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

The protocol describes a method for culturing avian T cells in vitro by isolating memory T cells from infected ducks, allowing for the generation of highly purified specific duck T cells. Additionally, an intracellular cytokine staining (ICS) method was established to accurately measure IFN-γ secretion in duck T cells.

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.

The process of T cell activation, proliferation, differentiation, and conversion into memory T cells is central to antigen-specific immune responses¹. Initially, T cells are activated when their T cell receptors (TCR) recognize antigenic peptides presented on the surface of antigen-presenting cells (APCs) by major histocompatibility complex (MHC) molecules^2,3. This activation process requires not only the binding of TCR to MHC molecules but also the coordination of co-stimulatory signals, which are crucial for fully activating T cell functions⁴. Once activated, T cells quickly enter the proliferation phase, generating numerous clones that share the same antigen specificity as the original T cell. During this proliferation phase, T cells further differentiate based on their function, primarily into CD8⁺ T cells and CD4⁺ T cells⁵. These differentiated cells mediate cytotoxic killing and assist immune responses, respectively. Following the initial infection, a portion of the activated T cells transform into long-lived memory T cells⁶. These memory T cells can persist in the immune system for extended periods, rapidly responding to the same pathogen upon re-exposure. As a result, they generate a stronger and more efficient immune response, providing long-term immune protection for the host⁷. This entire process is critical for the design of T-cell vaccines, as it helps improve vaccine efficacy and durability⁸.

In this context, cultivating antigen-specific T cells in vitro becomes essential. This protocol was developed based on established methods for in vitro culture of human T cells, with necessary adaptations for the avian immune system⁹. By growing these cells outside of the body, researchers can study the T cell response under different immune states, such as activation, proliferation, differentiation, and memory formation¹⁰. This in vitro approach is invaluable for understanding the roles T cells play in immune responses. Furthermore, under in vitro culture conditions, the secretion of cytokines (such as IFN-γ) by T cells can be monitored^11,12. This monitoring is crucial for assessing the quality, intensity, and persistence of immune responses, as well as studying interactions between T cells and other immune cells. Additionally, in vitro expansion of antigen-specific T cells enables large-scale enrichment, increases the sensitivity of detection, and enhances the assessment of T cell response levels¹³. Thus, in vitro culture of antigen-specific T cells serves as a powerful tool to better understand the role of poultry T cells in immune responses, which increases the sensitivity of detection and enhances the assessment of T cell response levels. While similar in vitro methods have been well established in mammalian systems, the application of such techniques in avian species remains limited. This is especially relevant in poultry immunology, where tools for analyzing T cell responses are underdeveloped. Ducks, in particular, serve as natural reservoirs for several avian influenza viruses, yet little is known about their antigen-specific T cell immunity. Therefore, developing a standardized in vitro T cell culture protocol tailored to avian systems is essential for advancing both basic research and applied immunology in poultry.

A key cytokine involved in these processes is IFN-γ, a type II interferon primarily produced by CD8⁺ T cells, type 1 CD4⁺ T cells, and NK cells¹⁴. IFN-γ plays a crucial role in inhibiting viral replication and regulating immune responses¹⁵. The expression level of IFN-γ reflects the immune status and serves as a marker for T cell activation, enabling researchers to assess T cell response levels through its expression^16,17. One common method for detecting cytokine expression in immune cells is intracellular cytokine staining (ICS)^18,19. However, due to limitations in experimental materials and techniques, research on ducks has lagged behind that of mammals⁵. At present, many researchers rely on qPCR to measure IFN-γ expression levels, though this method has certain limitations¹¹. In our lab, we successfully developed an antibody for duck IFN-γ that is compatible with flow cytometry. Building on that success, in this study, we established an ICS method to detect IFN-γ protein expression in duck T cells, providing a reliable tool for further study on duck T cell responses.

