Elsevier

Molecular Immunology

Volume 51, Issue 2, June 2012, Pages 136-142
Molecular Immunology

Secreted HLA recapitulates the immunopeptidome and allows in-depth coverage of HLA A*02:01 ligands

https://doi.org/10.1016/j.molimm.2012.02.117Get rights and content

Abstract

HLA molecules are cell-surface glycoproteins that present peptides, derived from intracellular protein antigens, for surveillance by T lymphocytes. Secreted HLA (sHLA) technology is a powerful approach for studying these peptides, since it facilitates large-scale production of HLA-bound peptides. We compared secreted and membrane-bound forms of HLA A2 in terms of intracellular trafficking and their bound peptide repertoire (termed the immunopeptidome). We demonstrate that sHLA and membrane bound HLA (mHLA) negotiate intracellular compartments with similar maturation kinetics. Moreover, mass spectrometry revealed a substantial overlap in the immunopeptidome was observed when HLA A2-bound peptides were purified from various sources of sHLA and mHLA. By combining machine based algorithms with manual validation, we identified 1266 non-redundant peptides. Analysis of these peptides revealed a number bearing post-translational modifications, although some of these may arise spontaneously others represent modifications performed within the cell that survive antigen processing. Peptides bearing some of these modifications have not previously been described for HLA ligands, therefore, this compendium of 1266 non-redundant peptide sequences adds greatly to the existing database of HLA A2 ligands. Peptides from all sources displayed comparable HLA A2 consensus binding motifs, peptide lengths, predicted HLA A2 binding affinities and putative source antigens. We conclude that sHLA is a valid and useful technique for studying the immunopeptidome.

Highlights

► We validate secreted HLA technology for epitope discovery studies by comparing the assembly, transport and peptide cargo of membrane-bound and secreted HLA. ► Each form navigates the intracellular compartments with similar maturation kinetics. ► The 1266 unique HLA A2-bound peptides sequenced show large overlap between HLA forms. ► This represents the largest single HLA A2 peptide repertoire analysis and includes several novel post-translationally modified peptides.

Introduction

Human Leukocyte Antigen (HLA) molecules are cell-surface glycoproteins that present a vast array of small peptides, liberated via the intracellular proteolysis of diverse protein antigens, to T lymphocytes. This affords the immune system a sensitive means of monitoring cell health and detecting infection and cancer. It has been estimated that HLA molecules encoded by a single allele can present over 10,000 different peptides, which share a conserved motif representing ‘anchor residues’ responsible for binding to the specific MHC allele (Engelhard et al., 2002). The peptide repertoire is often referred to as the immunopeptidome (Admon and Bassani-Sternberg, 2011, Caron et al., 2005, Caron et al., 2011, Hickman and Yewdell, 2010, Perreault, 2010).

With advances in the speed, sensitivity and resolution of mass spectrometers, direct analysis of the immunopeptidome via peptide sequencing has become a viable option for studying HLA-bound peptides. By directly sequencing the peptides, we can identify epitopes arising from infectious agents, better characterise HLA peptide binding motifs, observe post-translationally modified HLA-bound peptides, and gain insight into changes in antigen presentation in various disease states. To facilitate such studies, cell lines have been engineered to secrete the ectodomain of class I HLA molecules (sHLA) (Prilliman et al., 1997). The transmembrane and cytosolic domains of the class I molecule are genetically removed, so that transfectants secrete HLA into the culture supernatant, from which it may be purified with high yields.

Investigators justify the use of sHLA as an experimental substitute for membrane-bound HLA with observations that eluted peptides usually agree with published motifs (Barnea et al., 2002, Berg et al., 2004, Prilliman et al., 1997) and the binding affinities of particular peptides seem similar (Buchli et al., 2004). More recent studies have more directly compared secreted HLA and membrane bound HLA peptide repertoires (Bassani-Sternberg et al., 2010, Ben Dror et al., 2010) but there has yet to be a direct biochemical comparison of secreted to membrane-bound HLA. Therefore to investigate if their peptide repertoires may differ we compare the intracellular maturation and peptide repertoire of secreted and membrane-bound forms of HLA A2 to validate the use of sHLA for studies of naturally presented peptides. We observe that membrane and secreted forms of HLA A*02:01 mature with comparable kinetics. We also sequenced a large number of HLA-bound peptides (1266 non-redundant sequences) and demonstrate that secreted and membrane-bound forms of HLA have very similar peptide repertoires. Analysis of these peptides revealed several peptides bearing novel post-translational modifications not previously reported as HLA ligands and this compendium of sequences adds greatly to the existing database of HLA A2 ligands.

Section snippets

HLA A*02:01 gene constructs, transfection and large scale production

The HLA A*02:01 gene was truncated after exon 4 to generate a sHLA construct lacking transmembrane and cytoplasmic domains (as described (Prilliman et al., 1997)). Truncated and full-length genes were cloned into pcDNA3.1/V5-His–TOPO eukaryotic expression vectors (Invitrogen). Vectors were stably transfected into the human B-lymphoblastoid cell line 721.221, which is deficient in endogenous HLA class I (Storkus et al., 1989). Clonal transfectants expressing soluble (sA2.221) or membrane bound

Results

Secreted HLA is an increasingly popular tool for producing large quantities of HLA to facilitate studies of the immunopeptidome. However, it is not well established whether removal of the transmembrane anchor has subtle effects on the peptide repertoire. In order to compare membrane-bound and secreted forms of HLA, we generated two clonal transfectant cell lines, termed mA2.221 and sA2.221, transfected with the full-length or truncated HLA A*02:01 gene such that they express membrane-bound and

Discussion

Secreted HLA technology presents clear practical advantages for researchers. Instead of needing to lyse cells to extract HLA from the membrane, with the concomitant lengthy centrifugation steps and loss of yield, sHLA is simply purified from cell culture supernatant. The yield of soluble material may be even higher since sHLA accumulates as cells continually secrete it, whereas there is a limit to the amount of HLA cells can express on the cell surface, due to continual turnover at the cell

Conflict of interest

The authors have declared no conflict of interest.

Role of the funding source

This work was supported by the National Health and Medical Research Council of Australia (Project Grants 508929 and 508927, Senior Research Fellowship to AWP) and the Australian Research Council (LP0883541).

The funding sources had no other involvement in any part of the study, such as design, data analysis and report writing for publication.

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