Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss

Yehfang Hu, Ayham Alnabulsi, Abdo Alnabulsi, Callum Scott, Carolina Tafalla, Christopher J. Secombes, Tiehui Wang

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16 Citations (Scopus)


IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3–5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.
Original languageEnglish
Pages (from-to)328-338
Number of pages11
JournalFish & Shellfish Immunology
Early online date31 Jul 2021
Publication statusPublished - 31 Oct 2021

Bibliographical note

This research was funded by the European Commission under the 7th Framework Programme for Research and Technological Development (FP7) of the European Union (grant agreement No. 311993 TARGETFISH). YH was supported by a PhD Studentship from the Ministry of Education, Republic of China. TW was supported by the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (grant reference number HR09011) and contributing institutions.


  • Interferon-γ
  • Cell-mediated immunity
  • Monoclonal antibody
  • Rainbow trout
  • Cytokine expression


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