Recombinant il 2

To evaluate the effects of resveratrol on the ex vivo expansion of human γδ T cells, PBMCs from healthy individuals were seeded onto 12-well plates (1 × 10⁶ cells/mL) and cultured for seven days in Optimizer CST medium (Gibco-Thermo Scientific) supplemented with 100 IU/mL of recombinant IL-2, 10 ng/mL of recombinant IL-15 (PeproTech, Rocky Hill, NJ, USA), and 10% FBS (referred hereafter referred to as T-cell medium), in the presence of E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and several concentrations of resveratrol (Sigma-Aldrich) or vehicle (0.5% DMSO). The cells were then stained with fluorochrome-labeled anti-CD3, anti- γδ TCR, anti-NKG2D, and anti-CD197 antibodies and analyzed by flow cytometry. The ex vivo growth of regulatory T cells (Treg) was assessed by culturing PBMCs in T-cell medium supplemented with anti-CD3/CD28 magnetic beads (Invitrogen, Waltham, MA, USA) for seven days in the presence or absence of several concentrations of resveratrol or vehicle. The cells were stained with fluorochrome-labeled anti-CD3, anti-CD25, anti-CD4, and anti-CD127 antibodies and analyzed by flow cytometry and were further confirmed as classical Treg cells by staining them with anti-CD3, anti-CD4, and anti FoxP3 antibodies, which was performed using a Foxp3 staining kit (eBiosciences).

IL-2/JES6-1 immunocomplexes were prepared by pre-incubating recombinant IL-2 (PeproTech) with anti-IL-2 mAb (JES6-1; Bioxcell) at a 2:1 cytokine:antibody molar ratio for 30 min at room temperature (23 (link), 65 (link)). Mice were injected with anti-IL-5 antibodies (200 μg) and/or IL-2/JES6-1 immunocomplexes (1 mg every 2 days) by i.p injection.

Mouse CD4⁺ T cells were isolated from the spleen by positive sorting using the CD4 (L3T4) MicroBeads (Miltenyi Biotec, Germany). The purity of the isolated cells was assessed by flow cytometry (shown in Additional file 1: Figure S1, purity of CD4⁺  > 95%). Purified CD4⁺ T cells were cultured in RPMI-1640 medium (CORNING, USA) containing 10% FBS (CORNING, New Zealand) and 50 IU/mL recombinant IL-2 (Peprotech, USA) with 5 μg/mL pre-coated anti-mouse CD3 (17A2, Biolegend) and 3 μg/mL anti-mouse CD28 (37.51, BioLegend).
In co-culture assay, ERCs, NC-ERCs, and CD73-KO-ERCs were seeded, respectively, at a density of 5 × 10⁴ cells/well and CD4⁺ T cells were seeded at 25 × 10⁴ cells/well in 24 well plates. When indicated, AMP (50 µM, MedChemExpress, Shanghai, China) was added. At the time of harvest (72 h), the cell membrane expression of the activation markers CD69 and CD154, and the dilution of CFSE as a measure of proliferation were assessed by flow cytometry. Besides that, the population of CD4⁺IFN-γ⁺ cells were detected by flow cytometry, and the IFN-γ levels in cell culture supernatants were determined by ELISA (DAKEWE, Beijing, China).