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.

The following data sets were generated

Article and author information

Author details

  1. David George Saliba

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  2. Pablo F Cespedes-Donoso

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1641-4107
  3. Štefan Bálint

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4470-5881
  4. Ewoud B Compeer

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3050-7633
  5. Kseniya Korobchevskaya

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  6. Salvatore Valvo

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  7. Viveka Mayya

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  8. Audun Kvalvaag

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  9. Yanchun Peng

    MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  10. Tao Dong

    MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3545-3758
  11. Maria-Laura Tognoli

    Department of Oncology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  12. Eric O'Neill

    Department of Oncology, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  13. Sarah Bonham

    Discovery Proteomics Facility, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  14. Roman Fischer

    Discovery Proteomics Facility, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9715-5951
  15. Benedikt M Kessler

    Discovery Proteomics Facility, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  16. Michael L Dustin

    Kennedy Institute of Rheumatology, Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom
    For correspondence
    michael.dustin@kennedy.ox.ac.uk
    Competing interests
    Michael L Dustin, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4983-6389

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.

Reviewing Editor

  1. Facundo D Batista, Ragon Institute of MGH, MIT and Harvard, United States

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).

Version history

  1. Received: April 9, 2019
  2. Accepted: August 28, 2019
  3. Accepted Manuscript published: August 30, 2019 (version 1)
  4. Version of Record published: September 17, 2019 (version 2)

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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  1. David George Saliba
  2. Pablo F Cespedes-Donoso
  3. Štefan Bálint
  4. Ewoud B Compeer
  5. Kseniya Korobchevskaya
  6. Salvatore Valvo
  7. Viveka Mayya
  8. Audun Kvalvaag
  9. Yanchun Peng
  10. Tao Dong
  11. Maria-Laura Tognoli
  12. Eric O'Neill
  13. Sarah Bonham
  14. Roman Fischer
  15. Benedikt M Kessler
  16. Michael L Dustin
(2019)
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

Share this article

https://doi.org/10.7554/eLife.47528

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