New light on the HLA-DR immunopeptidomic landscape.

The set of peptides processed and presented by MHC class II molecules define the immunopeptidome, and its characterisation holds keys to understanding essential properties of the immune system. High-throughput mass spectrometry techniques enable interrogation of the diversity and complexity of the immunopeptidome at an unprecedented scale. Here, we analysed a large set of MS-immunopeptidomics data from 40 donors, 221 samples, covering 30 unique HLA-DR molecules. We identified likely co-immunoprecipitated HLA-DR irrelevant contaminants using state-of-the-art prediction methods and unveiled novel light on the properties of HLA antigen processing and presentation. The ligandome (HLA binders) was enriched in 15-mer peptides, and the contaminome (non-binders) in longer peptides. Classification of singletons and nested sets showed that the first were enriched in contaminants. Investigating the source protein location of ligands revealed that only contaminants shared a positional bias. Regarding subcellular localisation, nested peptides were found to predominantly be of endo-lysosomal whereas singletons shared an equal distribution between the cytosolic and endo-lysosomal origin. According to antigen processing signatures, no significant differences were observed between the cytosolic and endo-lysosomal ligands. Further, the sensitivity of MS-immunopeptidomics was investigated by analysing overlap and saturation between biological MS-replica, concluding that at least 5 replicas are needed to identify 80% of the immunopeptidome. Moreover, the overlap in immunopeptidome between donors was found to be very low both in terms of peptides and source proteins, the latter indicating a critical HLA bias in the antigen sampling in the HLA antigen presentation. Finally, the complementarity between MS and in-silico approaches for comprehensively sampling the immunopeptidome was demonstrated.