All experiments with all available avian influenza A (H5N1) viruses were carried out in an animal biosafety level 3 laboratory and animal facility according to the protocols of South China Agricultural University (CNAS BL0011). All animal research projects were approved by the Institutional Animal Care and Use Committee (identification code 2021f154, July 29, 2021) of the South China Agriculture University. All animal procedures were performed according to the regulations and guidelines established by this committee and international standards for animal welfare. The animals used in this study were 4-week-old domestic mallard ducks (Anas platyrhynchos domestica), including both males and females, with body weights ranging from 500 to 600 g.

1. Duck PBMCs isolation and preparation of a single-cell suspension

1. Collect 2 mL of heparinized blood from the jugular vein of each duck and transfer into EDTA-containing tubes to prevent coagulation from each individual duck
2. Dilute the blood using the provided sample diluent from the lymphocyte isolation kit or PBS. Gently mix by pipetting with a Pasteur pipette. The usual dilution ratio is 1:1 for blood to diluent.
3. Transfer an equal volume of lymphocyte separation solution into a centrifuge tube as the diluted blood. Carefully layer the blood suspension on top of the lymphocyte separation solution. Centrifuge the mixture at 400 x g, 25 °C for 15 min, setting the falling acceleration to 1.
   NOTE: The amount of lymphocyte separation solution should not be less than 4 mL. The components of the lymphocyte separation solution kit can be found in the Table of Materials.
4. After centrifugation, observe the four distinct layers in the centrifuge tube from top to bottom. The top layer is the sample diluent, followed by the annular milky white lymphocyte layer, then the separation solution, and the bottom layer consists of red blood cells.
5. Carefully use a pipette to collect the second layer, the annular milky white lymphocyte layer, and transfer it into a new centrifuge tube. Add 10 mL of cleaning solution to mix with the cells.
6. Centrifuge the sample at 440 x g for 5 min at room temperature. Discard the supernatant, then resuspend the cell pellet in 10 mL of RPMI 1640 medium for cell counting.
7. Optional) To further purify the sample, after centrifuging at 440 x g for 5 min, use red blood cell lysis buffer to remove any remaining mixed blood cells.

2. In vitro H5N1 AIV-specific T cell culture

1. Purchase 2-week-old healthy mallard ducks (Sheldrake) from a duck farm and house them in negative-pressure isolators. Confirm that the ducks are negative for AIV by using hemagglutination inhibition (HI) assays prior to the experiment. Infect 4-week-old ducks with H5N1 AIV 1 x 10⁶, with 50% egg infectious dose [EID50]/0.2 mL intranasally.
2. Isolate memory PBMCs from H5N1-infected ducks 28 days post-infection, following the method described in step 1. Count cells using a hemocytometer. Dilute the cell suspension at a 1:1 ratio with 0.08% trypan blue and count viable (unstained) cells manually under a light microscope.
   NOTE: As supported by our previously published study²⁰, peripheral blood mononuclear cells (PBMCs) isolated at 28 days post-infection are considered to be enriched in memory T cells. At this stage, the effector phase has subsided, and memory cell populations are predominant.
3. Set the concentration to 3 x 10⁶ cells/mL, ensuring 1 mL of medium is added to each well of the 48-well plate.
4. Co-infect the isolated PBMCs with H5N1 avian influenza virus (AIV) to serve as APCs. The viral dose is MOI = 5. Co-culture the PBMCs with the virus for 1 h at 37 °C. Gently shake by hand every 15 min.
5. After 1 h of infection, wash the cells 2x with PBS to remove unbound viral particles. Resuspend the washed PBMCs in 1 mL of T cell culture medium and further incubate the suspended cells at 37 °C for 5 h.
6. Centrifuge the cells at 440 x g for 5 min at room temperature. Afterward, resuspend the incubated antigen-presenting cells (APCs) in 100 µL of T cell culture medium and add them to the effector cells. The ratio of antigen-presenting cells to effector cells should be 1:5.
7. Incubate the cells in a 5% CO₂ atmosphere at 37 °C for 14 days. Every 2 days, replace half of the cell culture supernatant with fresh T cell medium by carefully pipetting to ensure even distribution. Discard half of the medium containing the cells, then add an equal volume of new medium.
8. On day 7, observe the cells under an optical microscope at 100x.
9. Use PBS-treated memory PBMCs as the unstimulated control group.

