Quantification and phenotypic characterisation of peripheral IFN-? producing leucocytes in chickens vaccinated against Newcastle disease.

ABSTRACT: The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-? responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-? using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-? production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-? positive cells. ConA stimulation induced extensive IFN-? production in both CD3⁺TCR??⁺ (???T cells) cells and CD3⁺TCR??^- cells (???T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-? producing cells were CD3^- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-? production but to a lower extent than ConA and with a large variation between individuals. The CD3⁺TCR1??^-CD8?⁺ (CTL) population produced the highest NDV specific IFN-? responses, with significantly elevated levels of IFN-? producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3⁺TCR1??^-CD4⁺ (Th) population did not show a significant increase in IFN-? production in NDV stimulated samples which was in part due to a high number of IFN-? producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-? producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

SUBMITTER: Andersen SH 

PROVIDER: S-EPMC5697524 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature