TCL1-specific cytotoxic T cells can be generated from normal donors. (A) PBMCs from HLA-A2⁺ normal donors were stimulated with overlapping 15-mer peptides spanning the entire length of the TCL1 protein (supplemental Table 1). T cells from each condition were washed and incubated with autologous CD3-depleted PBMCs as antigen-presenting cells (APC) in the presence or absence of the corresponding peptide. After 18 hours, the production of IFN-γ was determined in the supernatants by ELISA. Representative data from one of 3 normal donors tested is shown. (B) T-cell lines generated from HLA-A2⁺ normal donors using TCL1_65-79 peptide were incubated with APC in the presence or absence of the TCL1_65-79 peptide or control HIV Gag_77-85 peptide, and intracellular cytokine assay was performed. The percentage of CD4⁺ and CD8⁺ T cells producing IFN-γ are shown. (C) TCL1_65-79 peptide-specific T cells were incubated with T2 cells or EBV-transformed B lymphoblastoid cell lines (IHW1003, IHW1019, IHW1089, and IHW1135) mismatched at their MHC class I locus in the presence of the TCL1_65-79 peptide or control HIV Gag_77-85 peptide. IFN-γ production was determined as in panel A. The HLA-A alleles for each of the cell lines are shown. (D,E) A 4-hour ⁵¹Cr-release cytotoxicity assay was performed. TCL1_65-79 peptide-specific T cells were incubated with T2 cells alone or T2 cells pulsed with TCL1_65-79 peptide or control HIV Gag_77-85 peptide, at various effector:target ratios. For MHC blocking assay, an effector: target ratio of 20:1 was used in the presence or absence of isotype control antibody or blocking antibodies (10 μg/mL for each antibody) against MHC class I, MHC class II, HLA-A2, or HLA-B and -C. All cytotoxicity assays were performed in triplicate wells, and mean and standard deviation are shown.