Mice
Female BALB/c mice were 6-8 weeks of age and purchased from the Chinese Academy of Sciences Shanghai Laboratory Animal Center. The mice were raised in horizontal laminar flow cabinets and provided sterile food and water in a specific pathogen-free facility. This animal study was approved by the Institutional Animal Care and Use Committee of Fudan University (Ethic No. 2019 Huashan Hospital JS-071). These animals were randomly divided into four groups (n = 6 for each group).
AR models
According to the published protocols [13], a group of mice were administered 0.5 mg/mL of ovalbumin (OVA, grade V; Sigma-Aldrich, St. Louis, Missouri, USA) and 20 mg/mL of aluminium hydroxide (Sinopharm Chemical Reagent Co Ltd., Shanghai, China) in normal saline at a dosage of 0.2 mL/mouse through the intraperitoneal injection. The sensitization process was repeated three times at weekly intervals (days 1, 8 and 15). After that, these mice were challenged by the daily instillation of OVA solution droplet (40 mg/mL in normal saline) into the nostrils (0.02 mL/mouse) with a micropipette on days 22 to 29 (Fig. 1). This group of mice was used as allergic models (AR group). As a negative control, another group of mice received the challenge treatment of normal saline alone (normal group). A group of allergic mice received the adoptive transfer of CD8+ Tregs from normal mice (normal CD8+ Tregs group) or allergic mice (AR CD8+ Tregs group) cultured in vitro intravenously in the tail vein on challenging days. Nasal symptoms were assessed by counting numbers of sneezing and nasal rubbing during 10 minutes immediately after the last OVA intranasal provocation on day 29.
Samples preparation
After mice were sacrificed, the fore teeth were cut off. The lower jaw and cheek muscles were removed. Nasal mucosa samples were collected and cut into two portions: One was for flow cytometry analysis, and the other was cultured in vitro.
Flow cytometry analysis
Nasal mucosa samples were processed into cell suspensions. Then the cell suspensions were centrifuged for 10 minutes at 200 × g at 4°C, and cells were harvested for flow cytometry analysis. CD8+ cells were isolated using mouse CD8 microbeads according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+CD25+ T cells were separated using an EasySep™ mouse CD8+ T cells enrichment kit (StemCell Technologies, Vancouver, BC, Canada), followed by isolation with CD25 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For intracellular staining, these cells were stained with Foxp3 staining kit (MyBioSource, Inc., San Diego, CA, USA). Finally, above cells were resuspended and analyzed using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA) and FlowJoSoftware (TreeStar Inc., Ashland, OR, USA).
Cell cultures
CD8+ Tregs were cultured in vitro at 5 × 105/well in 96-well plates coated with anti-CD3. IL-2 was then added at 1000 IU/mL. The cell cultures were kept for three days. The cell supernatants were used to measure levels of cytokines IL-10 and transforming growth factor (TGF)-β with enzyme-linked immunosorbent assay (ELISA), and the cell lysates were used to assess the contents of messenger (m) RNAs of IL-10 and TGF-β by using real-time reverse transcription–polymerase chain reaction (RT-PCR). After that, CD8+ Tregs were resuspended in normal saline, and were adoptively transferred at 100 µL per mouse (5 × 105cells/mouse) intravenously in the tail vein on challenging days.
Nasal lavage fluid (NLF) preparation
After mice were killed, one blunted 18-gauge needle was used to obtain NLF. One injection of 2000 μL normal saline was performed and the fluid was collected with a tube under both nares of the mouse nose for NLF [14]. A portion of NLF was centrifuged for 10 minutes at 150 × g at 4°C. The supernatants were stored at −70°C for eosinophil cation protein (ECP), IL-4, IL-5, IL-13, IL-10 and TGF-β assays. Another portion was collected for detection of eosinophils. Differential cell counts on 150 cells were performed on cytospins (Cytospin 4 Shandon Ltd., Runcorn, UK) stained with Giemsa [15].
Nasal mucosal cultures
Nasal mucosa from normal and AR mice were cultured in vitro in accordance with published protocols [16]. To assess the impact of CD8+ Tregs treatment on allergic condition, nasal mucosa from AR mice were saturated for 1 hour in culture medium with DMEM and 10% calf serum and 10 μg/mL gentamicin in the presence of CD8+ Tregs (5 × 105) from normal or AR mice cultured in vitro for three days, and then placed on a hydrated 1 × 1cm gelatin sponge with the submucosa downward and the mucosa facing upward. Nasal mucosa from normal mice were not treated using CD8+ Tregs. Finally, all the cultured samples were collected and stored at –20 °C for further examinations. The tissue culture supernatants were used to measure mediator concentrations of ECP, IL-4, IL-5 and IL-13.
ELISA
IL-10 and TGF-β in cell cultures, ECP, IL-4, IL-5 and IL-13 in tissue cultures, and all these mediators in NLF were evaluated using corresponding ELISA kits which were all purchased from MyBioSource, Inc., San Diego, CA, USA. The ELISA procedures were strictly performed according to the manufacturers' protocols.
Real-time RT-PCR
Real-time RT-PCR was performed to evaluate the mRNA of IL-10 and TGF-β in CD8+ Tregs cultures. Briefly, the total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase. For reverse transcription, 2 μg of the above RNA was reversely transcribed with random hexamers (Invitrogen, Carlsbad, CA, USA) and cDNA was amplified in accordance with the manufacturer’s instructions. IL-10 mRNA primers were as follows: forward primer 5'-TCCATCATG CCTGGCTCA-3', and reverse primer 5'-GGTGTTTTAGCTTTTCATTTT-3'. TGF-β mRNA primers were as follows: forward primer 5'-ATTCCTGGCGTTACCTTGG-3', and reverse primer 5'-AGCCCTGTATTCCGTCTCCT-3'. GAPDH mRNA primers were as follows: forward primer 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer 5'-TCCACCACCCTGTTGCTGTA-3'. After initial denaturation at 95°C for 10 minutes, the amplification profile was 15 seconds of denaturation at 95°C, 1 minute of annealing and extension at 60°C for 45 cycles. For the measurement 2 μL of diluted cDNA was amplified in a total reaction volume of 20 μL by using an 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA) with 20 × SYBR Green mixture (Invitrogen, Carlsbad, CA, USA). Evaluation of data was performed using the ΔCT method with GAPDH as the internal standard.
Statistical analysis
Statistical analysis was performed by using a commercially available statistical software prism 6.0 (GraphPad Software Inc., San Diego, California, USA). The analysis between individual in vivo groups was examined by ANOVA, followed by the Student’s t test ± Welch’s correction, and presented as mean ± SEM (standard error of the mean). The significance of a difference was accepted at the 5% level of confidence. p < 0.05 was considered statistically significant.