Anti mcd28

Myocardial M-MDSCs or G-MDSCs from CVB3-infected mice were seeded at 1 × 10⁶ per well. Sorted CD4⁺CD25^-CD62L^highCD44^low T cells (1 × 10⁶ per well) were added in the presence of purified anti-mCD3 (5 μg/ml, Biolegend) and anti-mCD28 (5 μg/ml, Biolegend) and culture for 72 h. And then cells were subjected to proliferation assays using cell proliferation ELISA BrdU kit (Roche) following the manufacturer’s instructions. Absorbance at 370 nm was detected on a microplate reader (Bio-Tek).

Murine T cells (3 × 10⁵ cells/well) were cultured over 3 days at 37 °C in migration medium (RPMI 1640 medium [Life Technologies, Darmstadt, Germany]) supplemented by 10% heat-inactivated IgG free FCS (PAA Laboratories, Pasching, Austria), 100 U/ml Pen-Strep (PAA Laboratories), 2 mM l-glutamine and 5 μg/ml anti-mCD28 (Biolegend, clone 37.51) in the bottom chambers of 24-well transwell plates (pore size 5 μm) coated with 5 μg/ml anti mCD3 (Biolegend, clone 145-2C11). After 3 days, the supernatant of the T cells was either left inside the well or transferred into a separate well for analysis. The upper chambers were filled with freshly isolated neutrophils (10⁶ cells/well) in migration medium, and the entire plate was incubated for 45 min at 37 °C for migration.