ELISPOT and intracellular cytokine staining: Novel assays for quantifying T cell responses in the chicken

Abstract

SummaryThe measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferongamma (ChIFN-g) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4 + and CD8 + T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-g production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-g production by CD4 + and CD8 + cells.

Investigations on the protective role of passively transferred antibodies against avian metapneumovirus infection in turkeys

“…Infectious bronchitis virus-specific T lymphocytes of infected chickens have been detected via cytotoxicity assay (Seo & Collisson, 1997), and transfer of infectious bronchitis virus-specific T lymphocytes to naïve chicks resulted in protection against virulent challenge infection (Seo et al, 2000). Newcastle disease virus-specific T-cell activity has been quantified by ex vivo recall stimulation of leukocytes and subsequent detection of antigeninduced interferon gamma production via ELISA, ELI-SPOT or intracellular cytokine staining (Lambrecht et al, 2004;Ariaans et al, 2008). However, methods to detect aMPV-specific T lymphocytes in poultry and especially in turkeys remain to be developed.…”

Towards a universal vaccine for avian influenza: Protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus

“…Pools of irrelevant peptides were used to control for background responses. IFN-γ ELISpot was carried out using the Chicken IFN-γ CytoSet™ (Life Technologies, UK) as described previously [10]. Briefly, 96-well multiscreen plates with Protein Binding Immobilon-P-Membrane (Millipore, Billerica,USA) were coated with 100 μL/well of 5 mg/ml mouse anti-chicken IFN-γ (Life Technologies, UK) in coating buffer (Sigma–Aldrich, UK) overnight at 4 °C.…”

Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8+ T lymphocytes

“…Indirect concentrations were determined using a nitric oxide detection assay following incubation with a chicken macrophage line and the direct concentrations were determined using a commercial ELISA (Invitrogen, La Jolla, CA) (Ariaans et al, 2008; Lambrecht et al, 2004). Each ex vivo stimulation assay was conducted in duplicate.…”