba, T Korytar, H Kock, B Köllner, B Jaureguiberry, T Nakanishi & U Fischer (2011a) The expression of CD8alpha discriminates distinct T cell subsets in teleost fish Dev Comp Immunol, 35, 752-63 Takizawa, F., E O Koppang, M Ohtani, T Nakanishi, K Hashimoto, U Fischer & J M Dijkstra (2011b) Constitutive high expression of interleukin-4/13A and GATA-3 in gill and skin of salmonid fishes suggests that these tissues form Th2-skewed immune environments Mol Immunol, 48, 1360-8 The Scottish Association for Marine Science and Napier University 2002 Review and Synthesis of the Environmental Impacts of Aquaculture Scottish Executive Central Research Unit Tian, J Y., B J Sun, Y P Luo, Y A Zhang & P Nie (2009) Distribution of IgM, IgD and IgZ in mandarin fish, Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation Aquaculture, 288, 14-21 Toda, H., Y Saito, T Koike, F Takizawa, K Araki, T Yabu, T Somamoto, H Suetake, Y Suzuki, M Ototake, T Moritomo & T Nakanishi (2011) Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish Dev Comp Immunol, 35, 650-60 Toda, H., Y Shibasaki, T Koike, M Ohtani, F Takizawa, M Ototake, T Moritomo & T Nakanishi (2009) Alloantigen-specific killing is mediated by CD8-positive T cells in fish Dev Comp Immunol, 33, 646-52 Tonissen, K F & P A Krieg (1993) Two neural-cell adhesion molecule (NCAM)-encoding genes in Xenopus laevis are expressed during development and in adult tissues Gene, 127, 243-7 145 References Toranzo, A E., J L Romalde, B Magarinos & J L Barja (2009) Present and future of aquaculture vaccines against fish bacterial diseases Options Méditerranéennes, 86 Tort, L., J C Balasch & S Mackenzie (2003) Fish immune system A crossroads between innnate and adaptive responses Inmunologia, 22, 277-286 Uribe, C., H Folch, R Enriquez & G Moran (2011) Innate and adaptive immunity in teleost fish a review Veterinarni Medicina, 56, 486-503 Utke, K., S Bergmann, N Lorenzen, B Ko ̈llner, M Ototake & U Fischer (2007) Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus Fish & Shellfish Immunology, 22, 182-196 Utke, K., H Kock, H Schuetze, S M Bergmann, N Lorenzen, K Einer-Jensen, B Köllner, R A Dalmo, T Vesely, M Ototake & U Fischer (2008) Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus Dev Comp Immunol, 32, 239-52 Van Kaer, L., V V Parekh & L Wu (2011) Invariant natural killer T cells: bridging innate and adaptive immunity Cell Tissue Res, 343, 43-55 Villey, I., R D Chasseval & E D Villartay (1999) RORγT, a thymus-specific isoform of the orphan nuclear receptor RORγ  /  TOR, is up-regulated by signaling through the pre-T cell receptor and binds to the TEA promoter Eur J Immunol, 29, 4072–4080 Vivier, E., E Tomasello, M Baratin, T Walzer & S Ugolini (2008) Functions of natural killer cells Nat Immunol, 9, 503-10 Volff, J N (2005) Genome evolution and biodiversity in teleost fish Heredity (Edinb), 94, 280-94 Walsh, F S & G Dickson (1989) Generation of multiple N-CAM polypeptides from a single gene Bioessays, 11, 83-8 Wang, R., H Xie, Z Huang, J Ma, X Fang, Y Ding & Z Sun (2011a) T cell factor regulates thymocyte survival via a RORgammat-dependent pathway J Immunol, 187, 5964-73 Wang, T., P Diaz-Rosales, M M Costa, S Campbell, M Snow, B Collet, S A Martin & C J Secombes (2011b) Functional characterization of a nonmammalian IL-21: rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFNgamma, IL-10, and IL-22 J Immunol, 186, 708-21 Wang, T., P C Hanington, M Belosevic & C J Secombes (2008) Two Macrophage Colony-Stimulating Factor Genes Exist in Fish That Differ in Gene Organization and Are Differentially Expressed The Journal of Immunology, 181, 3310-3322 Wang, T., J W Holland, S A Martin & C J Secombes (2010a) Sequence and expression analysis of two T helper master transcription factors, T-bet and GATA3, in rainbow trout Oncorhynchus mykiss and analysis of their expression during bacterial and parasitic infection Fish Shellfish Immunol, 29, 705-15 Wang, T., W Huang, M M Costa & C J Secombes (2011c) The gamma-chain cytokine/receptor system in fish: more ligands and receptors Fish Shellfish Immunol, 31, 