Twenty-nine specific-pathogen-free (SPF) White Leghorn chickens (layer chickens) of 20-to-40-week-old (Charles River Laboratories, MA, USA) were housed in the SPF area of FARVET company and fed ad libitum with sterilized feed and water. Prior to the experiments, the animals were maintained healthy and were employed to supply the embryonated eggs used in vaccine manufacturing. On the day of the experiment, the animals were euthanized by cervical dislocation without anesthesia following the American Veterinary Medical Association (AVMA) guidelines. The procedure was performed by a trained veterinarian.
Experiments that required statistical analysis were performed 3 or 4 times (mentioned in the figure legends). The cells from 1 animal were analyzed per experiment.
Isolation and culture of mononuclear spleen cells
Spleens were collected aseptically from the chickens and immediately placed in a tube containing 5 ml of sterile RPMI-1640 medium (Sigma-Aldrich, MO, USA, Catalog # R7755-10 L). Subsequently, the tube was transported on ice to the laboratory. The spleen was perfused with 10 ml of RPMI-1640 supplemented with 10% fetal bovine serum (FBS - HyClone, GE Healthcare, UT, USA, Catalog # SV30180.03). To prepare single-cell suspensions, the splenocytes were strained through a 40 μm mesh into RPMI-1640 culture medium containing 5% FBS. The resulting cell suspension was pelleted by centrifugation for 5 min at 300 ×g and resuspended in 4 ml of D-PBS (Sigma Aldrich, Catalog # D5773-50 L). Mononuclear cells were isolated by density gradient centrifugation for 30 min at 400 ×g using Histopaque 1.078 (Sigma Aldrich, Catalog # 10771). Then, the cells washed twice with D-PBS (300 ×g for 10 min), were resuspended in 2 ml of FARMEM medium (Industrial Secret-FARVET company). An aliquot of cell suspension was mixed with 0.4% trypan blue solution (Sigma-Aldrich, Catalog # 93595-50ML). Through the trypan blue exclusion method, and using a Neubauer chamber the cells were counted, being the cellular viability between 90 and 95%. The cellular concentration was then adjusted to 10 × 106 cells/ml in the FARMEM medium. One hundred microliters of cells were seeded on P96 round-bottom plates and cultured with 5% CO2 atmosphere at 41 °C for 3 days in the presence or absence of 100 μl of 1 μg/mL of ConA (Sigma Aldrich, Catalog # C5275). All procedure was performed under sterile conditions in a biosafety cabinet (class II cabinet).
EdU powder was purchased from Thermo Fisher Scientific (MA, USA, Catalog # A10044), dissolved in dimethyl sulfoxide (Sigma Aldrich, Catalog # D4540) at 10 mM concentration, aliquoted, and stored at − 20 °C. EdU previously diluted in cell culture medium was added at a final concentration of 10, 25, or 50 mM at 4, 8, or 16 h before the end of the culture.
Recovery, fixation, and cell permeabilization
To detach the cells from the plastic, 20 μL of 20 mM EDTA (Calbiochem, CA, USA, Catalog # 324503) in D-PBS buffer (pH 7.4), was added and incubated for 20 min at room temperature . The cells, recovered by pipetting and aspiration, were fixed in 100 μL of 2% formaldehyde (Sigma-Aldrich, Catalog # 1040032500) in D-PBS buffer (pH 7.4) for 15 min at 4 °C and washed twice with 1 ml of D-PBS containing 5% FBS followed by centrifugation of 400 ×g for 5 min. To permeabilize the cells with Triton X-100 (Calbiochem, Catalog # 9400), the cells were resuspended in 100 μl of 0.5% or 0.05% Triton X-100 (prepared in D-PBS buffer, pH 7.4) and incubated for 15 min at room temperature. Subsequently, the cells were washed twice with 1 ml of D-PBS and centrifuged at 500 ×g for 5 min. Finally, the cells were resuspended in 50 μl of the click staining solution. The saponin reagent (Sigma-Aldrich, Catalog # S7900-100G) was part of the click staining solution, as described in the next section. In the optimized protocol, the fixed cells were permeabilized with 0.02% saponin by 1 h at room temperature. The cells were then washed with 1 ml of 0.02% saponin, centrifuged at 500 ×g for 5 min, and resuspended in 50 μl of the click staining solution.