3. Carboxy fluorescein Diacetate Succinimidyl Ester (CFSE) to monitor T cell proliferation

1. Adjust the cell density to 0.5-1 x 10⁷ cells/mL and resuspend the cells with 5 mL of PBS after centrifugation at 440 x g for 5 min.
2. Dilute 10 mM stock to 1 µM CFSE with 5 mL of pre-chilled PBS; operate in the dark.
3. Tilt the two centrifuge tubes at 45°, add the CFSE dilution to the cell suspension with a Pasteur pipette, mix well, and place in a 37 °C water bath for 15 min in the dark.
4. Wash cells by diluting them in 10 volumes of pre-cooled PBS containing 5% FBS, sediment by centrifugation at 440 x g for 5 min, and discard the supernatant. Repeat the wash 2x.
5. Stimulate cells labeled with CFSE in vitro as per step 2. At the completion of the proliferation assay, harvest cells and analyze by flow cytometry.

4. Flow cytometric analysis for proliferation of CD8⁺ T and CD4⁺ T cells

1. After 7 days of culture, collect all the cells from each well and divide them into two flow tubes for analysis.
2. Transfer the cells to a new centrifuge tube, centrifuge at 440 x g for 5 min at room temperature, and discard the supernatant.
3. Add 100 µL of antibody cocktail (mouse anti-duck CD8, 1:50 or mouse anti-duck CD4, 1:50) and incubate the cells for 30 min in the dark at 4 °C.
4. Transfer the cells to a new centrifuge tube, centrifuge at 440 x g for 5 min at room temperature, and discard the supernatant.
5. Add 100 µL of antibody cocktail (FITC-conjugated Goat Anti-Mouse IgG2b, 1:50) and incubate the cells for 30 min in the dark at 4 °C.
6. Wash the cells once with 1 mL of PBS by centrifuging at 400 x g for 5 min at 4 °C. Analyze the data using FlowJo software. The gating strategy is shown in Supplementary Figure 1.

5. T-cell response by signature gene expression assays using qPCR

1. Count cells at the end of T-cell culture, collect in 1.5 mL centrifuge tubes, and centrifuge at 400 x g for 5 min. Discard the supernatant carefully.
2. RNA extraction
   NOTE: Extraction can be performed in a fume hood or ultra-clean bench to prevent RNA contamination.
   1. Lyse the cells using the lysis buffer provided in the kit as per the manufacturer's instructions. Transfer lysed sample to gDNA-Filter Columns (pre-placed in collection tubes), centrifuge at 13,400 x g for 30 s, discard columns, and collect the filtrate. Add 0.5 volume of absolute ethanol to the filtrate. Mix thoroughly.
      NOTE: A cloudy solution or precipitate after ethanol addition is normal. Shake and proceed to the next step.
   2. Transfer the mixture to RNA columns, centrifuge at 13,400 x g for 30 s. Discard filtrate, add 700 µL of Buffer RW1, and centrifuge again.
   3. Discard filtrate, add 700 µL of Buffer RW2 (with absolute ethanol), and centrifuge. Discard filtrate, add 500 µL of Buffer RW2 (with absolute ethanol), and centrifuge for 2 min. Carefully remove the column from the tube to avoid contamination from the filtrate. Ensure all rinse solutions are removed to prevent interference in downstream reactions.
   4. Transfer column to a new RNase-free collection tube, add 50-200 µL of RNase-free ddH₂O, let it sit at room temperature for 1 min, then centrifuge at 13,400 x g for 1 min to elute RNA. Measure RNA purity and concentration.
      NOTE: Preheat RNase-free ddH₂O to 65 °C for increased yield and perform a second elution if needed.
3. Synthesize cDNA following the kit instructions. Synthesize the cDNA strand as per the manufacturer's instructions, adding 5x the amount of reverse transcriptase. Reverse transcribe at 37 °C for 15 min, then 85 °C for 5 s, and cool to 4 °C.
4. Set up PCR reaction according to the manufacturer's protocol.
   1. In a qPCR tube, combine 10 µL of 2x PCR reaction enzyme mix, 0.4 µL of primer 1, 0.4 µL of primer 2, 1 µL of cDNA template, and 8.2 µL of ddH₂O to make a 20 µL mix.
   2. Run PCR in the real-time quantitative PCR instrument with the following program:
      Stage 1: 95 °C for 30 s, Rep x 1
      Stage 2: 95 °C for 3 s, 60 °C for 30 s, Rep x 35
      Stage 3: 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 s, Rep x 1

