Recombinant human il 15 protein

Seven-week-old male BALB/c mice (n = 10) were killed by cervical dislocation, and spleens were removed and crushed to obtain splenocytes. NK cells were isolated from splenocytes by negative selection using an NK cell isolation kit (110-115-818, Miltenyi Biotec, Bergisch Gladbach, North Phine-Westphalia, Germany) according to the manufacturer’s instructions [31 (link)]. Isolated NK cells were cultured at a density of 5 × 10⁶ cells/mL in T-25 flasks in Roswell Park Memorial Institute-1640 medium (Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA), 1% L-glutamine (Gibco), 1% Antibiotic-Antimycotic (Gibco), 50 µM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 50 ng/mL recombinant human IL-15 protein (R&D systems, Minneapolis, MN, USA). Fresh medium was added to the cells on days 4, 5, and 6. Cells were maintained in a humidified atmosphere containing 5% CO₂ and 95% air at 37 °C.

CD4⁺CD25⁺CD127^dim/− Tregs were purified from PBMCs by CD4⁺CD25⁺CD127^dim/− Regulatory T Cell Isolation Kit II, human (Miltenyi Biotec, Bergisch Gladbach, Germany; Catalog# 130-094-775) using magnetic activated cell separation method following manufacturer’s instructions. CD4⁺CD25⁺CD127^dim/− Tregs were stimulated with recombinant human IL-15 protein (R&D Systems, Minneapolis, MN, USA; Catalog# 247-ILB-025/CF) at the final concentration of either 10 ng/mL or 100 ng/mL for 48 hours. Purified Tregs amount of 5 × 10⁴ was co-cultured in direct contact with 2 × 10⁵ of autologous PBMCs for another 72 hours in the presence of anti-CD3/CD28 (1 µg/mL) as previously reported [26 (link)]. The total cell number in the co-culture system was determined with the Cell Counting Kit 8 (WST-8/CCK-8) (Abcam, Cambridge, MA, USA; Catalog# ab228554) following manufacturer’s instructions. A 20 µL of CCK-8 solution was added to each well in the past 4 hours of culturing. Absorbance of the samples was measured at 450 nm. Wells containing a known number of viable PBMCs were used to create a calibration curve for calculation of tested cell numbers.

PVRIG expression in inactivated and activated PBMCs was determined. For resting PBMCs, PBMCs (AllCells Biotech Co., Ltd., Shanghai, China) were thawed, washed and resuspended in media (RPMI-1640 + 10% fetal calf serum). PBMCs were cultured without stimulation for 20 h, after which PVRIG expression was determined in different cell subpopulations of PBMCs via flow cytometry. For activated PBMCs, PBMCs were stimulated with Dynabeads™ human T-activator CD3/CD28 (Gibco, cat. # 11131D) and recombinant human IL-15 protein (R&D Systems, cat. # 247-ILB) for 24 h, 3 days, 6 days and 10 days. After culture, the cells were collected and stained for cell surface markers before flow cytometry.