First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio)

PLoS One. 2015 Jun 17;10(6):e0126378. doi: 10.1371/journal.pone.0126378. eCollection 2015.

Abstract

Adaptive immunity in homeotherms depends greatly on CD4+ Th cells which release cytokines in response to specific antigen stimulation. Whilst bony fish and poikilothermic tetrapods possess cells that express TcR and CD4-related genes (that exist in two forms in teleost fish; termed CD4-1 and CD4-2), to date there is no unequivocal demonstration that cells equivalent to Th exist. Thus, in this study we determined whether CD4-1+ lymphocytes can express cytokines typical of Th cells following antigen specific stimulation, using the zebrafish (Danio rerio). Initially, we analyzed the CD4 locus in zebrafish and found three CD4 homologues, a CD4-1 molecule and two CD4-2 molecules. The zfCD4-1 and zfCD4-2 transcripts were detected in immune organs and were most highly expressed in lymphocytes. A polyclonal antibody to zfCD4-1 was developed and used with an antibody to ZAP70 and revealed double positive cells by immunohistochemistry, and in the Mycobacterium marinum disease model CD4-1+ cells were apparent surrounding the granulomas typical of the infection. Next a prime-boost experiment, using human gamma globulin as antigen, was performed and revealed for the first time in fish that zfCD4-1+ lymphocytes increase the expression of cytokines and master transcription factors relevant to Th1/Th2-type responses as a consequence of boosting with specific antigen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptive Immunity
  • Amino Acid Sequence
  • Animals
  • Antibodies / chemistry
  • CD4-Positive T-Lymphocytes / classification
  • CD4-Positive T-Lymphocytes / immunology*
  • CD4-Positive T-Lymphocytes / microbiology
  • CD4-Positive T-Lymphocytes / pathology
  • Cytokines / biosynthesis*
  • Cytokines / immunology
  • Genetic Loci / immunology
  • Histocompatibility Antigens Class II / genetics
  • Histocompatibility Antigens Class II / immunology*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mycobacterium Infections, Nontuberculous / genetics
  • Mycobacterium Infections, Nontuberculous / immunology*
  • Mycobacterium Infections, Nontuberculous / microbiology
  • Mycobacterium Infections, Nontuberculous / veterinary*
  • Mycobacterium marinum / immunology
  • Phylogeny
  • RNA, Messenger / genetics
  • RNA, Messenger / immunology*
  • Sequence Alignment
  • Th1-Th2 Balance
  • ZAP-70 Protein-Tyrosine Kinase / genetics
  • ZAP-70 Protein-Tyrosine Kinase / immunology
  • Zebrafish / classification
  • Zebrafish / genetics
  • Zebrafish / immunology
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / immunology
  • gamma-Globulins / administration & dosage

Substances

  • Antibodies
  • Cytokines
  • Histocompatibility Antigens Class II
  • RNA, Messenger
  • Zebrafish Proteins
  • gamma-Globulins
  • ZAP-70 Protein-Tyrosine Kinase

Associated data

  • GENBANK/HE983357
  • GENBANK/HE983358
  • GENBANK/HE983359

Grants and funding

Both first authors were supported by the Scottish Overseas Research Scholarship Awards Scheme (SORSAS) to undertake a PhD program at the University of Aberdeen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.