673-87 146 References Wang, T., M M Monte, W Huang, P Boudinot, S A Martin & C J Secombes (2010b) Identification of two FoxP3 genes in rainbow trout (Oncorhynchus mykiss) with differential induction patterns Mol Immunol, 47, 2563-74 Wei, S., J M Zhou, X Chen, R N Shah, J Liu, T M Orcutt, D Traver, J Y Djeu, G W Litman & J A Yoder (2007) The zebrafish activating immune receptor Nitr9 signals via Dap12 Immunogenetics, 59, 813-21 Weisberger, J., D Cornfield, W Gorczyca & Z Liu (2003) Down-Regulation of Pan-T-Cell Antigens, Particularly CD7, in Acute Infectious Mononucleosis American Journal of Clinical Pathology, 120, 49-55 Wen, Y., W Fang, L X Xiang, R L Pan & J Z Shao (2011) Identification of Treg-like cells in Tetraodon: insight into the origin of regulatory T subsets during early vertebrate evolution Cell Mol Life Sci, 68, 2615-26 Whyte, S K (2007) The innate immune response in finfish: a review of current knowledge Fish Shellfish Immunol, 23, 1127-51 Wikipedia (2015) Rainbow trout online wikipedia.org Wilson, M R., H Zhou, E Bengtén, L W Clem, S T.B., G W Warr & N W Miller (1998) T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes Molecular Immunology, 35, 545-557 Xing, Y & C Lee (2007) Relating alternative splicing to proteome complexity and genome evolution Adv Exp Med Biol, 623, 36-49 Yang, X O., B P Pappu, R Nurieva, A Akimzhanov, H S Kang, Y Chung, L Ma, B Shah, A D Panopoulos, K S Schluns, S S Watowich, Q Tian, A M Jetten & C Dong (2008) T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma Immunity, 28, 29-39 Yoder, J A (2004) Investigating the morphology, function and genetics of cytotoxic cells in bony fish Comp Biochem Physiol C Toxicol Pharmacol, 138, 271-80 Yoder, J A., T M Orcutt, D Traver & G W Litman (2007) Structural characteristics of zebrafish orthologs of adaptor molecules that associate with transmembrane immune receptors Gene, 401, 154-64 Yoder, J A., P M Turner, P D Wright, V Wittamer, J Y Bertrand, D Traver & G W Litman (2010) Developmental and tissue-specific expression of NITRs Immunogenetics, 62, 117-22 Zapata, A., B Diez, T Cejalvo, C Gutierrez-de Frias & C A (2006) Ontogeny of the immune system of fish Fish Shellfish Immunol 20, 126-36 Zapata, A G & E L Cooper (1990) The immune system: comparative histophysiology Chichester: John Wiley and Sons Zarkadis, I K., M R Sarrias, G Sfyroera, J O Sunyer & J D Lambris (2001) Cloning and structure of three rainbow trout C3 molecules a plausible explanation for their functional diversity Dev Comp Immunol, 25, 11-24 147 References Zhang, Y A., I Salinas, J Li, D Parra, S Bjork, Z Xu, S E LaPatra, J Bartholomew & J O Sunyer (2010) IgT, a primitive immunoglobulin class specialized in mucosal immunity Nat Immunol, 11, 827-35 Zhang, Y A., I Salinas & J O Sunyer (2011) Recent findings on the structure and function of teleost IgT Fish Shellfish Immunol, 31, 627-34 Zhu, L., Z Yan, M Feng, D Peng, Y Guo, X Hu, L Ren & Y Sun (2014a) Identification of sturgeon IgD bridges the evolutionary gap between elasmobranchs and teleosts Dev Comp Immunol, 42, 138-47 Zhu, L Y., A F Lin, T Shao, L Nie, W R Dong, L X Xiang & J Z Shao (2014b) B cells in teleost fish act as pivotal initiating APCs in priming adaptive immunity: an evolutionary perspective on the origin of the B-1 cell subset and B7 molecules J Immunol, 192, 2699-714 Zimmerman, A M., J P Evenhuis, G H Thorgaard & S S Ristow (2004) A single major chromosomal region controls natural killer cell-like activity in rainbow trout Immunogenetics (2004) 55:, 55, 825–835 Zimmerman, A M., F M Moustafa, K E Romanowski & L A Steiner (2011) Zebrafish immunoglobulin IgD: unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes Mol Immunol, 48, 2220-3 Zorn, A M & P A Krieg (1992) Developmental regulation of alternative splicing in the mRNA encoding Xenopus laevis neural cell adhesion molecule (NCAM) Dev Biol, 149, 197-205 Zou, J., S Bird, J Truckle, N Bols, M Horne & C Secombes (2004) Identification and expression analysis of an IL-18 homologue and its alternatively spliced form in rainbow trout (Oncorhynchus mykiss) Eur