New light on the HLA-DR immunopeptidomic landscape

Area of Science:

• Immunology
• Proteomics
• Mass Spectrometry

Background:

• The immunopeptidome, peptides presented by MHC class II, is crucial for immune system understanding.
• High-throughput mass spectrometry (MS) allows large-scale analysis of immunopeptidome complexity.

Purpose of the Study:

• To analyze a large dataset of MS immunopeptidomics data from 40 donors.
• To identify contaminants and understand HLA antigen processing and presentation properties.
• To assess MS sensitivity and inter-donor immunopeptidome overlap.

Main Methods:

• Analysis of 221 samples across 30 unique HLA-DR molecules.
• Utilized prediction methods to identify HLA-DR contaminants.
• Investigated peptide properties, source protein location, and subcellular localization.
• Assessed MS replica saturation and inter-donor peptide overlap.

Main Results:

• Ligandome (binders) enriched in 15-mer peptides; contaminome (non-binders) in longer peptides.
• Contaminants showed positional bias; nested peptides were endolysosomal, singletons had mixed origins.
• At least 5 MS replicas are required to identify 80% of the immunopeptidome.
• Very low inter-donor overlap in immunopeptidome, indicating significant HLA bias.

Conclusions:

• MS and in silico methods are complementary for immunopeptidome sampling.
• Findings provide novel insights into HLA antigen processing and presentation.
• Low inter-donor overlap underscores the importance of individual HLA type in antigen presentation.