Anti mcd3

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).

TCRγδ or NKT cells were isolated from murine spleens using the TCRγ/δ + T Cell Isolation Kit (using BALB/cJ mice) or the NK1.1 + iNKT Cell Isolation Kit (using C57BL/6 J mice), respectively (Miltenyi), following the manufacturer’s protocol. The enrichment of cells was determined by FACS. Isolated TCRγδ cells were 78% positive by staining with CD3-PE/TCRγδ−BV421 (Biolegend), isolated NKT cells had 77% positive staining using CD3-PE/CD49b-APC (Biolegend). Isolated TCRγδ or NKT cells were restimulated for 18 h using 5 μg/ml anti mCD3 (Biolegend, clone 145-2C11). The cultured cells, the supernatant or the combination of both was subsequently co-cultured with freshly isolated murine neutrophils50 (link) in a ratio of 1:4 for additional 4 h. The neutrophils were stained for FACS using CD18-FITC, CD62L-PE-Vio770, Ly6G-APC-Vio770 and CD45-VioGreen (Miltenyi) following standard procedures to evaluate the activation status. Dead cells were excluded using PI. The supernatant of cultured cells was analyzed for cytokine release using the LEGENDplex™ Mouse Proinflammatory Chemokine Panel (Biolegend).