Fig 1: Activation of subpurified CD8+ T-cell proliferation in coculture with Myosin heavy chain peptide. Splenocytes from the mice immunized by intramuscular injection of 50 µL of influenza HA vaccine, 50 µL of HBV vaccine (Bimmugen), or PBS were harvested, and red blood cells were removed. CD8+ T-cells were then sorted using a magnetic-activated cell sorting kit (CD8+ T-cell isolation kit II, Miltenyi Biotech) according to the manufacturer’s protocol. The isolated CD8+ T-cells were resuspended in PBS containing 2% FBS. CD8+ T-cells (1 × 106 cells/mL) were cocultured with 100 µL of PBS containing a range of cardiac myosin heavy chain-a amino acids 334–352 peptide (Cosmo Bio Co., Ltd.) at a concentration of 5 or 10 µg/mL, mouse IL-2 at a concentration of 10 or 50 IU/mL (PeproTech) for 48 h. The cultured cells were harvested and the CD8+ T-cell proliferation was assessed using the BrdU cell proliferation enzyme-linked immunosorbent assays (ELISA) kit (catalog no. ab126556; Abcam, Cambridge) based on the manufacturer’s instructions. The supernatant was collected and analyzed for IFN-? release. IFN-? levels in the culture supernatants were measured via ELISA using a commercial kit (R&D Systems, Inc.) based on the manufacturer’s instructions. Coculture of cardiac myosin heavy chain-a aa334–352 peptide with CD8+ T-cells subpurified from the F15 NOD Nf?b1 heterozygote mice inoculated with influenza HA or HBV vaccine markedly activated the CD8+ T-cell proliferation and increased serum IFN-? levels. Experiments were conducted with 5 animals in each group.