We first investigated whether the H9N2 virus infected and replicated in RPMECs using immunofluorescence staining and a plaque-forming assay. Then, we quantified PD-L1 expression in RPMECs induced by H9N2 virus infection using RT-PCR and flow cytometry. Then, the effect of the induction of PD-L1 expression on the function of T cells was observed by using a coculture system established in transwell chambers with 6-well inserts. Finally, we investigated the levels of TNF-α and IFN-γ in the culture supernatants of RPMECs infected with the H9N2 virus.
Isolation of RPMECs and T cells
RPMECs were isolated from the lungs as previously described with some modifications . Briefly, the excised lung tissues from seven-day-old specific pathogen-free (SPF) F344 rats were rolled on dry filter paper to remove the mucosal layer, washed with phosphate buffered saline (PBS), and minced in fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). The tissue mass was then seeded in a culture plate, and the excess serum was discarded. The cells that migrated from the tissue blocks were digested with trypsin, washed with PBS, and incubated with a FITC-conjugated anti-CD31 antibody (Abcam, Cambridge, UK). RPMECs were purified by flow cytometry and cultured in EC basal medium (EBM; Lonza, Basel, Switzerland) supplemented with 20% FBS (Gibco, Carlsbad, CA, USA) and 10 ng/mL VEGF165 (PeproTech, NJ, USA). The purity of the endothelial cells was verified by staining for vascular endothelial growth factor receptor 2 (VEGFr2) under observation of a laser scanning confocal microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany). In brief, the cells were seeded on the bottom of a glass dish and fixed with methanol-acetone (1:1) for 20 min at room temperature. After being rinsed with PBS, the cells were incubated with a polyclonal rabbit antibody against rat VEGFr2 (Abcam, Shanghai, China) at 37 °C for 45 min, washed three times with PBS. and incubated with a FITC-labeled goat anti-rabbit secondary antibody (Origene, Rockville, MD, USA) at 37 °C for 30 min. The cell nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride; Cell Signaling Technology, Danvers, MA, USA).
To isolate rat T cells, blood was collected from F344 rats, and peripheral blood mononuclear cells (PBMCs) were isolated by polysucrose and sodium diatrizoate density gradient separation (Sigma Aldrich, Shanghai, China) as previously described . The PBMCs were incubated in RPMI 1640 medium containing 10% FBS at 37 °C under 5% CO2 for 2 h to facilitate monocyte adhesion. Negative selection enrichment columns (R&D Systems, MN, USA) were then used to enrich T cells according to the manufacturer’s protocol. The isolated T cells were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS for 72 h, and cells isolated from the unvaccinated rats were cultured in the same medium containing 5 μg/mL CD3 (Santa Cruz Biotechnology, Dallas, USA), 5 μg/mL CD28 (Abcam, Cambridge, UK) and 10 μg/ml PHA (Sigma Aldrich, Shanghai, China) for 72 h. To activate the isolated T cells, F344 rats were infected with 100 μL virus solution (2 × 107 plaque-forming units, PFUs) by nasal drip, and then T cells were isolated on the 7th day postinfection.
In vitro virus infection
The H9N2 virus (Ck/HB/4/08) was inoculated into 9-day-old SPF chicken embryos, and virus titers were determined by measuring PFUs. Madin-Darby canine kidney (MDCK, CCL-34, ATCC) cells used for PFUs were cultured in DMEM media supplemented with 5% FBS. AIV-specific sialic acid α-2,3-galactose receptor (SA2-3Gal) expression was confirmed by biotinylated Maackia amurensis lectin II (VECTOR, CA, USA) staining and then followed by staining with FITC-conjugated avidin D (green) and DAPI (blue) for nuclei. To assess H9N2 virus infection, RPMECs were washed with PBS, inoculated with virus at different multiplicities of infection (MOIs) and incubated for 1 h. Then, the cells were washed with PBS and incubated with DMEM, 0.2% bovine serum albumin (Gibco, Carlsbad, CA, USA) and 0.2 μg/mL TPCK-treated trypsin . Viral titers in the supernatants were measured using PFUs. To investigate the PD-L1 level induced by inactivated H9N2 virus, viral particles were inactivated using 0.094% β-propionolactone (BPL; SERVA Electrophoresis, Heidelberg, Germany) according to a previously described protocol .
