Insights into MHC class I antigen processing gained from large-scale analysis of class I ligands

Abstract

Short peptides derived from intracellular proteins and presented on MHC class I molecules on the cell surface serve as a showcase for the immune system to detect pathogenic or malignant alterations inside the cell, and the sequencing and analysis of the presented peptide pool has received considerable attention over the last two decades. In this review, we give a comprehensive presentation of the methods employed for the large-scale qualitative and quantitative analysis of the MHC class I ligandome. Furthermore, we focus on insights gained into the underlying processing pathway, especially involving the roles of the proteasome, the TAP complex, and the peptide specificities and motifs of MHC molecules. The identification of post-translational modifications in MHC ligands and their implications for processing are also considered. Finally, we review the correlations of the ligandome to the proteome and the transcriptome.

Fig. 1

Flowchart illustrating the different approaches for large-scale analysis of MHC class I ligands. The purified ligands can then be further analyzed quantitatively or qualitatively. The qualitative evaluation of MHC class I ligands is nowadays almost exclusively done via LC-MS/MS. The spectra obtained from the mass spectrometer data can reveal epitopes, binding motifs, and post-translational modifications of the peptides. The quantitative analysis can be performed either by comparing the relative mass changes of a labeled control peptide with the corresponding natural ligand, or by spiking of labeled peptides into the sample for an absolute quantification

Fig. 2

Amino acid positions of relevance in peptide motifs that are accessible by the analysis of the HLA ligandome, using nonamers as example. The C-terminus of ligands results from proteasomal cleavage. Positions of main and auxiliary binding anchors for MHC molecules differ among allotypes, main anchors being most frequently positions 2 and 9. TAP can transport longer peptides than suitable for MHC binding, in which case ERAP1 cleaves surplus N-terminal amino acids. Residues relevant for TAP binding are the three N-terminal amino acids of the transported peptide as well as the antepenultimate and the C-terminal ones

Fig. 3

Number of reported ligands for each HLA allotype. Ligands from the SYFPEITHI database and unpublished data of our group were considered. Allotypes with more than 50 ligands, for which confident peptide motifs can be derived, are highlighted