Cytotoxic CD8+ T lymphocytes (CTLs) recognize
peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as
cancer. Difficulty in predicting HLA class I
ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I
ligands on a large scale and analyzed the source-
protein sequences flanking the
ligands. This comprehensive analysis revealed that the frequency of
proline at
amino acid positions 1-3 upstream of the
ligands was selectively decreased. The depleted
proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of
proline at upstream positions 1-3 attenuated CTL responses against a model
epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the
proline signature. Our results highlight, to our knowledge, a unique role and position of
proline for inhibiting downstream
epitope presentation, which provides a rule for defining natural
peptide-HLA class I repertoire formation and CTL responses.