Dataset Information

PI31 modulates the quantity of MHC class I loaded peptides in DCs

ABSTRACT: We used siRNAs to knockdown PI31 in primary DCs, followed by capturing HLA class I complexes using W6/32-conjugated immunoresin. Acid treatment abolished noncovalent interactions among the complex components. Using reverse-phase HPLC, we separated eluted peptides from the α-chain and β2-microglobulin of the HLA complexes and analysed eluted peptide fractions by LC-MS/MS.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Dendritic Cell, Blood

SUBMITTER: Nicola Ternette

LAB HEAD: Nicola Ternette

PROVIDER: PXD013247 | Pride | 2026-05-28

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CntrlA_peptides_1_1_0.mzid.gz	Mzid
	CntrlA_peptides_1_1_0.pride.mztab.gz	Mztab
	CntrlAb_peptides_1_1_0.mzid.gz	Mzid
	CntrlAb_peptides_1_1_0.pride.mztab.gz	Mztab
	CntrlB_peptides_1_1_0.mzid.gz	Mzid

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Publications

NOD2 and TLR2 Signal via TBK1 and PI31 to Direct Cross-Presentation and CD8 T Cell Responses.

Corridoni Daniele D Shiraishi Seiji S Chapman Thomas T Steevels Tessa T Muraro Daniele D Thézénas Marie-Laëtitia ML Prota Gennaro G Chen Ji-Li JL Gileadi Uzi U Ternette Nicola N Cerundolo Vincenzo V Simmons Alison A

Frontiers in immunology 20190430

NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8<sup>+</sup> T cells are important for immunity to such pathogens but how NOD2 and TLR2 induce antigen specific CD8<sup>+</sup> T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation i ...[more]

PMID: 31114588

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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.

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.