Comparison of culture conditions for expanding virus-specific T cells from CD45RA⁻CD62L⁺CD8⁺ precursors. (A) Representative flow plots showing tetramer staining of the enriched CD8⁺CD62L⁺ T_CM before and 10 days after stimulation with a B*0702_CMVpp65 peptide. T_CM cells were plated either in a 24-well or 96-well plate with B*0702_CMVpp65 peptide-pulsed γ-irradiated PBMCs. Shown are data from individual wells of the 24-well cultures and pooled wells of the 96-well cultures. (B) T_CM cells from 7 donors were cultured the same as in panel A and tested for tetramer positivity on day 10. T_CM from 3 donors were stimulated with an A*0201_CMVpp65 peptide, and T_CM from the other 4 donors were stimulated with a B*0702_CMVpp65 peptide. Symbols represent the frequency of tetramer-positive T cells in cultures from each individual donor and the horizontal lines indicate the mean ± SEM. (C) T_CM cells were plated onto 96-well plates and stimulated with either peptide-pulsed PBMC (■) or peptide-pulsed DCs (□). After 10 days of culture, 6 to 12 wells with growth were randomly selected and analyzed for the frequency of virus-specific T cells by tetramer staining. For each of the 3 donors, a B*0702_CMVpp65 peptide was used for stimulation. The graph shows the mean frequency of tetramer positive cells in each donor and SEM at day 10 after each stimulation condition. (D) T_CM cells from 4 donors were plated with autologous PBMCs pulsed with CMV or EBV peptides known to be presented by HLA alleles of the donor. The frequency of virus-specific T cells was determined by tetramer staining in the starting population and 10 days after stimulation. Data are representative of 5 independent experiments from each donor.