Comprehensive Analysis of the Naturally Processed Peptide Repertoire: Differences between HLA-A and B in the Immunopeptidome

RESEARCH ARTICLE Comprehensive Analysis of the Naturally Processed Peptide Repertoire: Differences between HLA-A and B in the Immunopeptidome Ingrid M. M. Schellens1,2,4☯, Ilka Hoof3☯, Hugo D. Meiring4,5☯, Sanne N. M. Spijkers1, Martien C. M. Poelen4, Jacqueline A. M. van Gaans-van den Brink4, Kees van der Poel1, Ana I. Costa1, Cecile A. C. M. van Els4, Debbie van Baarle1,2,4‡*, Can Kesmir3‡ 1 Laboratory of Translational Immunology, Department of Immunology, University Medical Center Utrecht, Utrecht, the Netherlands, 2 Department of Internal Medicine and Infectious Diseases, University Medical Center Utrecht, Utrecht, the Netherlands, 3 Theoretical Biology and Bioinformatics, Utrecht University, Utrecht, The Netherlands, 4 Centre for Immunology of Infectious Diseases and Vaccines, National Institute for Public Health and the Environment, Bilthoven, The Netherlands, 5 Institute for Translational Vaccinology (Intravacc), Bilthoven, The Netherlands ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * OPEN ACCESS Citation: Schellens IMM, Hoof I, Meiring HD, Spijkers SNM, Poelen MCM, van Gaans-van den Brink JAM, et al. (2015) Comprehensive Analysis of the Naturally Processed Peptide Repertoire: Differences between HLA-A and B in the Immunopeptidome. PLoS ONE 10(9): e0136417. doi:10.1371/journal.pone.0136417 Editor: Scheherazade Sadegh-Nasseri, Johns Hopkins University, UNITED STATES Received: December 29, 2014 Accepted: August 4, 2015 Published: September 16, 2015 Copyright: © 2015 Schellens et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a HiPo (High Potential) grant from Utrecht University, and NWO grant 823.02.014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abstract The cytotoxic T cell (CTL) response is determined by the peptide repertoire presented by the HLA class I molecules of an individual. We performed an in-depth analysis of the peptide repertoire presented by a broad panel of common HLA class I molecules on four B lymphoblastoid cell-lines (BLCL). Peptide elution and mass spectrometry analysis were utilised to investigate the number and abundance of self-peptides. Altogether, 7897 unique self-peptides, derived of 4344 proteins, were eluted. After viral infection, the number of unique selfpeptides eluted significantly decreased compared to uninfected cells, paralleled by a decrease in the number of source proteins. In the overall dataset, the total number of unique self-peptides eluted from HLA-B molecules was larger than from HLA-A molecules, and they were derived from a larger number of source proteins. These results in B cells suggest that HLA-B molecules possibly present a more diverse repertoire compared to their HLA-A counterparts, which may contribute to their immunodominance. This study provides a unique data set giving new insights into the complex system of antigen presentation for a broad panel of HLA molecules, many of which were never studied this extensively before. Introduction The function of Human leukocyte antigen (HLA) class I molecules is to present intracellular peptides to CD8+ T cells. Cytosolic and nuclear proteins as well as proteins of intracellular pathogens are degraded by (cytosolic) proteases, and the resulting peptide fragments are PLOS ONE | DOI:10.1371/journal.pone.0136417 September 16, 2015 1 / 18 Differences in Immunopeptidomes Presented by HLA-A and B Molecules transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) complex. In the ER the peptide fragments bind to available HLA class I molecules after which the peptide-HLA (pHLA) complexes leave the ER and reach the cell surface [1]. Each nucleated cell can express several hundred thousand copies of up to six different classical HLA class I molecules, each molecule containing a single peptide [2]. The repertoire of peptides presented by HLA molecules on the cell surface is often referred to as the immunopeptidome [3,4]. The HLA gene cluster is the most polymorphic cluster in the human genome, with already over 6500 different geneproducts described for the three major groups of HLA class I molecules (HLA-A, HLA-B, and HLA-C) [5], all having unique peptide binding properties. Therefore, each individual, depending on the inherited combination of up to six different HLA class I molecules, may present a unique immunopeptidome and therefore respond differently to infectious diseases, inflammatory conditions, autoimmune diseases and malignancies. Many genetic associations between HLA class I molecules and infectious diseases have been reported, including but not limited to, HIV/AIDS, hepatitis, leprosy, tuberculosis, malaria, leishmaniasis and schistosomiasis (reviewed in [6]). HLA-B molecules, the most polymorphic of the HLA allotypes, are frequently associated with disease outcome, either beneficial or detrimental. Moreover, HLA-B restricted T-cell responses have been shown to be immunodominant as compared to HLA-A and -C restricted responses, both within individuals [7–9], and at a population level [10]. This immunodominance is not well understood but might be explained by potential differences in the presentation of peptides between HLA loci or by differences in the quality and/or quantity of the restricted CD8+ T-cell responses. As the association between HLA-B and disease outcome surpasses single infectious agents, we hypothesised that general characteristics of HLA-B molecules, e.g. the diversity and/or the abundance of the peptides presented at the cell surface, may be driving these associations. Therefore, we here analysed the repertoire of naturally processed peptides presented on 4 different B lymphoblastoid cell-lines (BLCL), together expressing 5 different HLA-A, 6 HLA-B and 5 HLA-C molecules. The immunopeptidomes of these molecules have been analysed before and after infection with measles virus (MV) to investigate potential locus-specific preferences as well as changes upon infection in the presented repertoire. MV was chosen as a model pathogen, for several reasons. First, MV infects B cells, also in large scale cultures, being of use in immunopeptidome studies [11]. Second, T-cell mediated immunity has been shown to be important for clearance of MV infection and protection against re-infection. And third, several HLA-B molecules have been shown to be associated with higher immune levels after MV vaccination [12,13]. Peptide-HLA complexes were affinity-purified (...truncated)