Up-regulation of transgenic TCR and other genes after ex vivo restimulation. Peripheral blood from 4 patients who received MART-1–specific TCR-transduced lymphocytes was harvested 2 to 10 months after adoptive transfer. Lymphocytes were isolated and cultured in complete media alone (MOCK), with exogenous IL-2 (600 IU/mL), or with IL-2 and anti-CD3/anti-CD28 beads (1 bead/cell). Total RNA was isolated at the time of in vitro culture (FRESH) or after 48 hours of ex vivo culture, reverse transcribed, and subjected to real-time PCR for (A) the MART-1–specific TCR, (B) β-actin, (C) the endogenous TCR α-chain, and (D) CD3ϵ. Each bar represents an individual patient. Peripheral blood from 2 of the 4 patients were maintained in ex vivo culture for 3 days with or without stimulation conditions and compared with the freshly thawed peripheral blood. Only those cells receiving ex vivo restimulation secreted interferon-γ and demonstrated specific recognition of MART-1 peptide-pulsed target cells (E-F).