Interleukin 2 (il 2)

The hUC-MSCs were seeded in 12-well plates at a density of 5 × 10⁴ cells/well and pretreated with IFN-γ (40 ng/ml) and ATP (2 mM), as described above. hPBMCs (1 × 10⁶ cells/ml) were activated using anti-CD3/CD28 antibodies for 48 h and then cultured with IL-2 (200 U/ml) alone for 48 h. The hPBMCs were incubated with 5 μM CFSE (Beyotime) for 20 min at a concentration of 10⁶ cells/ml. They were protected from light during the entire procedure. After labeling, hPBMCs were stimulated with 200 IU/ml IL-2 (Acrobiosystems) in T-cell culture medium (RPMI 1640 medium supplemented with 10% FBS, 1% penicillin, and 1% glutamine) and co-cultured with or without pretreated hUC-MSCs (hUC-MSC: hPBMC ratio = 1:10) at 37°C and 5% CO₂. After 3 d, hPBMCs were collected, and CD3⁺ T-cell proliferation was measured by flow cytometry as a decrease in CFSE intensity because of cell division from the original population. APC-CD3 antibody was purchased from BD Biosciences.

The levels of the following pro-inflammatory factors were measured: tumor necrosis factor-α (TNF-α; cat no. PT512; Beyotime Biotechnology), interleukin (IL)-2 (cat no. PI575; Beyotime Biotechnology), IL-4 (cat no. PI612; Beyotime Biotechnology), IL-5 (cat no. PI620; Beyotime Biotechnology), IL-13 (cat no. PI627; Beyotime Biotechnology), IL-17 (cat no. PI545; Beyotime Biotechnology), interferon-γ (IFN-γ; cat no. PI508; Beyotime Biotechnology), OVA-specific IgE (cat. no. 439807-1; BioLegend, Inc.) and leukotriene C4 (LTC4; cat. no. E-EL-M0753 km⁻¹; Shanghai Zhenyu Chemical Technology Co., Ltd). The measurement was performed in mice of different groups by ELISA specific kits according to the manufacturer’s instructions. Each experiment was repeated at least three times.

IL-2 (Beyotime Biotechnology Co., Ltd., Shanghai, China) was dissolved into sterile physiological saline at a dose of 1500UI/100 μL/ day [43 (link)].
First, 6–8 week-old male ICR mice were purchased from Huafukang Biotechnology Co., Ltd. (Beijing, China), with the production license No. SCXK (Yue) 20110015. The mice were housed as three per cage under SPF standard room conditions (12 h light/dark cycle, 23 ± 2 °C, relative humidity of 50%–60%, free access to food and water). The experimental protocol for animals was in accordance with the relevant regulations and was approved by the Animal Care and Use Committee of Guangdong Ocean University, China (IACUC-20160922-037). After 14 days of adaptation to the new conditions, the mice were divided into three groups (n = 12): the control (CT), CUMS and CUMS + IL-2 groups.