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

• DOI: 10.1016/j.dci.2008.05.007
• Publication Date: 2008-06-12T13:55:39Z
• Authors: M.P. Ariaans; P.M. van de Haar; J.W. Lowenthal; W. van Eden; E.J. Hensen; L. Vervelde
• Keywords: ELISPOT; Polyclonal antibodies

The 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 interferon-gamma (ChIFN-gamma) 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-gamma 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-gamma production by CD4+ and CD8+ cells.