Functional characterization of mTERT_198-205-specific T lymphocytes. (A) Recognition of different syngenic tumor cells by mTERT-specific bulk T cells after repeated in vitro stimulations. T cells from different in vitro passages were incubated with an equal number of tumor cells for 24 hours and supernatants tested for released IFN-γ by ELISA. (B) Comparison of functional avidity of mTERT_198-205-specific bulk CTLs with high- and low-avidity CTL clones. The functional avidity was evaluated in ⁵¹Cr release assay against MBL-2 target cells loaded with different mTERT_198-205 peptide concentrations at an effectors to target ratio of 10:1. (C) The mTERT-specific bulk T cells (left), the high-avidity (middle), and the low-avidity (right) clones were tested at different effectors to target cells ratios for cytotoxic activity against several different murine tumor cell lines by use of a ⁵¹Cr release assay. The representative of 3 experiments is shown. (D) Therapeutic effectiveness of mTERT_198-205-specific CTLs. ACT was performed by the use of a melanoma metastatic model. Histograms show the mean ± SD of percentage reduction in the number of pulmonary metastases for melanoma-bearing mice inoculated with either mTERT-specific (polyclonal, high-avidity, and low-avidity) or β-gal–specific CTLs. Data are from 2 cumulative independent experiments. (Student t test: polyclonal mTERT vs β-gal, P = .001; low-affinity mTERT vs β-gal, P = .001; high-affinity mTERT vs β-gal, P < .001.)