Time course of CCR7 expression on antigen-stimulated CTLs. (A) BAGM9 CTLs (2 × 105) were stimulated with 2 × 105 MZ2-MEL.43 tumor cells pulsed with the MAGE-3 peptide in medium containing IL-2 (50 U/mL). 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 an FITC-conjugated anti-CCR7 antibody (black histograms) or a control isotype-matched antibody (gray histograms). Only the living CD8+ cells are shown. Surface expression of CCR7 is indicated as the percentage of the cells having a fluorescence intensity above 99% of the cells labeled with the control isotype-matched antibody. The number of CCR7 transcripts was evaluated by quantitative PCR, and the results were normalized to the CD8 transcripts. (B) Clone BAGM9 was stimulated with either beads coated with anti-CD3 and anti-CD28 antibodies, mature dendritic cells, or EBV-B cells. If indicated, cells were pulsed with the MAGE-3 peptide. Samples were collected from the cocultures at different time points and the CCR7 surface expression was evaluated. (C) CTL clone WEIS3 had a CD27–CD28– phenotype. Some CTLs were stimulated with their antigen 25 days before and some others 12 days before. CTLs (105) were stimulated with MZ2-MEL.43 tumor cells pulsed with the MAGE-3 peptide at the indicated stimulating-cell/CTL ratio. The surface expression of CCR7 was measured 2 days after stimulation. Cells were labeled with an FITC-conjugated anti-CCR7 antibody. The percentage of the cells having a fluorescence intensity above 99% of the cells labeled with the control isotype-matched antibody is indicated on the y-axis. The expansion of the clones was evaluated in a flow cytometer by counting the number of CD8+ cells at day 2 and at day 5.
