Dataset Information

IDENTIFICATION OF AN UNCONVENTIONAL SUBPEPTIDOME BOUND TO THE BEHÇET’S DISEASE - ASSOCIATED HLA–B*51:01 THAT IS REGULATED BY ENDOPLASMIC RETICULUM AMINOPEPTIDASE 1 (ERAP1)

ABSTRACT: Human leukocyte antigen (HLA) - B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are two strong predisposing genetic factors to Behçet's disease (BD). Previous studies have focused on two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry. The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2. This unconventional group of HLA-B*51:01-bound peptides was enriched for 8-mers (with fewer 9-mers) compared to Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides with the exception of the less abundant Leucine at position Ω. Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type - dependent manner. The HLA-B*51:01 peptidome includes a surprisingly high proportion of unconventional non-Pro/Ala2 peptides which are increased by ERAP1 silencing, mimicking the loss-of-function BD risk variant.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Nicola Ternette

LAB HEAD: Nicola Ternette

PROVIDER: PXD013064 | Pride | 2020-03-18

REPOSITORIES: Pride

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Identification of an Unconventional Subpeptidome Bound to the Behçet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1).

Chen Liye L Shi Hui H Koftori Danai D Sekine Takuya T Nicastri Annalisa A Ternette Nicola N Bowness Paul P

Molecular & cellular proteomics : MCP 20200311 5

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA- ...[more]

PMID: 32161166

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Project description:Infection or reactivation with human cytomegalovirus (HCMV) is still a major cause of morbidity and mortality in patients undergoing solid organ transplantation or allogeneic stem cell transplantation (alloSCT). Recent studies have shown that protection against latent HCMV infection relies on a much broader antigen spectrum than previously expected. The identification of novel immunogenic HCMV-derived peptides that facilitate HCMV control is therefore essential to improve immune monitoring of HCMV-specific T cells and HCMV vaccine development. In particular, only a few HCMV-derived peptides have been described for less common HLA alleles, such as HLA-A*03:01 and HLA-B*15:01, limiting the implementation of these techniques for a substantial number of patients. Here, we identified novel HCMV-derived HLA-A*03:01- and HLA-B*15:01-restricted immunogenic peptides by an innovative combined in vitro and in silico approach. We utilized the computational tools Peptide-PRISM (1) and PRICE (2) to analyze mass spectrometric (MS) and ribosome sequencing (ribo-seq) datasets, respectively. To refine our in silico approach, we utilized machine learning to rate the identified peptide candidates based on their translational activity, binding affinity and positioning within the small open reading frames (sORFs), with the goal of assessing their likelihood to be presented on HLA-A*03:01 and HLA-B*15:01 molecules. After identifying the highest-scoring 49 canonical and 49 cryptic potentially immunogenic peptides for each HLA allele, we assessed their immunogenicity by screening HCMV-seropositive and -seronegative healthy donors, as well as alloSCT patients for their peptide-specific T-cell responses. We used a stimulation procedure in two consecutive steps: peripheral blood cells (PBMCs) were stimulated starting with peptide pools and subsequently with potentially reactive single peptides. In vitro T-cell stimulation resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T cell response of ≥ 0.02%. Highest T-cell responses were identified against two HLA-A*03-restricted and three HLA-B*15-restricted canonical peptides with up to 3.42% of IFNγ+/CD8+ T cells in patients 180 days post alloSCT. In conclusion, we identified specific T-cell responses against eight canonical and two cryptic HLA-A*03:01- and HLA-B*15:01-restricted peptides in healthy individuals and post-transplanted patients and which were classified as immunogenic. Of these, six peptides are novel HCMV-derived, immunogenic peptides according to the UniProt database, broadening the spectrum of immunogenic HCMV-derived peptides for personalized immune monitoring and vaccine development.

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