CD8 T-cell memory characteristics are reversible. (A) Cell size (forward scatter [FSC]) and pyronin Y RNA) staining of intermediate and high-density CD8 T cells before and after in vitro incubation for 24 and 48 hours. (B) IFN-γ production by intermediate and high-density CD8 T cells before and after in vitro incubation for 48 hours. Shaded histograms represent IFN-γ production by unstimulated cells. (C) Intermediate, high, and intermediate-density CD8 T cells that had been cultured in medium for 48 hours were labeled with CFSE. Cells were stimulated with plate-bound anti-CD3 for the indicated times, at which point CFSE dilution was determined by flow cytometry. (D) Cell-cycle analysis of intermediate-density, high-density, and 48-hour in vitro incubated intermediate-density (48-hour–intermediate) CD8 T cells stimulated with or without anti-CD3/CD28 for the indicated periods of time. (E) Cell cycle of high-density, intermediate-density gp33-41 tetramer-positive, and intermediate-density 48-hour cultured gp33-41 tetramer-positive CD8 T cells from mice previously infected with LCMV. (F) IFN-γ production by stimulated intermediate-density gp33-41 tetramer-positive and high-density CD8 T cells before and after incubation in medium for 48 hours. Shaded histograms represent isotype control. (G) The cultured intermediate-density cells shown in (E) were adoptively transferred into naive B6 recipients. Two weeks later, tetramer-positive CD8 T cells were analyzed for cell-cycle state (right panel). Freshly isolated high-density CD8 T cells are shown as a control (left panel).