6. Intracellular Cytokine Staining (ICS)

NOTE: This protocol was developed to assess the effector response of H5N1-specific CD8⁺ T cells by detecting intracellular IFN-γ secretion in ducks.

1. Culture H5N1-specific T cells in vitro following the procedure described in step 2. Harvest all cells (from each well) after 7 days of culture initiation.
2. Incubate the antigen-presenting cells (APCs) as outlined in step 2. Add the incubated APCs and Brefeldin A (1:1,000) to the effector cells and co-incubate for 6 h in a 39 °C incubator.
3. Transfer the cells to a new centrifuge tube, centrifuge at 440 x g for 5 min at room temperature, and discard the supernatant.
4. Add 100 µL of antibody cocktail (mouse anti-duck CD8, 1:50) to the cells and incubate for 30 min in the dark at 4 °C.
5. Wash the cells by centrifuging at 400 x g for 5 min at 4 °C and resuspend them in 1 mL of PBS. Add an antibody cocktail (FITC-conjugated Goat Anti-Mouse IgG2b, 1:50) and incubate for 30 min in the dark at 4 °C.
6. Wash the cells again by centrifuging at 400 x g for 5 min at 4 °C. Discard the supernatant, resuspend the cells in 100 µL of fixation buffer, and incubate for 20-25 min in the dark at 4 °C.
7. Wash the cells once more by centrifuging at 400 x g for 5 min at 4 °C. Add 1 mL of 1x permeabilization buffer and wash the cells 2x by centrifugation at 440 x g for 5 min at 4 °C.
8. Discard the supernatant, resuspend the cells in 100 µL of permeabilization buffer, and add the antibody (Mouse Anti-Duck IFN-γ, 1:10) to the cells. After fixation and membrane permeabilization, incubate the cell-antibody mixture for 30 min in the dark, occasionally shaking during the incubation at 4 °C.
   NOTE: The Permeabilization/Wash solution is a 10x stock solution and must be diluted with PBS before use.
9. Wash the cells once more with 1 mL of PBS by centrifuging at 400 x g for 5 min at 4 °C. Add 100 µL of antibody cocktail (PE-conjugated Goat Anti-Mouse IgG3, 1:250) and incubate for 30 min in the dark at 4°C.
10. Analyze the data using FlowJo software. The gating strategy is shown in Figure 1.

This protocol was developed based on an earlier study on the detection of antigen-specific T cell effector responses in ducks²⁰. The first step of the experiment involves the in vitro culture of virus-specific T cells, which serve as the foundation for subsequent effector response studies. Initially, APCs were incubated and co-cultured with effector cells. Morphological observations revealed that after proliferation, the cells exhibited cluster growth (Figure 2A). CFSE labeling further confirmed successful proliferation by showing multiple peaks of cell division (Figure 2B). Finally, we used duck-specific cell markers CD8/CD4 to assess the proportion and absolute numbers of cells²⁰, confirming the successful in vitro culture of antigen-specific T cells (Figure 2C).

