The MEL5 TCR exhibits a strict preference for 10mer peptides. C1R-HLA A*0201 cells (6 × 10⁴) were pulsed with the indicated 10mer (A-B), 9mer (C), 11mer (C), or 8mer (E) peptides at the concentrations depicted for 2 hours at 37°C. Subsequently, 3 × 10⁴ MEL5 CD8⁺ T cells were added and incubated overnight. The supernatant was then harvested and assayed for MIP1β by ELISA. Error bars represent SDs. (D,F) MEL5 CD8⁺ T cells (5 × 10⁴) were incubated with PE-conjugated HLA A*0201 tetramer (25 μg/mL) folded around ELAGIGILTV (black), FWLLPAWAL (red), FWLLGAWAL (blue), FFAGGIGIRTI (cyan), FLAGGIGIRTL (green), WLLPAWGV (yellow), or WLLPTWGV (pink) for 15 minutes at 37°C and then stained with 5 μL of 7-aminoactinomycin D for 30 minutes at 4°C, washed twice, and resuspended in PBS. Negative control staining is shown in dark purple.