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Spontaneous immune responses to the cancer testis antigen NY-ESO-1 are frequently found in cancer patients bearing antigen-expressing tumors. In HLA-A2-expressing patients, naturally elicited NY-ESO-1-specific, tumor-reactive cytotoxic T lymphocytes (CTLs) are mostly directed against an immunodominant epitope corresponding to peptide NY-ESO-1 157-165. NY-ESO-1-specific CTLs can also be induced by synthetic peptide vaccines, but they are heterogeneous in terms of functional avidity and tumor reactivity. The authors investigated the structural bases of this phenomenon by analyzing the TCR features of natural and vaccine-induced NY-ESO-1-specific CTLs. The results indicate that CTLs from the two groups exhibit highly structurally conserved but distinct TCR features, suggesting that the synthetic peptides used for vaccination may fail to faithfully mimic the naturally processed antigen. Together, the results of this study underline the strength of TCR molecular monitoring and will be instrumental for the development and monitoring of vaccines aimed at eliciting CTLs with high tumor reactivity.