Isolation and Activation of Cardiac Immune Cells

To obtain CD4⁺ T lymphocytes and antigen-presenting cells (APCs), single-cell suspensions of splenocytes were sorted using BD FACSAria II (BD Biosciences, San Jose, CA, USA). CD4⁺ T cells and APCs were co-cultured in a 10:1 ratio. CD4⁺ T cells were activated with anti-CD3/CD28 beads (ThermoFisher, USA) or 10 ng/mL of α-MyHC (Caslo), and the conditioned medium was collected on Day 3 for further experiments. Experiments were performed on primary cardiac microvascular endothelial cells (cMVECs) isolated from hearts of 4-week-old Tnf^+/+ and Tnf^−/− mice as described previously.^{19 (link)} cMVECs were cultured in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and 2 ng/mL recombinant mouse epidermal growth factor (BioLegend, USA). cMVECs were activated with recombinant mouse TNF-α (5 ng/mL, BioLegend) or conditional medium. A detailed description is provided in the Supplemental Methods section.