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The Behcet's disease risk variant HLA-B51/ ERAP1-Hap10 alters human CD8 T cell immunity

ABSTRACT: The ERAP1 haplotype Hap10 encodes for a variant allotype of the ER-resident peptide-trimming aminopeptidase ERAP1 with low enzymatic activity resembling functional KO. This haplotype recessively confers the highest risk for Behcet's diseases (BD) currently known, but only in carriers of HLA-B*51, the classical risk factor for the disease. The mechanistic implications and biological consequences of this epistatic relationship are unknown. Here, we aimed to determine its biological relevance and functional impact, including its effect on the HLA-class I peptidome. We generated CRISPR-Cas9 ERAP1 KOs from HLA-B51+ LCL and analyzed the HLA I-bound peptidome for peptide length differences. ERAP1 KO cells showed peptidomes with longer peptides above 9mer than WT LCL (which carried heterozygous ERAP1 Hap7/Hap2 encoding for an allotype with medium-high trimming activity), pointing to a disproportionately large number of under-trimmed peptides. Such under-trimmed peptides may alter immunogenicity likely through a change in immunodominance of the ensuing CD8 T cell response signifying the potential relevance of this finding to disease risk and adaptive immune responses in human ERAP1 Hap10/ HLA-B*51 carriers with and without BD. For a more detailed discussion, immunogenicity and immune-phenotype data, please see the related manuscript: Cavers et al. The Behcet's disease risk variant HLA-B51/ ERAP1-Hap10 alters human CD8 T cell immunity.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Beatrix Ueberheide

PROVIDER: MSV000089312 | MassIVE | Mon Apr 25 11:07:00 BST 2022

REPOSITORIES: MassIVE

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Project description: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.

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The Behcet's disease risk variant HLA-B51/ ERAP1-Hap10 alters human CD8 T cell immunity

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