Figure 6.

V5 peptide containing T-cell receptor (TCR) vectors demonstrate superior biological activity. (a) Antigen-specific proliferation. Peripheral blood lymphocytes (PBLs) from melanoma patient were transduced with lentiviral vectors harboring the V5 tag-modified gp100 (154) TCR and TCR expression confirmed by fluorescence-activated cell sorting (FACS) analysis (right panel). Four days post-transduction, PBLs were co-cultured with different tumor cell lines (ratio 1:1) in media without interleukin IL-2 for 72 h, and16 h before harvest 1 μCi of (3H) thymidine was added. Radionucleotide incorporation was determined by liquid scintillation counter. Results representing mean c.p.m. of triplicate cultures were plotted as shown (left panel). (b) Robust expression of TCR in CD4/CD8 subsets. CD4/CD8 cells were negatively selected from PBL, and following anti-CD3/CD28 bead activation, cells were transduced with lentiviral vector fuV5F2A. Seven days post-transduction, TCR expression in CD4 and CD8 cells was determined by FACS analysis. (c) Induction of interferon (IFN)-γ by melanoma cells. Transduced CD4/CD8 cells were co-cultured with equal ratio of tumor cells for 16 h, and the concentration of IFN-γ determined by enzyme-linked immunosorbent assay (ELISA) (data are the mean of triplicate cultures). (d) Lytic activities of transduced PBL. Transduced CD4/CD8 cells were co-cultured with ⁵¹Cr-labeled tumor cells at the indicated ratio and the release of ⁵¹Cr was measured. The percentage of cell lysis was calculated using the formula ((specific release−spontaneous release)/(total release−spontaneous release)) × 100. Results representing mean of triplicate cultures were plotted. Melanoma cells included MART-1-positive human lymphocyte antigen (HLA)-A2⁺ 624, and 526, plus two MART-1-positive HLA-A2⁻ cells 888 and 938. Data shown represent the mean of triplicate cultures.