Mice were sacrificed by cervical dislocation at 28 days after infection. The lungs and spleens were collected and cut with scissors, then digested by pancreatin at 37°C with continuous agitation for 1 hour in a temperature-controlled water bath. After digestion, digested tissues were filtered through a sheet of mesh (70 μm) screen into a 15 ml centrifuge tube for single-cell suspension; the tissues left were homogenized by grinding and also filtered into the 15 ml centrifuge tube. After washed by 1 ml RPMI-1640 (Gibco Invitrogen, Carlsbad, USA), the cells were resuspended with RPMI-1640 (supplemented with 10% calf serum, 100 U/l penicillin, and 100 U/l streptomycin) and diluted to 5 × 106 cells/ml.

The isolated single-cell suspension was incubated at 37°C in a 5% CO2 incubator with 1 μl/ml Brefeldin A (BFA, BD) for 5 hours. Then, cells were washed with RPMI-1640 and resuspended in flow cytometry staining buffer for surface marker staining. The cells were incubated with antimouse CD3-Pacific Blue (Clone 17A2, BioLegend) (1 : 200), CD4-BV510 (Clone RM4-5, BD Horizon™) (1 : 200), and CD69-BV605 (Clone H1.2F3, BioLegend) (1 : 100) antibodies for 30 minutes in the dark at 4°C. After surface marker staining, cells were permeabilized with fixation/permeabilization buffer (Transcription Factor Buffer Set, BD Pharmingen™) for 40 minutes at 4°C, then incubated with antimouse IFN-γ-BV711 (Clone XMG1.2, BD Horizon™) (1 : 100), β-catenin-Alexa 488 (Clone 14/Beta-Catenin, BD Transduction Laboratories™) (1 : 100) and TNF-α-PE-Cy7 (Clone MP6-XT22, BioLegend) (1 : 100) antibodies for 30 minutes in the dark at 4°C for intracellular cytokines staining; then, cells were washed by perm/wash buffer (included in Transcription Factor Buffer Set).

On account, β-catenin is a nuclear transcription factor, which expresses in both the cell cytoplasm and nucleus. Transcription Factor Buffer Set was used in this study for intracytoplasmic and intranuclear staining of β-catenin.

BD Canto II flow cytometer (BD Biosciences) was used to analyze the expression of surface markers and intracellular cytokines, and at least 10,000 events were collected. The results were analyzed using the FlowJo version 10.0.7 software (Tree Star, Ashland, OR, USA).

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