TCR gene transfer and tumor recognition assays. (A) Efficiency of gene transfer. A mouse TCR with specificity for an HLA-A2–restricted epitope of p53 was transferred to LC15 cells by either RNA electroporation or retroviral transduction. The cells were stained with anti–mouse α/β TCR antibody (heavy lines) or the matched isotype control antibody (light lines) 24 hours after electroporation or 2 days after transduction. Cells were analyzed by flow cytometry. The number in the upper right corner denotes the percentage of cells positive for mouse α/β TCR. (B) p53⁺ tumor antigen recognition assay. LC15 effector cells were electroporated with GFP mRNA, electroporated with the p53 TCR mRNA, or transduced with the p53 TCR retroviral vector. Effector cells (1 × 10⁵) were plated in 96-well plates with 1 × 10⁵ tumor targets that were either p53 positive (MDA231 and H2087) or p53 negative (Saos2 and 938mel). After an 18-hour coculture, the cell culture media was collected and assayed for IFN-γ content by ELISA. (C) MART tumor antigen recognition assay. LC15 effector cells were untreated or electroporated with MART TCR mRNA. Tumor targets were melanoma lines either HLA-A2 positive (526mel and 624mel) or HLA-A2 negative (888mel and 938mel). After an 18-hour coculture, the cell culture medium was collected and assayed for IFN-γ and GM-CSF content by ELISA.