Tnf α

Isolation kits for CD8⁺ T cells and CD4⁺CD25⁺CD127^low/− regulatory T cells and isolation LD and MS columns were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Rabbit polyclonal antibody to human A2aR and FoxP3 was obtained from Abcam (Cambridge, USA), while mouse polyclonal antibody to human CD8, CD39, CD73 and human lymphocyte separation solution was acquired from LianKe MultiSciences (Hangzhou, China). ARL67156 (CD39 antagonist) was obtained from Tocris Bioscience (Bristol, UK). α,β-Methylene-ADP (CD73 antagonist) was obtained from Santa Cruz (Delaware Ave, USA). MRS1754 (A_2bR antagonist) and ZM241385 (A_2aR antagonist) were purchased from Sigma (St. Louis, USA). All the antagonists were dissolved in DMSO and had a final concentration less than 3/1000. ATP was dissolved in DMSO and then further diluted in physiological saline. ATP and IFN-γ assay kits were acquired from Jiancheng (Nanjing, China). TNF-α and perforin assay kits were obtained from KeyGen Biotech (Nanjing, China). The adenosine assay kit was obtained from BioVision (Milpitas, USA). The cAMP assay kit was obtained from Cloud-Clone Corp. (Wuhan, China). The CFSE Cell Proliferation Assay and Tracking Kit was purchased from BestBioScience (Shanghai, China). PE Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (Franklin Lakes, USA).

The ventricle tissues were homogenized, and total proteins obtained using Tissue and Cell Total Protein Extraction Kits (Jiangsu KeyGEN BioTECH Corp., Ltd) were used to analyse BAFF and TNF‐α expression. Total protein (20 μg) was resolved via 15% SDS‐PAGE (Beyotime Biotech Co., Ltd., Shanghai, China) and then transferred to polyvinylidene fluoride membranes. Nonspecific protein binding on the membranes was blocked with 5% non‐fat milk for 2 h, followed by incubation with anti‐BAFF (Abcam plc.), anti‐TNF‐α (Proteintech Group, Inc.) and anti‐β‐actin (Cell Signalling Technology Inc.) antibodies overnight at 4°C. The anti‐β‐actin was selected as reference according previous study¹⁸ and due to the molecular weight of BAFF being close to that of anti‐GAPDH. After three washes with TBST buffer, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody at room temperature for 2 h. Proteins were detected using an enhanced chemiluminescence system and quantified using image J software.