The components of the Click reaction were as follows: EdU (described above) Copper (II) sulfate (Sigma-Aldrich, Catalog # C3036) diluted in water at 200 mM, Alexa Fluor™ 488 Azide (Thermo Fisher Scientific, Catalog # A10266) reconstituted in dimethyl sulfoxide at 6 mM, and fresh ascorbic acid (Sigma-Aldrich, Catalog # A5960-25G) dissolved in water at 1 M. The optimized staining solution was composed of 0.01% saponin prepared in D-PBS, pH 7.4 (3591 μl of the stock), 0.3 mM copper (II) sulfate (6 μl of the stock), 4 μM Alexa Fluor™ 488 Azide (2.7 μl of the stock), and 100 mM ascorbic acid (400 μl of the stock). The reagents were added in the same order as mentioned above, and the solution was mixed between additions. Subsequently, the fixed cells were resuspended in 50 μL of the staining solution and incubated for 20 min in the dark at room temperature. Finally, the cells were washed twice with 1 ml of D-PBS (500 ×g, 5 min). To identify the T cell subset, the cells were resuspended in 300 μl of D-PBS containing 5% FBS and incubated by 30 min in the dark at room temperature. Subsequently, the cells were centrifuged (500 ×g, 5 min) and prepared for the staining step.
Antibodies and flow cytometry reagents
Mouse Anti-Chicken CD4-Alexa Fluor® 647 (clone CT-4, Catalog # 8210–31), Mouse Anti-Chicken CD8α PE (clone 3–298, Catalog # 8405–09) were purchased from SouthernBiotech company (AL, USA). The viability determination reagent, 7AAD, was purchased from BD Biosciences (CA, USA, Catalog # 559925).
Fluorescent cell staining
Cells were labeled with directly conjugated monoclonal antibodies. Before cell staining, the cells were blocked for 10 min at 4 °C with 15 μl of 2.5% normal mouse serum (Abcam, MA, USA, Catalog # ab7486) prepared in ice-cold D-PBS containing 5% of FBS (FACS buffer). Subsequently, the cells were incubated with 35 μl of Anti-Chicken CD4 or Anti-Chicken CD8α antibodies diluted in ice-cold FACS buffer for 20 min at 4 °C. Cells were then washed with 500 μl of ice-cold FACS buffer followed by centrifugation at 300 ×g for 5 min (staining before click reaction) or 500 ×g for 5 min (staining after click reaction). Previously for flow cytometric acquisition, the cells were resuspended in 500 μl of FACS buffer and filtered through a 44 μM nylon mesh (Sigma-Aldrich, Catalog # NY4100010) into Falcon® 5 ml polystyrene round-bottom tubes (Corning, NY, USA, Catalog # 352054). All antibodies were titrated to the optimal concentration before use.
Flow cytometry was performed on FACSMelody (BD Biosciences, CA, USA), which is equipped with two lasers: 488 nm and 635 nm, and on FACSCanto II (BD Biosciences, CA, USA), which is equipped with three lasers: 488 nm, 635 nm and 405 nm. Cytometer Performance was checked with CS&T beads (BD) before each acquisition, according to the manufacturer’s instructions.
To establish the EdU protocol 30,000 events were acquired. In antigen-specific stimulation experiments 50,000 events were collected. The data were analyzed using FlowJo software v10.6.1 (BD Biosciences).
Four chickens from 16 weeks of age were inoculated with the vectorized vaccine FARMUNE® (HVT-IBDV-ILTV) on day 0 of age. To prepare the recall antigen, infectious bursal disease virus (IBDV) was obtained from infected cell cultures and was concentrated using a PEG kit (Abcam, Catalog # ab102538) according to the manufacturer’s instructions. Subsequently, the virus was heat-inactivated in a water bath (56 °C, 1 h) aliquoted and stored at − 20 °C. To evaluate the antigen-specific proliferation, spleen mononuclear cells isolated as described above were cultured in presence of 1 × 107 copies/ml of the inactivated IBVD. The IBDV concentration was previously determined by qPCR. To evaluate the basal proliferation the cells were cultured with only medium and as a positive control we used ConA at a final concentration of 1 μg/ml.
All quantitative data were analyzed using the software GraphPad Prism version 6.1 (GraphPad Software, San Diego, CA). Mann-Whitney test was utilized to assess the differences between groups. p ≤ 0.05 was considered statistically significant.