Selection of a subpopulation having lost the ability to switch on CCR7 upon stimulation. CTL clone WEIS3, which had a CD27^–CD28^– phenotype, was stimulated with MZ2-MEL.43 tumor cells pulsed with the MAGE-3 peptide and IL-2. Cells were stained 40 hours after each stimulation with an anti-CCR7 antibody, with a biotinylated anti–mouse IgG2a antibody, and with a PE-Cy5–conjugated streptavidin (black histograms). Control cells were stained with an isotype-matched antibody instead of the anti-CCR7 antibody (empty histograms). Cells with the highest fluorescence (CCR7^high) and cells with the lowest fluorescence (CCR7^neg) were selected by flow cytometry, cultured with IL-2, and restimulated approximately 2 weeks later. The percentages indicated in the figure correspond to the fraction of the cells having a fluorescence intensity above 99% of the cells labeled with the control isotype-matched antibody. After 2 rounds of selection and a further stimulation, 60% of the cells were surviving at day 2 for both subpopulations. Expansion was estimated at days 2 and 5 after stimulation by counting the CD8⁺ cells by flow cytometry.