Anti cd28

PBMCs were isolated from the blood of RA patients and healthy donors as previously described.^[20] Briefly, after gradient centrifugation by using lymphocyte separation medium (TBD science), mononuclear cells were collected, washed in RPMI 1640 medium (Gibco, ThermoFisher Scientific), and adjusted to 10⁶ cells mL⁻¹ in 1640 supplemented with 50 IU mL⁻¹ penicillin (Gibco, ThermoFisher Scientific), 50 µg mL⁻¹ streptomycin (Gibco, ThermoFisher Scientific), and 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific). PBMCs (10⁶ cells mL⁻¹) were seeded in a 48‐well tissue culture plates (Corning, New York, USA), and then co‐incubated with anti‐CD3 (2 µg mL⁻¹) plus anti‐CD28 (2 µg mL⁻¹) antibody (biogems) with or without salivaricin A2 (12.5, 50, or 200 µg mL⁻¹) or salivaricin B (12.5, 50, or 200 µg mL⁻¹)^[12] at 37 °C in air with 5% CO₂. After 66 h culturing, the supernatants were collected, clarified by centrifugation (3000 rpm, 10 min, room temperature). Cytokines were measured by enzyme‐linked immunosorbent assay (ELISA) kits for IL‐10, IL‐17A, IL‐21, IFN‐γ, TNF‐α, and IL‐6 according to the manufacturer's instructions (Multisciences, Hangzhou, China). Informed consent was obtained from all subjects. This study was approved by Peking University People's Hospital Ethics Committee.

Naïve CD4⁺T cells were obtained from the spleens of healthy BALB/c mice using a naïve CD4⁺T cells isolation kit (Miltenyi Biotec) following the manufacturer's instruction. The cells were then labelled with carboxyl fluorescein diacetate succinimide (CFSE) and cultured in 24-well plates that were coated with anti-CD3 (10μg/ml, BioGems) and anti-CD28 (2μg/ml, BioGems) antibodies for 12 hours. Meanwhile, MDSCs were isolated from the spleen of in situ H₂₂ hepatocellular carcinoma-bearing mice using MACS technique (Miltenyi Biotec) and pretreated with JHD (500μg/ml) or isochoric PBS for 36 hours. Subsequently, CD4⁺ T cells and MDSCs were collected respectively, and co-cultured at ratios of 1:1and 1:2. For the positive group, naive CD4⁺ T cells were co-cultured with DCs (CD11b⁺CD11c⁺) which were obtained by fluorescence-activated cell sorting (FACS). Naïve CD4⁺T cells, which were activated by anti-CD3 and anti-CD28 antibodies, were used as the negative control. After 48 hours of culture, the cells were collected and Fc-receptor blocking reagent (Miltenyi Biotec) was used to block the Fc-receptor of cells at 4°C for 10min. Finally, cells were incubated with a Percp/Cy5.5-CD4 antibody (eBioscience) at 4°C for 10min. The percentage of CFSE was detected by Flow cytometry (Beckman Coulter Cytoflex), with gate of Percp/Cy5.5-CD4⁺ cells.

Naive CD4+ T cells from WT mice were extracted by magnetic bead negative selection using the EasySep™ mouse naive CD4+ T cell isolation kit (Catalog # 19765, Stem Cell Technology, Vancouver, Canada). Extracted Naive CD4+ T cells were cultured in 10% FBS + RPMI 1640 medium with 100 U/mL of penicillin and streptomycin and induced in plates cultured with 2 mg/mL anti-CD3 (05112–25–100, Biogems, NJ, USA) and 2 mg/mL anti-CD28 (10312–25–100, Biogems). The conditions for Th17 cell polarization in vitro: IL-6 (10 ng/mL, 78052, Stem Cell Technology), TGF-β1 (5 ng/mL, 100-21C-10, PeproTech, NJ, USA), IL-23 (25 ng/mL, 200-23-10, PeproTech), IL-1β (10 ng/mL, 78035, Stem Cell Technology). After naive CD4+ T cells were stimulated, cells were collected, and Th17 cells were quantified by flow cytometry.