On day 7 of the culture, we collected the cells and assessed the expression of immune-related genes. We found that the proliferating T cells predominantly expressed cytotoxic-associated genes such as Granzyme A and IFN-γ^11,20 (Figure 3), suggesting that they primarily contribute to cytotoxic responses. To further investigate this effector response, we established a duck-specific staining protocol to quantitatively detect IFN-γ secretion by duck T cells. Flow cytometry was used to fix, permeabilize, and label CD8 T cells from the proliferated population with an anti-IFN-γ antibody. The results showed a significant upregulation in the proportion of IFN-γ⁺ cells in both CD8^high+T and CD8^low+T populations following antigen stimulation (Figure 4), indicating a robust effector response in the CD8⁺ T cells generated by proliferation.

Figure 1: Gating strategy of CD4⁺/CD8⁺T cell. (A) Gating and analysis of the percentage and phenotype of CD4⁺ T cells between H5N1-stimulated and unstimulated cells after 14 days of culturing. (B) Gating and analysis of the percentage and phenotype of CD8⁺ T cells between H5N1 stimulated and unstimulated cells after 14 days of culturing. This figure has been modified from²⁰. Please click here to view a larger version of this figure.

Figure 2: In vitro culture of H5N1 AIV-specific duck T cells. (A) Morphological analysis of memory PBMCs with or without stimulation by H5N1 AIV. Two independent experiments were conducted using two different duck memory PBMC donors. (B) Proliferation of CFSE-labeled memory PBMCs was evaluated by CFSE dilution in H5N1-stimulated cells from H5N1-infected ducks after 2 weeks of culture. The red sample represents CFSE-labeled memory PBMCs without stimulation, while the yellow, green, and black samples represent CFSE-labeled memory PBMCs stimulated with H5N1 after 7, 8, and 14 days of culture, respectively. (C) The percentage of CD4⁺ and CD8⁺ T cells was analyzed between H5N1-stimulated and unstimulated cells after 14 days of culture. Statistical significance was determined using an unpaired t-test. (D) The number of CD4⁺ and CD8⁺ T cells was compared between H5N1-stimulated and unstimulated cells after 14 days of culture. Data on T cell percentages and numbers were obtained from two independent experiments with two replicates each. Statistical analysis was performed using an unpaired t-test ns p > 0.05, *p < 0.05, **p < 0.01. This figure has been modified from²⁰. Please click here to view a larger version of this figure.

Figure 3: Detection of the H5N1 AIV-specific duck T cell response by qRT-PCR. The data was collected from three replicates in H5N1 stimulated group and unstimulated group, respectively. The results were presented as means ± SEM, and the paired t-test was used for statistical comparison. *p < 0.05, **p < 0.01. This figure has been modified from²⁰. Please click here to view a larger version of this figure.

Figure 4: Flow cytometry analysis of IFN-γ expression in CD8⁺ T cells from duck PBMCs stimulated. (A) ICS gating strategy for the stimulated group and the control group. (B) Statistical analysis of IFN-γ expression in CD8^low+ and CD8^high+ cells from duck PBMCs after stimulation. The difference in IFN-γ expression between groups was assessed by t-test, and comparisons were considered significant at p≤ 0.05. This figure has been modified from²⁰. Please click here to view a larger version of this figure.

Supplementary Figure 1: Gating strategy used for CD4⁺ and CD8⁺ T cells. Please click here to download this File.

This protocol provides an efficient method for the in vitro culture of duck antigen-specific T cells and also establishes an intracellular cytokine staining protocol for ducks, which can be used to assess the effector responses of T cells. Currently, there are no published reports on in vitro culture protocols for duck T cells. We primarily referenced protocols for human antigen-specific T cells but are committed to optimizing the incubation conditions for APCs. We are considering further optimization of our differentiation protocol.