RPMEC/T cell coculture system
The T cell/RPMEC coculture system was established in transwell chambers with 6-well inserts (Corning, Shanghai, China). The RPMECs were seeded in the upper chambers at a concentration of 1 × 105 cells/well, and the confluence of the RPMEC monolayers was detected on days 0, 1, 2 and 3 by measuring permeability to FITC-labeled dextran (Sigma Aldrich, Shanghai, China). Then, RPMECs were infected with the H9N2 virus or inoculated with viral particles. After 24 h, T cells were plated over the infected monolayer and incubated for 8 h, Afterwards, the migrated T cells in the lower chamber were harvested and analyzed further. A viral particle control was used since normal MECs expressed very low levels of adhesion molecules, which caused a decreased proportion of migrating T cells. The transmigrated T cells in samples from the bottom chamber were counted by a TC-20 cell counter (Bio-Rad, CA, USA).
RPMECs infected with live H9N2 virus or inoculated with viral particles were harvested at 6, 12 and 24 h postinfection, and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The fold change of the PD-L1 mRNA level in different groups relative to the control group was calculated using GAPDH as the housekeeping gene. The primer sequences were as follows: GAPDH: F, 5′ ACAACTTTGGTATCGTGGAAGGAC3’ and R, 5’AGGGATGATGTTCTGGAGAGCC3’; PD-L1: F, 5’GGAGGACCTGAAGCCTCAAC3’ and R, 5’CGTCCTGCAGCTTGACATCT3’.
The RPMECs were harvested, and a single cell suspension was obtained . Then, the cells were incubated with a PE-labeled antibody against PD-L1 (BioLegend, CA, USA) and a FITC-labeled antibody against H9N2 virus hemagglutinin (Sino Biological, Beijing, China). To detect intracellular perforin, the T cells were fixed, permeabilized with 4% paraformaldehyde in PBS for 10 min at room temperature and stained with an anti-perforin primary antibody (Santa Cruz Biotechnology, Dallas, USA) and a FITC-labeled secondary antibody (ORIGENE, Rockville, MD, USA). All labeled cells were analyzed by flow cytometry, and 10,000 events were acquired per sample. The protein expression levels were evaluated in terms of the percentage of positively labeled cells.
To determine the proliferation rate of the migrated T cells, T cells harvested from the coculture system were resuspended in medium and seeded in 96-well plates at a density of 1 × 104 cells/well. After adding PHA to a final concentration of 5 μg/mL, the cells were cultured for 24, 48 or 72 h. Then 10 μL 3-(4, 5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide (MTT, 5 mg/mL, Sigma, Shanghai, China) solution was added to each well for 4 h, followed by 150 μL DMSO (Sigma, Shanghai, China). Finally, the optical density at 490 nm was measured at each time point.
Annexin V-FITC and propidium iodide staining
To detect the apoptosis rate of the migrated T cells, cells were stained with Annexin V-FITC and PI for 20 min according to the manufacturer’s protocol (Santa Cruz Biotechnology, Dallas, USA). The percentage of apoptotic cells was measured by flow cytometry.
To evaluate the levels of IL-2, IFN-γ and granzyme B, T cells that migrated to the lower chamber were harvested, reseeded in a 12-well plate, and cultured for 48 h. The supernatants were collected and measured by ELISA kits (R&D Systems Inc., MN, USA) according to the manufacturer’s instructions. To determine the levels of IFN-γ and TNF-α induced by the H9N2 virus, T cells were infected with the H9N2 virus at an MOI of 1 or 5. Supernatants were collected from each group at 12 h and 24 h postinfection and analyzed using ELISA kits (R&D Systems Inc., USA).
PD-L1 CRISPR activation plasmid transfection
For the overexpression assay, RPMECs seeded in upper chambers at a concentration of 1 × 105 cells/well were transfected with the control plasmid or PD-L1 CRISPR activation plasmid (the details of the plasmids were provided in the supplementary material) using a Lipofectamine 3000 transfection reagent kit (Invitrogen, Carlsbad, CA, USA). According to the kit instructions, mixtures of plasmid (2 μg) and Lipofectamine 3000 transfection reagent (7.5 μL) using Opti-MEM were prepared. Then, the mixture was added to the RPMECs for 72 h at 37 °C in a 5% CO2 incubator. The overexpression level of PD-L1 was detected at 72 h after transfection by western blotting, and the ratio of PD-L1 to β-actin was determined by ImageJ software (NIH, USA).
The data were analyzed using GraphPad Prism software 6.0 (GraphPad, La Jolla, CA, USA). The results were expressed as the mean ± standard deviation (SD) of at least 3 independent experiments. The different groups were compared using Student’s t-test or one-way analysis of variance (ANOVA) as appropriate. A p-value < 0.05 was considered statistically significant.