Mice and infection
Female specific-pathogen-free (SPF) BALB/c mice (6–8 weeks of age) were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Liaoning, China). Mice had fresh water and autoclaved food on a weekly schedule and maintained in individual filter cages at the Laboratory Animal Center, China Medical University. The animal protocol was approved by the Institutional Animal Care and Use Committee of China Medical University. Human RSV type A2 (RSV A2) strain was grown and assayed for infectivity (expressed as a 50% tissue culture infectious dose, TCID 50) in Hep2 cells (ATCC) as previously described[14]. Mice were inoculated intranasally with RSV at an inoculum dose of 2 × 106 TCID 50 in 20 μL of sterile PBS per mouse. Mice were inoculated intranasally with sterile PBS as a control group (expressed as day 0 in figures).
Lung cell preparation
Mice were anesthetized and the pulmonary circulation was flushed with ice-cold PBS to remove intravascular blood pool. Then, lung tissue was minced and incubated in RPMI 1640 (with 200 μg/mL collagenase D and 40 μg/mL DNase I) at 37 °C for 90 min on a rocker. The digested lung tissue was passed through a 70-μm stainless steel mesh, then red blood cells were lysed with 0.15 M ammonium chloride and 1 mM potassium bicarbonate, the lung cells were resuspended in RPMI 1640 medium.
Isolation of pulmonary ILC2s and splenic CD4+ T cells
Pulmonary ILC2s, defined as CD45+ linage- ST2+ cells were obtained using a MoFlo high-speed cell sorter (Beckman Coulter). Naïve CD4+ T Cells were purified using naïve CD4+ T Cell Isolation kit (130-104-453, Miltenyi). The purity of these isolated cells was > 85%.
Adoptive transfer experiment
For adoptive transfer experiment, lung cells from mice that were infected with RSV at day 3, were pooled, and CD45+ Lin− ST2+ cells, which were identified as ILC2s in this study, were isolated. Then 1 × 105 of ILC2s (purity was > 86%) in 100 μL of PBS were transferred by intravenous injection 2 h before RSV infection. Samples were collected at day 6 after RSV infection.
Real-time PCR
ILC2s were sorted from the lungs of RSV-infected mice at various time points. The total RNA was isolated from the lung homogenates or purified ILC2s using TRIzol reagent (Life Technologies) and converted to cDNA with a SuperScript III Reverse Transcriptase using Oligo (dT) primers (Life Technologies). Quantitative real-time PCR was performed using SYBR Green Master Mix (Life Technologies). Primers used for the detection of expression of mRNAs for IL-4, IL-5, IL-13, IFN-γ, MHC II, CD80 and CD86 were below:
IL-4-F: 5´- TGTACCAGGAGCCATATCCA-3´, IL-4-R: 5´- TTCTTCGTTGCTGTGAGGAC-3´; IL-5-F: 5´- GGCTTCCTGTCCCTACTCAT-3´, IL-5-R: 5´- TCCTCGCCACACTTCTCTTT-3´; IL-13-F: 5´- AGCATGGTATGGAGTGTGGA -3´, IL-13-R: 5´- TTGCAATTGGAGATGTTGGT -3´; IFN-γ-F: 5´- TATCTGGAGGAACTGGCAAA -3´, IFN-γ-R: 5´- GGTGTGATTCAATGACGCTT -3´; MHC II-F: 5´-TCTGATTCTGGGGGTCCTCG -3´, MHC II -R: 5´-ATAGGTGCCTACGTGGTCGG -3´; CD80-F: 5´-ACCCCCAACATAAACTGAGTCT -3´, CD80-R: 5´-TTCCAACCAAGAGAAGCGAGG-3´; CD86-F: 5´-TGTTTCCGTGGAGACGCAAG -3´, CD86-R: 5´-TTGAGCCTTTGTAAATGGGCA-3´; β-actin-F: 5´-CAACGAGCGGTTCCGATG-3´, β-actin-R: 5´-GCCACAGGATTCCATACCCA-3´.
Reactions were run using a 7500 Real-Time PCR System ( Applied Biosystems ) under identical amplification conditions. Results are normalized to β-actin and presented as fold mRNA expression (fold change = 2-△△CT).
Flow cytometry
Lung cells were first blocked for Fc receptors with Fc Block (CD16/32, BD Bioscience) and then surface-stained with fluorescein-conjugated antibodies in PBS for 30 min at 4 °C under light protection. For intracellular cytokine staining, naïve CD4+ T cells were isolated from the lungs of mice and stimulated with PMA (25 ng/mL, Sigma) and ionomycin (1 μg/mL, Sigma) for 4.5 h at 37 °C. Brefeldin A (10 μg/mL, Sigma) was added for the last 4 h of culture. After incubation, intracellular staining was performed according to the manufacturer's instructions (BD Pharmingen). Antibodies used were as follows: anti-IFN-γ-PE (Biolegend), anti-MHC II-APC/cy7 (Biolegend), anti-CD80-BV421 (Biolegend), anti-CD86-PE/cy7 (Biolegend), anti-CD3-FITC (Biolegend), anti-CD4-PerCP (Biolegend), anti-CD69-APC/cy7 (Biolegend), anti-IL-4-PE (Biolegend), anti-IL-5-BV421 (eBioscience), anti-IL-13-PE/cy7 (eBioscience), anti-CD45-APC (eBioscience), anti-ST2-PE (eBioscience), and FITC-conjugated anti-lineage antibodies (eBioscience). The lineage marker antibody cocktail was composed of antibodies against: DX5 (or NK1.1), CD3, CD4, CD5, CD8, CD11b, CD19, B220, Gr-1 and TCRδ (BD Bioscience).
Neutralizing Antibody: Anti-mouse MHC Class II (clone: M5/114; InVivoMAb grade; BioXcell, BE0108), anti-mouse CD4 (α-CD4; clone: GK1.5; InVivoMAb grade; BioXcell, BE0003).
ELISA
To measure the protein levels of cytokines in BALF. Supernatant of BALF were measured with ELISA kits (R&D Systems) including IL-4, IL-5, IL-13 and IFN-γ according to the manufacturer's instruction. The absorbances at 450 nm were determined by a microplate reader.
Histopathological examination
Bronchoalveolar lavage fluids (BALF) were collected and centrifuged. Differential counting of cells in the pellet was performed on cytospins. For lung histology, lung tissue sections were processed for hematoxylin and eosin (H&E) staining.
Co-culture experiment
2 × 105 of pulmonary ILC2s from the mice on day 3 after RSV infection and equal numbers of splenic naïve CD4+ T cells from normal mice, were co-cultured in vitro in the presence or absence of 0.5 μg/mL anti-MHC II mAbs. The cells were cultured in a 24-well plate in 500 μL RPMI-1640 medium containing 10% FBS with the presence of IL-2 (20 ng/mL) 、IL-7 (20 ng/mL) and IL-33 (20 ng/mL) for 72 h, the flow cytometry was used to detect the proliferation of CD4+ T cell in the co-cultures.
Statistical analysis
Differences between control and experimental groups were compared using one-way analysis of variance (ANOVA) to calculate the statistical significance (GraphPad Prism software, version 8.0). P values < 0.05 were considered significant.