Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T-cells
- University of Oxford, United Kingdom
Abstract
Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signalling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.
Data availability
The Mass spec data set has been deposited in ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD007988.
Article and author information
Author details
Pablo F Cespedes-Donoso
Funding
European Commission (AdG 670930)
- David George Saliba
- Pablo F Cespedes-Donoso
- Štefan Bálint
- Ewoud B Compeer
- Michael L Dustin
Wellcome (PRF 100262)
- Michael L Dustin
Cancer Research UK (UK A19277)
- Eric O'Neill
Chinese Academy of Sciences (2018-I2M-2-002)
- Yanchun Peng
- Tao Dong
National Institutes of Health (N/A)
- Michael L Dustin
Kennedy Trust for Rheumatology Research (N/A)
- Michael L Dustin
European Molecular Biology Organization (ALTF 1420-2015)
- Pablo F Cespedes-Donoso
The Research Council of Norway in conjunction with Marie Sklodowska-Curie Actions (275466)
- Audun Kvalvaag
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: Leukapheresis products (non-clinical and de-identified) from donor blood were used as a source of human T cells and monocytes. The Non-Clinical Issue division of National Health Service approved the use of leukapheresis reduction (LRS) chambers products at the University of Oxford (REC 11/H0711/7). Clone 35 was isolated from a healthy volunteer where written informed consent was given. Ethical approval was obtained from the University of Oxford Tropical Ethics Committee (OXTREC).
Copyright
© 2019, Saliba et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T-cells
eLife 8:e47528.
https://doi.org/10.7554/eLife.47528
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