Quantification and phenotypic characterisation of peripheral IFN-γ 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-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ 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-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3⁺TCRγδ⁺ (γδ T cells) cells and CD3⁺TCRγδ^- cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3^- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3⁺TCR1γδ^-CD8α⁺ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ 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⁺TCR1γδ^-CD4⁺ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Keywords: Chicken; Flow cytometry; Interferon-γ; Intracellular cytokine staining; T cells; Vaccines.

Fig. 1

Intracellular staining of IFN-γ in transfected CHO cells. A) CHO cells were either transfected with plasmids containing the chicken IFN-γ gene or empty plasmids (mock controls). Cells (day 22 after transfection) were cultured with or without BFA for 18 hours and subsequently stained intracellularly using monoclonal anti-chicken IFN-γ antibody (Mab80) conjugated with APC. Representative samples are show with frequency of IFN-γ+ cells indicated above gate. B) Comparison of transfected CHO cells (day 30 after transfection) cultured with 10 μg/ml BFA for 18 hours and subsequently stained intracellularly with either commercial ELISA capture antibody (5C.123.08) & A647 conjugated secondary anti-mouse IgG1 or monoclonal anti-chicken IFN-γ antibody directly conjugated with APC (Mab80) or commercial ELISA detection antibody (5C.123.02) directly conjugated with APC or polyclonal rabbit anti-chicken IFN-γ antibody & FITC-conjugated secondary goat anti-rabbit IgG. Representative samples are show with frequency of IFN-γ+ cells indicated above gate.

Fig. 2

NDV-specific antibodies in serum measured by ELISA. Chickens of two different MHC haplotypes B19 and B21 were used for the vaccination experiment. One group was left as naive controls (naive), one group was intramuscularly vaccinated with commercial inactivated vaccine (IA) twice (4 and 7 weeks of age) and the last group was orally vaccinated with commercial live attenuated vaccine (LA) twice (4 and 7 weeks of age). Vaccination times are indicated as week 0 PV1 and week 3 PV1. Each value represents a mean of 8 individual determinations ± 95% confidence intervals. Significant differences between MHC haplotypes within the same treatment group are indicated by * (P < 0.05).

Fig. 3

Gating strategies for flow cytometry analyses. A) The gating strategy used for Fig. 4A (staining panel 1) and Fig 5A (staining panel 2) was: FSC/SSC defined lymphocytes (not shown), live lymphocytes by viability dye (ViD) exclusion using Near-IR live/dead cells stain, IFNγ⁺, IFNγ ⁺CD3⁺ and TCRγδ⁺/TCRγδ^-. For Fig. 5A the IFNγ⁺CD3⁺TCRγδ^- cells were further divided into CD4⁺ and CD8α⁺ cells (not shown). Below the FMO-FITC negative control is shown. B) The gating strategy for Fig. 4B and 4C (staining panel 1) was: FSC/SSC defined lymphocytes (not shown), live lymphocytes by ViD exclusion (not shown), CD3⁺, TCRγδ⁺/TCRγδ^-, IFNγ⁺. C) The gating strategy for Fig. 5B and 5C (staining panel 2) was: FSC/SSC defined lymphocytes (not shown), live lymphocytes by ViD exclusion (not shown), CD3⁺ (not shown), TCRγδ^-, CD4⁺/CD8α⁺, IFNγ⁺.

Fig. 4

The phenotype and frequencies of IFN-γ producing cells upon ConA stimulation. A)The frequency of IFN-γ positive cells within the live lymphocyte gate (left) – mean values are shown (n = 16) +/- SD. The proportion of cells being CD3^-, CD3⁺TCRγδ^- or CD3⁺TCRγδ⁺ in IFN-γ⁺ population (right). The percentages shown are mean values of all chickens in the experiment (n = 48). B) & C) Frequencies of IFN-γ⁺ cells in the CD3⁺TCRγδ^- population or the CD3⁺TCRγδ⁺ population of PBMC samples from naive and NDV immune animals either immunised with inactivated (IA) or live attenuated (LA) vaccine with and without stimulation with 10 μg/ml ConA. Data from individual chickens are shown with stimulation indexes (SI) indicated above including 95% confidence intervals in brackets.

Fig. 5

The phenotype and frequencies of IFN-γ producing cells upon (NDV) antigen stimulation. A) The frequency of IFN-γ positive cells within the live lymphocyte gate (left) – mean values are show (n = 16) +/- SD. The proportion of cells being CD3⁺TCRγδ⁺, CD3⁺TCRγδ^-CD4⁺, CD3⁺TCRγδ^-CD8α⁺ or CD3^- in the IFN-γ⁺ population (right). The percentages shown are mean values of all chickens in the experiment (n = 48) +/- SD. B) & C). Frequencies of IFNγ⁺ cells in the CD3⁺TCRγδ^-CD4⁺ population or CD3⁺TCRγδ^-CD8α⁺ population of PBMC samples from naive and NDV immune animals either immunised with inactivated (IA) or live attenuated (LA) vaccine, with and without stimulation with NDV antigen. Data from individual chickens are shown with stimulation indexes (SI) indicated above including 95% confidence intervals in brackets. Significant SI differences are indicated by * (P < 0.05).