Be0001 2

Dynabeads M-450 Tosylactivated (Invitrogen) were used to covalently couple monoclonal antibodies. Briefly, beads were incubated in 1 ml of phosphate buffer 0.1 M pH 7.6 with each mAb at 37°C overnight in rotation. Unreacted tosyl groups were blocked by incubation with buffer tris 0.2 M 0.1%BSA pH 8.5 for 30 min at room temperature. Beads were then washed twice with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) 2 mM EDTA pH 7.4 and stored at 4°C. The following monoclonal antibodies were used to couple microbeads to simulate human T cells: anti-human CD3 (BE0001-2, Bio X cell) and anti-human CD3 (produced in house), anti-human CD137 (6B4, mouse IgG1, produced in house), mouse IgG2a (40202, Biolegend), mouse IgG1 (400102, Biolegend) (Supplementary Table 1). In the case of microbeads coupled to CD137L, the CD137L-Fc (Sino Biological) was used (Supplementary Table 2). In the case of microbeads coupled to anti-CD7, the anti-CD7 mAb (395602, Biolegend) was used. The following monoclonal antibodies were used for mAb coated beads to stimulate mouse T cells (Supplementary Table 3): antimouse CD3 (100238, Biolegend), antimouse CD137 (BE0239, Bio X cell), antimouse CD137 (3H3, produced in house), rat IgG2a (400533, Biolegend), rat IgG2b (400637, Biolegend). Details of concentrations for antibody coupling are provided in the tables.

Jejunum tissues of piglets were collected on the 8th day after infection with PEDV. After the pigs’ jejunums had been digested, CD8 (559584, BD, USA) and CD4 (PE-conjugated, 559586, BD, USA) T cells were isolated using magnetic beads, and the cell concentration was increased to 5×10⁵/ml using RPMI 1640 containing 10% FBS. Cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (4238011, Biolegend, USA), incubated for 10 min at 37 °C, washed and transferred to cell culture plates, activated by adding 5 μg/ml anti-CD3 (BE0001-2, BioX cell, USA) and 2 μg/ml anti-CD28 (BE0248, BioX cell, USA) mAbs, and treated with LCA (10/20 μM). Cells were incubated in a cell incubator at 37 °C, and cell proliferation was measured at 12-h intervals. Granzyme A, granzyme B, and perforin expression was also measured at 24 h. In addition, cell supernatants were collected at 24 h to detect IL-2 expression using ELISA.