<em>In Vitro</em> Culture for H5N1-Specific Duck T Cells and Detection of Immune Responses Using Intracellular Cytokine Staining Method

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no established protocols for culturing avian T cells in vitro have been reported to date. In this study, we present a protocol for the first time, utilizing the highly pathogenic avian influenza (HPAIV) H5N1 virus as a model. Here, 4-week-old ducks were infected with the virus, and memory T lymphocytes were isolated 28 days post-infection to culture H5N1-specific duck T lymphocytes, enabling the investigation of the specific immune response to the H5N1 virus. Key steps in the protocol include isolating memory peripheral blood mononuclear cells (PBMCs) from infected ducks, activating antigen-presenting cells (APCs) with an optimal multiplicity of infection (MOI = 5) for 6 h, and preparing T cell culture media supplemented with recombinant IL-2 and other specific additives. T-cell proliferation, cytokine secretion, and cytotoxic activity were closely monitored throughout the process. Additionally, we established an intracellular cytokine staining protocol to quantify IFN-γ secretion in duck T cells. This involved generating a hybridoma cell line expressing IgG3κ antibodies specific to duck IFN-γ, followed by successful antibody purification. The purified antibody was diluted 1:10 and applied in flow cytometry to precisely measure IFN-γ secretion. This method offers a reliable tool for evaluating duck T cell responses and lays the foundation for future investigations into avian viral immunology.