Duration of in vitro programming stimulus is a critical determinant of in vivo antitumor efficacy. (A) Sublethally irradiated WT mice bearing day 10 established subcutaneous B16 tumors were left untreated as controls (×) or received pmel T_EM cells (●), pmel T_EM cells programmed in vitro with 1 μM hgp100_25-33 peptide-pulsed irradiated APCs for indicated time points before transfer (■), or pmel T_EM cells plus rFPgp100 vaccination (□). For groups receiving pmel T_EM cells, mice received 10⁶ cells. For mice receiving peptide-programmed pmel T_EM cells, cultures were started with 10⁶ cells, and the total number of cells present at the time of transfer was divided evenly between the numbers of recipient mice and transferred. For all treatment groups receiving cell transfer, mice also received exogenous rhIL-2 (36 μg/dose × 6 doses). Results for tumor treatment experiments are presented as the mean measurements from 5 mice per group (± SEM). (B) Increased duration of in vitro programming does not impair the relative efficiency of engraftment after adoptive cell transfer. pmel T_EM cells were transferred without further stimulation (■) or received programming with 1 μM hgp100_25-33 peptide-pulsed irradiated APCs for 24 hours (▩) or 72 hours (□) before ACT. For all groups, cultures were started with 10⁷ pmel T_EM cells, and the total number of cells present at the time of transfer were divided evenly between recipient mice and transferred. The absolute number of pmel cells (identified by CD8⁺tetramer⁺ lymphocytes) was enumerated in the spleen of recipient mice 48 hours after transfer as outlined in “In vivo tumor treatment and enumeration of adoptively transferred cells.” Each data point represents the average ± SEM of 3 independent mice per treatment group. This experiment was repeated twice with similar results. *P < .01 for the comparisons of nonstimulated pmel T_EM cells and T_EM cells programmed for 24 hours to T_EM cells programmed for 72 hours before transfer. †P > .2 for the comparison of nonstimulated pmel T_EM cells to T_EM cells programmed for 24 hours before transfer.