J Biochem, 271, 1913-23 Zou, J & C J Secombes (2011) Teleost fish interferons and their role in immunity Dev Comp Immunol, 35, 1376-87 Zuckermann, F A (1999) Extrathymic CD4/CD8 double positive T cells Vet Immunol Immunopathol, 72, 55-66 Zwollo, P (2011) Dissecting teleost B cell differentiation using transcription factors Dev Comp Immunol, 35, 898-905 Zwollo, P., S Cole, E Bromage & S Kaattari (2005) B cell heterogeneity in the teleost kidney: evidence for a maturation gradient from anterior to posterior kidney J Immunol, 174, 6608-16 Zwollo, P., A Haines, P Rosato & J Gumulak-Smith (2008) Molecular and cellular analysis of B-cell populations in the rainbow trout using Pax5 and immunoglobulin markers Dev Comp Immunol 32, 1482-96 Zwollo, P., K Mott & M Barr (2010) Comparative analyses of B cell populations in trout kidney and mouse bone marrow: establishing B cell signatures Dev Comp Immunol, 34, 1291-9 148 Acknowledgement Acknowledgement On this place, I would like to use the opportunity to thank all my colleagues and friends that have supported me during my thesis This work was carried out in Friedrich Loeffler Institute from 2010 to 2014 During this time, I was funded by Vietnamese Government, KO393/1-1 DFG and DIREFO project and I am very grateful for the opportunity to work there First of all, I would like to give my sincerest thanks to my wonderful supervisor Dr Bernd Köllner for giving me the opportunity to be a member in his friendly team and an interesting, challenging and promising project in his lab in Friedrich Loeffler Institute I also appreciate very much for your help on my living in Greifswald and teaching me how to enjoy my life here since the first day I came I would also like to acknowledge Prof Dr Dr h.c Thomas C Mettenleiter for accepting me as a PhD student in Greifswald University Thank you very much for your support in the thesis writing and defense Many thanks go to Sabine Weber for her practical help in my lab work and the well organization of team work which guarantees everything is running well during my PhD Many thanks are also given to Dr Tomáš Korytář for his help in lab work, discussion and paper writing and being frankly friend I would also like to thank Joanna Jaros, Xiaocui He, Dr Sven Bergmann, Nussieba Osman for their helps during the five three years and being very good colleagues and very nice friends I also would like to give thanks to Katharina Rieger, Yaqing Zhu and all colleagues in FLI institute for giving me happy time and being my drivers from institute to my home in suddenly case I would like to thank Dr Uwe Fischer and Dr Fumio Takizawa for allowing me using CD8α antibody I would like to thank the members of the Joint Graduate Education Program and the 322 Project – Ministry of Education and Training of Vietnam, especially Prof Dr Le Tran Binh, Prof Dr Maria Theresia Schafmeister, Dr Jörn Kasbohm, Dr Le Thi Lai, Dr Luu Lan Huong for their responsibility in this program They had given me the opportunity to research in Germany I am deeply indebted to the Vietnamese Ministry of Education and Training, especially 149 Acknowledgement Nguyen Thi Thanh Tam for the scholarship that supported my first stage in Germany that provided me incomparable advantages for my life in Germany I would like to thank my administrators, my colleagues of Plant Biotechnology, Vietnamese Biotechnology Institute such as Dr Chu Hoang Ha, Dr Le Van Son, Nguyen Thi Nguyet Minh, Bui Phuong Thao at the IBT institute, who created good conditions, confirmed the permission for my studying in Germany and encouraged me to go ahead with my thesis I am also grateful to all my friends, in and out of Greifswald, are always invaluable to me, to name some, Bui Thanh Huong, Nguyen Giang Son, Bui Phuong Thuy, Nguyen Thanh Trung, Le Thi Mai Thanh, Le Diem Quynh, Nguyen Huyen, Bui Thanh Huong, Nguyen Giang Son, thanks for company all the time Any words would never be too exaggerated to express my gratefulness to my family, my beloved parents, brothers and my aunt in Berlin for always being so supportive and loving me