Screening of HLA-A*2402–binding peptides from amino acid sequences of CMV proteins for CD8⁺ T-cell stimulation by ELISPOT assay.

A total of 10 000 polyclonal CMV-specific CD8⁺ T cells established from PBMCs of a CMV-seropositive and HLA-A24⁺donor were cocultured with 50 000 T2-A24 cells in each well in the presence of each peptide at a concentration of 10 μM. When the spots were too many to count, 1000 CD8⁺ T cells were used as responder cells and the numbers of spots were shown after being multiplied by 10. Only peptides that had been shown to bind to HLA-A*2402 molecules by MHC stabilization assay were tested by ELISPOT. An HLA-A24–binding peptide, RYLRDQQLL, derived from the HIV envelope protein was used as control peptide (env). In some wells, autologous fibroblast cells that had been infected 2 hours previously with CMV AD169 strain (CMV) or mock-infected (mock) were used as antigen-presenting cells (10 000 cells/well). Each bar represents the average number of spots in duplicate wells.