CD137 enrichment includes tumor-lytic T cells. (A) A Melan-A–specific T-cell line was generated by 2 rounds of stimulation (i). The cells were restimulated with peptide-pulsed autologous PBMCs, and 24 hours later CD137⁺ cells were positively selected using antibody/bead complexes (ii). Selected T cells were cultured for additional 8 days until pMHC multimer staining was performed (iii). This enriched line was then coincubated for 24 hours (ratio 1:1) with the HLA-A2⁺ Melan-A⁻ melanoma cell line A325 (iv) or with the HLA-A2⁺ Melan-A⁺ melanoma cell line Mel526 (v), before staining for CD137. (B) The same enriched line (filled symbols) and the CD137⁻ flowthrough (open symbols) were also tested in a 4-hour chromium release assay using the melanoma cell lines as targets (circles indicate targets; Melan-A⁺, Mel526 melanoma cells; and triangles, Melan A⁻ A325 melanoma cells).