In vitro programming of T_EM cells obviates the requirement for in vivo vaccination. (A-B) Sublethally irradiated WT mice bearing day 10 established subcutaneous B16 tumors were left untreated as controls (×) or received in vitro differentiated pmel T_EM cells (○), pmel T_EM cells plus rFPgp100 vaccination (■), or pmel T_EM cells programmed in vitro with either hgp100_25-33 peptide-pulsed APCs (▲) or plate-bound anti-CD3/CD28 (♦) for 24 hours before transfer. 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) and are representative of 3 independent experiments per programming condition. (C) In vitro programming of T_EM cells causes cells to execute a proliferative response in vivo similar to that of vaccine-stimulated T_EM cells. Sublethally irradiated WT mice received nonrestimulated pmel T_EM cells (○), pmel T_EM cells programmed in vitro with plate-bound anti-CD3/anti-CD28 for 24 hours before cell transfer (♦), or pmel T_EM cells plus rFPgp100 vaccination (■). All treated mice received exogenous rhIL-2. The percentage of adoptively transferred pmel-1 cells (identified as CD8⁺Vβ13⁺ lymphocytes/lymphocyte gate) were enumerated in the spleens of treated animals as a function of time. Each data point represents the average ± SEM of 3 independent mice per treatment group. This experiment was repeated 3 times with similar results.