A novel method to analyze viral antigen-specific cytolytic activity in the chicken utilizing flow cytometry.

In order to overcome some of the main drawbacks that have emerged in the conventional assays for cytotoxic T-lymphocytes (CTLs) in the chicken, a novel approach to analyze viral antigen-specific cytolytic activity utilizing flow cytometry was developed. In this method, the target cells were distinguished from the effector cells by pre-labelling them with a fluorescent dye PKH67. Cell death was detected with propidium iodide which labels the DNA of damaged cells. Flow cytometric assay also enables phenotyping of the effector cells by direct or indirect immunofluorescence staining of lymphocyte surface molecules. The results showed that specific cytotoxic T cells were observed in the blood of chickens primed with fixed avian reticuloendotheliosis virus strain T transformed MHC-compatible B cells. Phenotypic analysis of the effector cells from blood demonstrated CTL activity both in CD8+ and CD4+ T cell populations and the majority CTLs were TCR2+ cells.