Dataset Information

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.

SUBMITTER: Mommen GP

PROVIDER: S-EPMC4824864 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Mommen Geert P M GP Marino Fabio F Meiring Hugo D HD Poelen Martien C M MC van Gaans-van den Brink Jacqueline A M JA Mohammed Shabaz S Heck Albert J R AJ van Els Cécile A C M CA

Molecular & cellular proteomics : MCP 20160113 4

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 ...[more]

PMID: 26764012

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Project description:Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.

Project description:BackgroundPolymorphic differences between human leukocyte antigen (HLA) molecules affect the specificity and conformation of their bound peptides and lead to differential selection of the T-cell repertoire. Mismatching during allogeneic transplantation can, therefore, lead to immunological reactions.Design and methodsWe investigated the structure-function relationships of six members of the HLA-B*41 allelic group that differ by six polymorphic amino acids, including positions 80, 95, 97 and 114 within the antigen-binding cleft. Peptide-binding motifs for B*41:01, *41:02, *41:03, *41:04, *41:05 and *41:06 were determined by sequencing self-peptides from recombinant B*41 molecules by electrospray ionization tandem mass spectrometry. The crystal structures of HLA-B*41:03 bound to a natural 16-mer self-ligand (AEMYGSVTEHPSPSPL) and HLA-B*41:04 bound to a natural 11-mer self-ligand (HEEAVSVDRVL) were solved.ResultsPeptide analysis revealed that all B*41 alleles have an identical anchor motif at peptide position 2 (glutamic acid), but differ in their choice of C-terminal pΩ anchor (proline, valine, leucine). Additionally, B*41:04 displayed a greater preference for long peptides (>10 residues) when compared to the other B*41 allomorphs, while the longest peptide to be eluted from the allelic group (a 16mer) was obtained from B*41:03. The crystal structures of HLA-B*41:03 and HLA-B*41:04 revealed that both alleles interact in a highly conserved manner with the terminal regions of their respective ligands, while micropolymorphism-induced changes in the steric and electrostatic properties of the antigen-binding cleft account for differences in peptide repertoire and auxiliary anchoring.ConclusionsDifferences in peptide repertoire, and peptide length specificity reflect the significant functional evolution of these closely related allotypes and signal their importance in allogeneic transplantation, especially B*41:03 and B*41:04, which accommodate longer peptides, creating structurally distinct peptide-HLA complexes.

Project description:Zwitterionic polysaccharide antigens (ZPSs) were recently shown to activate T cells in a class II major histocompatibility complex (MHCII)-dependent fashion requiring antigen presenting cell (APC)-mediated oxidative processing although little is known about the mechanism or affinity of carbohydrate presentation (Cobb BA, Wang Q, Tzianabos AO, Kasper DL. 2004. Polysaccharide processing and presentation by the MHCII pathway. Cell. 117:677-687). A recent study showed that the helical conformation of ZPSs (Wang Y, Kalka-Moll WM, Roehrl MH, Kasper DL. 2000. Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis. Proc Natl Acad Sci USA. 97:13478-13483; Choi YH, Roehrl MH, Kasper DL, Wang JY. 2002. A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides. Biochemistry. 41:15144-15151) is closely linked with immunogenic activity (Tzianabos AO, Onderdonk AB, Rosner B, Cisneros RL, Kasper DL. 1993. Structural features of polysaccharides that induce intra-abdominal abscesses. Science. 262:416-419) and is stabilized by a zwitterionic charge motif (Kreisman LS, Friedman JH, Neaga A, Cobb BA. 2007. Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism. Glycobiology. 17:46-55), suggesting a strong carbohydrate structure-function relationship. In this study, we show that PSA, the ZPS from Bacteroides fragilis, associates with MHCII at high affinity and 1:1 stoichiometry through a mechanism mirroring peptide presentation. Interestingly, PSA binding was mutually exclusive with common MHCII antigens and showed significant allelic differences in binding affinity. The antigen exchange factor HLA-DM that catalyzes peptide antigen association with MHCII also increased the rate of ZPS association and was required for APC presentation and ZPS-mediated T cell activation. Finally, the zwitterionic nature of these antigens was required only for MHCII binding, and not endocytosis, processing, or vesicular trafficking to MHCII-containing vesicles. This report is the first quantitative analysis of the binding mechanism of carbohydrate antigens with MHCII and leads to a novel model for nontraditional MHCII antigen presentation during bacterial infections.

Dataset Information

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

Publications

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

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