without any reserve 150 About the author About the author List of publication and oral presentation Publication and manuscripts from the thesis: Tomáš Korytář, Huong Dang Thi, Fumio Takizawa and Bernd Köllner (2013) A multicolour flow cytometry identifying defined leukocyte subsets of rainbow trout (Oncorhynchus mykiss) Fish & Shelfish immunology Huong Dang Thi, Tomáš Korytář, Joanna Jaros and Bernd Köllner Distribution of T cell subpopulations in lymphoid and mucosal organs of rainbow trout (Oncorhynchus mykiss) characterized by new lineage marker specific monoclonal antibodies (manuscript) Huong Dang Thi, Tomáš Korytář, Xiaocui He and Bernd Köllner CD56 (NCAM1) positive leukocyte population in rainbow trout – molecular and functional characterization (manuscript) Huong Dang Thi, Tomáš Korytář and Bernd Köllner (2012) A profile of peripheral and mucosal T-cell subpopulations in Rainbow Trout (Oncorhynchus mykiss) 12th Congress of The International Society for Developmental and Comparative Immunulogy (ISDCI), July to July 13, 2012 FuKuoka, Japan with the oral presentation Publications: Castro, R., Takizawa, F., Chaara, W., Lunazzi, A., Dang, T H., Koellner, B., Boudinot, P (2013) Contrasted TCRbeta diversity of CD8+ and CD8- T cells in rainbow troutPLoS One, 8(4) Tomáš Korytář*, Joanna Jaros, Huong Dang Thi, Marieke Verleih, Alexander Rebl, Tom Goldammer, Carolina Taffala and Bernd Köllner Peritoneal IgM+ B lymphocytes and myeloid cells exhibit distinct expression signatures during inflammation in rainbow trout (Oncorhynchus mykiss) (under review) H.T Dang, T Korytář, J Jaros, U Fischer, F Takizawa, and B Köllner (2011) T-cell subpopulations in lymphoid and mucosal organs in rainbow trout (Oncorhynchus mykiss)Investigation with newly established T-cell specific MAbs The 15th European Association of Fish Pathologists in Split, Croatia 151 About the author Joanna Jaros, Huong Dang Thi and Bernd Köllner (2011) Identification of virulent genes differentially expressed in Aeromonas Salmonicida genome under variable conditions The 15th European Association of Fish Pathologists in Split, Croatia with poster Huong Dang Thi, Tomáš Korytář and Bernd Köllner (2012) A profile of peripheral and mucosal T-cell subpopulations in Rainbow Trout (Oncorhynchus mykiss) 12th Congress of The International Society for Developmental and Comparative Immunulogy (ISDCI), July to July 13, 2012 FuKuoka, Japan with the oral presentation vv J.Jaros, T Korytar, D.T.Huong, M Weiss, B Köllner (2013) Rainbow trout (Oncorhynchus mykiss) thrombocytes are involved in MHC II dependent antigen Fish & Shelfish Immunology, 2013 with the oral presentation 10 H.T Dang, T Korytář, and B Köllner (2013) CD56-positive leucocyte populations in Rainbow trout (Oncorhynchus mykiss) – Molecular and functional characterization 16th International Conference on Disease of Fish and Shelfish, Tampere, Finland with poster 152 About the author Resume Personal Data Name: Huong Dang Thi Gender: Female Birth: 27/05/1982 Nationality: Viet Nam Tel: +493835171208 Email: danghuongslh@gmail.com Address: Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany Education May 2010- present Ph.D in Immunology, institute for immunology, FriedrichLoeffler-Institut (FLI), Germany Sep 2004-Mar 2007 Master in Biology, VietNam National university, Hanoi Sep 2000-June 2004 Bachelor in Biology, VietNam National university, Hanoi Work experience May 2007- 2010 Vietnamese Biotechnology Institute 18 Hoang Quoc Viet, Hanoi, Viet Nam 2010- present PhD student in Immunology, institute for immunology, Friedrich-Loeffler-Institut (FLI), Germany 153 Declaration Muster der Erklärung Hiermit erkläre ich, dass diese Arbeit von mir weder an der MathematischNaturwissenschaftlichen Fakultät der Ernst-Moritz-Arnd-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde Ferne erkläre ich, dass ich diese Arbeit selbständig verfasst und keine anderen als die darin angegebenen Hilfsmittel und Hilfen benutzt und keine Textabschnitte eines Dritten ohne Kennzeichnung übernommen habe Greifswald, den 27/04/2015 Huong Dang Thi 154