Time course of CD45RA expression on antigen-stimulated CTLs. (A) BAGM9 CTLs (2 × 105), which had a CD27–CD28– phenotype, were stimulated with 2 × 105 MZ2-MEL.43 tumor cells pulsed with the MAGE-3 peptide, in the presence of IL-2. Half of the medium was replaced at 2-week intervals by fresh medium and IL-2. Samples were collected from the coculture at different time points. Cells were labeled with an APC-conjugated anti-CD8 antibody and either a PE-conjugated anti-CD45RA antibody (black histograms) or a control isotype-matched antibody (gray histograms). Only the living CD8+ cells are shown in the figure. Surface expression of CD45RA is indicated as the MFI after subtraction of the MFI of the cells labeled with the control isotype-matched antibody. The number of CD45RA transcripts was evaluated by quantitative PCR, and the results were normalized to the CD8 transcripts. (B) Clone BAGM9 was stimulated with beads coated either with anti-CD3 and anti-CD28 antibodies, mature dendritic cells, or EBV-B cells. When indicated, cells were pulsed with the MAGE-3 peptide. Samples were collected from the cocultures at different time points and the surface expression of CD45RA was evaluated as described in panel A. (C) DIPE6/8 CTLs (3 × 105) were stimulated with 1.5 × 105 HLA-A2 EBV-B cells pulsed with the BMLF1 peptide, in the presence of IL-2. Cells were cultured, sampled, and analyzed in the same conditions as in panels A-B.
