Functional characterization of vaccine-induced circulating CD8+ T cells isolated by multimer-guided cell sorting. The functional avidity of antigen recognition and tumor reactivity of each population was assessed in CTL assays. For each tumor antigen-derived peptide, specific tumor-reactive clones LAU 156/5 (NY-ESO-1), LAU 156/34 (tyrosinase) and LAU 203/17 (Melan-A) were used as internal controls. (A) Functional avidity of antigen recognition was determined by assessing peptide recognition on T2 target cells in the presence of increasing concentrations of the peptides NY-ESO-1_157-165 (open circles), Melan-A_27-35 (closed diamonds), Melan-A_26-35 (closed squares), Melan-A_26-35 A27L (open squares), tyrosinase_368-376 (closed circles), or influenza matrix_58-66 (open triangles). (B) and (C) Tumor recognition was assessed on Me 275 (A2+, NY-ESO-1+, Melan-A+, tyrosinase+ and gp100+) and NA8-MEL (A2+, NY-ESO-1-, Melan-A-, tyrosinase- and gp100-) melanoma cell lines in the absence (closed symbols) or presence (open symbols) of the relevant peptide added exogenously.