There are two critical steps in this protocol that are essential for its success. First, the in vitro culture of duck T cells requires careful optimization of stimulation conditions, including antigen dose, APC activation, and culture duration, to ensure sufficient T cell expansion and functional preservation. Any deviation in these parameters may lead to suboptimal activation or cell death. Second, ICS assay is a key technique for functionally evaluating IFN-γ expression. Successful ICS requires precise control of stimulation time, fixation/permeabilization steps, and antibody specificity. Both steps are highly sensitive and must be carefully executed to achieve reliable and reproducible detection of antigen-specific T cell responses.

Here, we used flow cytometry to detect cell proliferation and qPCR to examine the production of hallmark cytokines. Most importantly, we successfully expressed and purified a hybridoma cell line producing mouse anti-duck IFN-γ antibodies, which were used to detect IFN-γ expression, thereby evaluating the effector response level of T cells. With a few minor modifications, this approach could also be adapted for cell proliferation assays in other species, such as chickens.

We used a peripheral blood mononuclear cell (PBMC) isolation kit to isolate duck PBMCs because the kit is both effective and time-saving. Other separation methods, such as the Precoll separation method, can also²¹ achieve the goal of isolating duck PBMCs through different steps. However, compared to the kit, these methods take more time.

Some issues may arise during the execution of this protocol. Firstly, T cell proliferation upon viral stimulation is primarily a response of memory T cells^22,23,24. To ensure proper T cell proliferation, we recommend using ducks that are 4-10 weeks post-infection. Currently, there are various methods available for isolating duck PBMCs, and the efficiency of PBMCs isolation may vary depending on the separation medium used. It is recommended to use a widely certified separation medium and to extract as many lymphocytes as possible²⁵.

Secondly, flow cytometry detected that the proportion of CD8⁺ T and CD4⁺ T cells in the proliferation was both less than 15%, which may be because the T cells cultured in this protocol mainly come from PBMCs, in which the proportion of CD8⁺ T and CD4⁺ T cells is inherently low. Future experiments using spleen tissue may yield more significant results.

Thirdly, during the culture process, many T cell clusters may not form, and some cells may die during T cell differentiation. This phenomenon is more pronounced in the unstimulated control group. This issue may be due to the fact that, as T cells are cultured, the unstimulated group does not receive activation and co-stimulation signals, directly leading to programmed cell death.

Fourth, we constructed a hybridoma cell line and successfully expressed and purified a large amount of mouse anti-duck IFN-γ antibodies. Using these antibodies, we developed an intracellular cytokine staining protocol for detecting duck IFN-γ. Next, we tested the sensitivity of the antibodies using ELISA and optimized the staining concentration for flow cytometry, ultimately determining the optimal concentration to be 1:10, paired with PE-Goat Anti-Mouse IgG3 as the secondary antibody at a dilution of 1:250. The experimental results showed that the expression of IFN-γ in the stimulated group was 2%-3%. The relatively low proportion of IFN-γ-producing cells may be attributed to the fact that the T cells were only stimulated once. A single round of antigen stimulation may not be sufficient to expand the population of antigen-specific T cells to a detectable level. Repeated rounds of antigen stimulation can enrich antigen-specific T cells and enhance their activation, thereby increasing the proportion of IFN-γ-positive cells. Furthermore, after just one stimulation, the responding T cell population is likely still polyclonal. Through repeated stimulation and selection, a more clonally enriched T cell population can be obtained, which tends to exhibit higher levels of IFN-γ production^26,27.

In conclusion, the current protocol describes a method for inducing the proliferation of duck antigen-specific T cells in vitro and establishes an intracellular staining protocol for detecting duck IFN-γ. This approach enhances our understanding of duck T cell immune responses, offers a reliable tool for evaluating T cell responses, and contributes to advancing research in avian viral immunology.

The authors have nothing to disclose.

This work was supported by National Natural Science Foundation of China (32473060 and 32461120064) (to MD and ML); Guangzhou Basic and Applied Basic Research Project (2025A04J5445) (to MD); the Young Scholars of the Yangtze River Scholar Professor Program (2024, Manman Dai); and the Young Peal River Scholar of "Guangdong Special Support Plan"(2024, Manman Dai). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.