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

We aim to establish an in vitro protocol to culture H5N1-specific duck T cells and quantify IFN-γ secretion using intracellular cytokine staining. Recent studies highlight the importance of avian T cell immunity in controlling viral infections, but standardized culture methods are still lacking for ducks. We successfully cultured H5N1-specific duck T cells and developed a novel flow segmental-based ICS protocol for detecting duck IFN-γ expression. We will explore antigen-specific T cell responses in other poultry species and develop avian immunological tools for vaccine evaluation and antiviral research.

[Narrator] To begin, isolate memory peripheral blood mononuclear cells, or PBMCs, from ducks that were infected with H5N1 virus 28 days earlier, and adjust the concentration of the isolated cells to 3 times 10 to the power of 6 cells per milliliter. Now, dispense 1 milliliter of the medium to each well of a 48-well plate. Then, add the H5N1 avian influenza virus to the PBMCs at a multiplicity of infection of 5 to co-infect the cells, and incubate the co-culture for one hour at 37° Celsius. Every 15 minutes, gently shake the plate by hand to ensure even distribution. After one hour of infection, wash the PBMCs twice with PBS to remove unbound virus particles. Resuspend the washed PBMCs in 1 milliliter of T-cell culture medium and incubate the suspension at 37° Celsius for five hours. Next, centrifuge the cells at 440 x g for five minutes at room temperature. Resuspend the pelleted antigen-presenting cells, or APCs, in 100 microliters of T-cell culture medium, and add the resuspended APCs to the effector cells at a 1 to 5 ratio. Now, incubate the co-culture in a 5% carbon dioxide atmosphere at 37° Celsius for 14 days. Every two days, remove half of the supernatant with a pipette. Discard the used medium containing cells and add an equal volume of fresh T-cell medium. On day seven, observe the cells under an optical microscope at 100x. Treat a separate set of memory APCs with PBS and use these as the unstimulated control group. On day seven, remove the cultures of H5N1-specific T cells from the incubator and harvest all cells from each well in a tube. Now, add the incubated APCs and brefeldin A at a 1 to 1000 dilution to the effector cells and co-incubate for six hours in a 39° Celsius incubator. Then, transfer the cells to a clean centrifuge tube and spin it at 440 x g for five minutes at room temperature. After centrifugation, discard the supernatant. Add 100 microliters of the antibody cocktail containing mouse anti-duck CD8 antibody to the cells, and incubate for 30 minutes in the dark at 4° Celsius. Resuspend the pellet in 1 milliliter of PBS. Centrifuge the cells at 400 x g for five minutes at 4° Celsius. After discarding the supernatant, add FITC-conjugated goat anti-mouse IgG2b antibody, and incubate for 30 minutes in the dark at 4° Celsius. Then, spin the cells again at 400 x g for five minutes at 4° Celsius. After discarding the supernatant, resuspend the cells in 100 microliters of fixation buffer and incubate the mixture for 20 to 25 minutes in the dark at 4° Celsius. Then, wash the cells twice with 1 milliliter of 1x permeabilization buffer as demonstrated earlier. Then, discard the supernatant and resuspend the cells in 100 microliters of permeabilization buffer. Incubate the cells with mouse anti-duck IFN-γ antibody diluted 1 to 10 for 30 minutes in the dark at 4° Celsius. After washing the cells, add 100 microliters of PE-conjugated goat anti-mouse IgG3 antibody diluted 1 to 250, and incubate for 30 minutes in the dark at 4° Celsius. Finally, analyze the processed samples using FlowJo software. After co-culture with antigen-presenting cells, virus-specific T cells exhibited cluster growth, indicative of successful proliferation. Carboxyfluorescein succinimidyl ester labeling revealed multiple peaks of cell division in stimulated samples, confirming extensive proliferation of duck memory T cells. The proportion of CD4+ and CD8+ T cells was significantly higher in H5N1-stimulated cultures than in unstimulated controls after 14 days. Proliferating T cells on day seven of culture showed elevated expression of cytotoxic-associated genes, such as granzyme A and interferon-gamma, indicating a cytotoxic phenotype. Flow cytometry revealed a significant increase in the proportion of interferon-gamma-positive cells within both CD8 high and CD8 low T cell populations following antigen stimulation.