Dataset Information

HLA B-27 rats peptidome - The HLA-B27 peptidome in vivo in spondyloarthritis-susceptible HLA-B27 transgenic rats and the effect of ERAP1 deletion

ABSTRACT: HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large-scale quantitative mass-spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of ERAP1, which reduced ERAP1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of ERAP1 affected approximately one third of the B27 peptidome, but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of ERAP1. Deletion on ERAP1 was permissive for the AS-like phenotype. Deletion of ERAP1 increased mean peptide length, and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser or Lys. The presence of ERAP1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Spleen

DISEASE(S): Ankylosing Spondylitis

SUBMITTER: Dganit Melamed Kadosh

LAB HEAD: Arie Admon

PROVIDER: PXD005502 | Pride | 2017-02-13

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MaxQuant_1.5.0.25.7z	Other
	RatA1-C10andHLA_B7andlewisforpride18.7.16.7z	Other
	RatDAZL-WT_Lewisforpride4.9.16.rar	Other
	Seq35416_QE.raw	Raw
	Seq35417_QE.raw	Raw

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Publications

The Human Leukocyte Antigen (HLA)-B27 Peptidome <i>in Vivo</i>, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

Barnea Eilon E Melamed Kadosh Dganit D Haimovich Yael Y Satumtira Nimman N Dorris Martha L ML Nguyen Mylinh T MT Hammer Robert E RE Tran Tri M TM Colbert Robert A RA Taurog Joel D JD Admon Arie A

Molecular & cellular proteomics : MCP 20170210 4

HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used larg ...[more]

PMID: 28188227

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Project description:Background The striking association of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years. However, its pathophysiological significance remains incompletely understood. In the HLA-B27/human-β2 microglobulin (hβ2m) transgenic rat (B27 rat) that spontaneously develops inflammatory phenotype characteristic of SpA, myeloid cells expressing the HLA-B27/h2m transgene are critical for disease induction and functionally impaired. Recently, we produced HLA-B27/hβ2m transgenic Drosophila to study non-canonical effects of HLA-B27. We showed that HLA-B27/h2m expressed in Drosophila wing imaginal disc deregulated BMP pathway by interacting physically with the type I bone morphogenetic protein (BMP) receptor (BMPR1) Saxophone (Sax), leading to a loss of crossveins. Methods Genetic interaction was studied between the activin/TGF pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs from transgenic Drosophila by high-performance liquid chromatography and mass spectrometry of the peptide fraction eluted from affinity purified HLA-B27. In mesenteric lymph node (mLN) T cells from B27 rats, physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s was assessed by proximity ligation assay and phosphorylation of SMADs and proteins of the non-canonical BMP/TGF pathway induced by its ligands was assessed by flow cytometry. Transcript level of several target genes of the TGF pathway was evaluated by real-time quantitative polymerase chain reaction in mLN subsets of T cells from B27 and control nontransgenic rats, with and without treatment by TGF. Results We showed that, in addition to the BMP pathway, inappropriate signaling through the activin/transforming growth factor β (TGFβ) pathway, involving Baboon (Babo) BMPR1, also contributed to the crossveinless wing phenotype. We identified a set of peptides bound to HLA-B27 with canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc, despite the lack of peptide loading complex. We then demonstrated specific physical interaction, between HLA-B27/h2m and both mammalian orthologues of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGF receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from adult and premorbid B27 rats, showing evidence for deregulated TGF pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFexposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly. Tgfb1 expression was reduced in naïve T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern. Conclusions This study highlights a complex interplay between HLA-B27 and the BMP/TGFβ pathways in both Drosophila and B27 rat. Given the importance of the TGF pathway in CD4+ T cells differenciation and regulation, the disturbance caused by HLA-B27 could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and the altered regulatory T cell phenotype observed in B27 rat.

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.

Project description:Background Clinical success of T cell receptor (TCR) gene therapy has previously been demonstrated for NY-ESO-1 TCR gene therapy. To increase numbers of cancer patients that can be treated with TCR gene therapy we aimed to identify a novel set of high-affinity cancer specific TCRs targeting different cancer testis (CT) antigens in prevalent HLA class I alleles. Methods In this study, we selected based on publicly available gene expression databases the most promising CT genes to target. From these selected genes we identified by HLA peptidomics the naturally processed and presented HLA class I peptides. With these peptide-HLA tetramers were generated, and by single cell sorting CT specific CD8+ T cells were selected, and expanded from the allo-HLA repertoire of healthy donors. By several functional assays high avidity CT-specific T cell clones with safe recognition pattern were selected. To evaluate the potential for clinical application in TCR gene therapy, TCRs were sequenced and transferred into peripheral blood derived CD8+ T cells. Results In total we identified, 7 novel CT-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6 and MAGE-A9 in the context of human leukocyte antigen(HLA) -A1, -A2, -A3, -B7, -B35 and -C7. TCR gene transfer into CD8⁺ T cells resulted in efficient cytokine production and cytotoxicity of variety of different tumor types without detectable cross-reactivity. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells was observed in an orthotopic xenograft model for established multiple myeloma, in which bone marrow located tumor cells were completely eradicated after T cell injection. Conclusion The identification of 7 novel CT-specific TCRs, reactive against CT antigens presented in a variety of different HLA class I alleles, allows selection of therapeutic TCRs for an increased number of cancer patients, and will improve development of personalized TCR gene therapy.