ELISPOT assay
IFN-γ secretion of effectors was assayed by enzyme-linked immunospot (ELISPOT). Multiscreen 96-well assay plates (Dakewe, Shenzhen, China) were precoated overnight at 4 °C with anti-IFN-γ antibody according to the manufacturer’s instruction. After washing with PBST (PBS-0.05 % Tween 20), plates were blocked for 1 h at 37 °C with PBS/1 % BSA. Cytotoxic T lymphocyte effectors from human HLA-A2+ donors were plated in triplicate wells at a density of 1 × 105/100 μl in RPMI-1640 medium. Plates were cultured overnight, washed extensively with PBST, and incubated with anti-IFN-γ mAb for 1 h at 37 °C. After washing, goat anti-biotin antibodies (Dakewe) were added, and the plates were incubated for 1 h at 37 °C. Thirty microliters of activator solution (Dakewe) was added to develop spots, and after 10–30 min, the plates were washed with distilled water to stop the reaction. After being air-dried, the number of spots in each well was counted using the Bioreader 4000 PRO-X (Bio-Sys; Germany).