Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

Abstract

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4+ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4+ T cell epitopes relevant in health and disease.

Carcinogenesis of renal cell carcinoma reflected in HLA ligands: A novel approach for synergistic peptide vaccination design

“…In light of recent findings, implementation of multiple fragmentation strategies may help overcome this problem in future studies. 53 Nevertheless, for both HLA class I and II we identified several tumor-associated enriched pathways conserved in malignant tissue samples. This shows for the first time that key proteins of intra-and extra-cellular biological processes of carcinogenesis are reflected in HLA ligands and can be used for targeting carcinomas in a more functional manner.…”

A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens

“…We earlier showed that EThcD is especially suited for the characterization of the peptidomes bound to HLA-I (10) and HLA-II molecules (11) and their PTMs (12). Using this workflow, we were able to generate a resource of over 260 phosphorylated HLA-I ligands.…”

Current tools for predicting cancer-specific T cell immunity

“…From the repertoire of thousands detected MHC II peptides, specific binding motifs can be identified, similar as was shown for class I. 66,76,80 As current tools for MHC II peptide binding predictions are relatively inaccurate, predictions based on these motifs may improve identification of patient-specific, mutation containing MHC II ligands (Fig. 3C).…”

“…Stringent control over false discovery rate (FDR) and peptide spectrum match (PSM) scoring ensures accurate and reliable results; typically one to five percent FDR thresholds are applied to peptidomic datasets using the target-decoy approach. 80,81 Inclusion of exhaustive lists of known mutations from repositories like the TCGA or COSMIC will increase the level of false positives and therefore better-personalized reference databases should be used. To quantify mutation containing HLA ligands, the sample can be spiked with synthetic peptides labeled with stable heavy isotopes (Fig.…”