Multiple doses of IL-7 administered over 24 hours are unable to recapitulate the effect of a single dose of IL-7/M25. CD8⁺ T cells were purified from LNs of B6.CD90.1⁺ IL-7tg⁺ donor mice, CFSE-labeled, and injected intravenously (7 to 10 × 10⁶ per host) into B6.CD90.2⁺ mice. (A) One day after adoptive transfer of donor cells, host mice received intraperitoneal injections of PBS, either a single injection of rhIL-7/M25 (1.5 μg/7.5 μg [middle left] or 10 μg/50 μg [middle right]), or a total of 12 doses of rhIL-7 alone (1.5 μg [bottom left] or 10 μg [bottom right]) administered every 2 hours for 24 hours. On day 7 after adoptive transfer, host LNs and spleen were harvested, and donor cell CFSE dilution was analyzed by flow cytometry. Shown are CFSE histograms of CD90.1⁺ CD8⁺ cells from host LNs that are representative of 2 to 3 experiments with 2 separately analyzed hosts per treatment. (B) Donor cells were allowed to park for 1 day following adoptive transfer, and then host mice received intraperitoneal injections of PBS, rhIL-7/M25 (3 μg/15 μg), or rhIL-7/M25_Fab (3 μg/equivalent of 15 μg M25) once (third panel), or at 2-hour intervals over 24 hours (bottom). Six days after adoptive transfer, host LNs and spleens were harvested, and the CFSE histograms of CD90.1⁺ CD8⁺ donor cells were determined by flow cytometry. Histograms shown are representative of 2 experiments with 2 separately analyzed mice per condition. (C) C57BL/6 mice were injected intraperitoneally with PBS (ctrl, solid columns), 1.5 µg rhIL-7 (top panel), 1.5 µg /7.5 µg rhIL-7/M25 (middle panel), or 10 µg rhIL-7 (bottom panel) and euthanized at the indicated intervals posttreatment. Injections were staggered such that all mice in the same experiment could be analyzed at the same time. Mean fluorescent intensity (MFI) or relative geometric MFI (RFI) of CD127 on CD8⁺TCRβ⁺ [T-cell receptor β⁺] lymphocytes normalized to the average CD127 MFI of the PBS-treated mice from each experiment, which was set at 100% RFI, is shown from 3 combined experiments with 2 to 7 separately analyzed mice per condition (± standard error of the mean [SEM]). Significant difference from PBS-treated mice is indicated as *P < .05; **P < .005; ***P < .0005; ****P < .0001. hrs, hours; ND, not determined.