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

Abstract

IFN-gamma 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-gamma 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-gamma 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-gamma peptides were developed and characterised to monitor IFN-gamma expression. The results show that the IFN-gamma mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-gamma protein and rainbow trout IFN-gamma produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-gamma at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-gamma 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-gamma(+) 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-gamma(+) 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-gamma in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-gamma producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.