Anti mouse cd28 37.51

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).

The human alveolar epithelial cell line A549 kindly provided by Dr. Dongsheng Pei of Xuzhou Medical University was cultured in DMEM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplied with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). The mouse fibroblast cell line NIH/3T3 cells were kindly presented by the Department of Thoracic Surgery of Xuzhou Medical University cultured in DMEM medium supplied with 10% FBS. For isolating primary mouse lung fibroblasts (MLFs), lung tissues were cut into small pieces followed by a first digestion with 5 mM EDTA, a second digestion with 1 mg/ml collagenase type I plus 0.5 mg/ml collagenase type IV plus 1 mg/ml DNase I, and a third digestion with 0.5 mg/ml dispase plus 1 mg/ml collagenase type I. Isolated primary MLFs were cultured in DMEM medium supplied with 10% FBS. T cells were isolated from spleen cells using a Mouse CD90.2 Positive Selection Kit II from STEMCELL Technologies (Shanghai, China). Isolated mouse T cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Shanghai, China) containing 10% FBS, 3 μg/mL anti-mouse CD3 (145-2C11), and 3 μg/mL anti-mouse CD28 (37.51) (BioLegend, San Diego, CA).