A novel approach for the identification of tumor antigen‐derived sequences recognized by CD8+ cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1×1011 nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma‐reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screeningwere efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX‐241–49, whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross‐reactivity of a human HIV‐1 gag p24‐specific CD4+ T cell clone obtained from an HIV‐1‐seronegative donor using a positional scanning synthetic combinatorial peptide library (PS‐SCL)‐based biometrical analysis. A number of decapeptides able to activate the HIV‐1 gag‐specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV‐1 gag p24 peptide. These findings show that an HIV‐1‐specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV‐1) could arise from exposure to other pathogens.