Figure 1. Functionality, TCR-αβ surface expression levels, and TCR/CD8 downregulation in CD8⁺ T cells engineered with self/tumor-specific TCR of incremental affinities.

(A) Ca²⁺ flux of TCR-transduced CD8⁺ T cells without (baseline) and with 1 μg/ml HLA-A2/NY-ESO–specific multimer stimulation. Maximal Ca²⁺ flux after ionomycin stimulation indicates equal capacity to mobilize calcium in all T cell variants. Data are expressed as Indo-1 (violet)/Indo-1 (blue) emission ratio. (B) Cytotoxic activity (% of maximal killing) against Me 275 and Me 290 (HLA-A2⁺/NY-ESO-1⁺) and Na8 (HLA-A2⁺/NY-ESO-1^–) tumor cell lines at an effector target ratio of 10:1. (C) Percentage of primary CD8⁺ T cells expressing the affinity-optimized NY-ESO-1–specific TCRs as detected by A2/NY-ESO-1_157–165–specific multimer staining (left panel). Cells with greater than 80% multimer labeling were used for further analysis. Surface expression levels (in MFI) of TCR β-chain BV13.1 (middle panel) and total αβTCR (right panel) are shown for all CD8⁺ transduced T cells. (D and E) Downregulation of CD8 coreceptor (D; in %) and TCR (E; in MFI) in engineered CD8⁺ T cells in the absence (baseline) or presence of 0.1 μg/ml unlabeled A2/NY-ESO-1–specific multimer. Data from T cells with reduced CD8 expression (D; CD8 low) are shown as percentage of total T cells (CD8 high and low). TCR downregulation following stimulation (E) is depicted as dark gray histograms or columns. Error bars represent mean ± SD.