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Characterization of the HLA class I Ligandome from Extracellular vesicles

ABSTRACT: Extracellular vesicles have shown promise in the field of anti-tumor vaccines. EVs are antigen-presenting entities capable of eliciting potent T-cell responses and have therefore being explored as cell-free based peptide vaccine vectors. However, whether the EV ligandome sufficiently resembles the cell ligandome as to preserve antigenicity and immunogenicity of the ligands is still a crucial aspect to be addressed. In particular, when aiming to use EVs as cell-free vaccine vectors. To understand whether HLA-I complexes homing to EVs display the same ligand sequence features and characteristics than those homing to the cell surface, we used a highly sensitive MS-based immunopeptidomics approach to generate a comprehensive side-by-side analysis of the EV and whole-cell (WC) ligandomes. In this work we identified thousands of ligands derived from both EVs and WCs, which allowed a deep characterization of important ligand properties such as the occurrence of PTMs in both ligandomes. Our results indicate that EVs are as good as cells when it comes to antigen presentation, since the sequence properties of the antigens were conserved in EVs and WCs ligandomes. Interestingly, EVs were enriched in HLA-B ligands and cysteinylated peptides, which can ultimately affect the antigenicity and immunogenicity of the ligands and should be taken into consideration when developing cell-free EV-based peptide vaccination approaches.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

SUBMITTER: Julia Bauzá Martinez

LAB HEAD: Wei Wu

PROVIDER: PXD021177 | Pride | 2021-07-05

REPOSITORIES: Pride

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

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Characterization of the HLA class I Ligandome from Extracellular vesicles

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