Characterization of the HLA class I Ligandome from Extracellular vesicles

Extracellular vesicles have shown promise in the field of anti-tumor vaccines. EVs are antigen-presenting entities capable of eliciting potent T-cell responses and have therefore being explored as cell-free based peptide vaccine vectors. However, whether the EV ligandome sufficiently resembles the cell ligandome as to preserve antigenicity and immunogenicity of the ligands is still a crucial aspect to be addressed. In particular, when aiming to use EVs as cell-free vaccine vectors. To understand whether HLA-I complexes homing to EVs display the same ligand sequence features and characteristics than those homing to the cell surface, we used a highly sensitive MS-based immunopeptidomics approach to generate a comprehensive side-by-side analysis of the EV and whole-cell (WC) ligandomes. In this work we identified thousands of ligands derived from both EVs and WCs, which allowed a deep characterization of important ligand properties such as the occurrence of PTMs in both ligandomes. Our results indicate that EVs are as good as cells when it comes to antigen presentation, since the sequence properties of the antigens were conserved in EVs and WCs ligandomes. Interestingly, EVs were enriched in HLA-B ligands and cysteinylated peptides, which can ultimately affect the antigenicity and immunogenicity of the ligands and should be taken into consideration when developing cell-free EV-based peptide vaccination approaches.