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.
Metrics
-
- 4,570
- views
-
- 646
- downloads
-
- 65
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Immunology and Inflammation
The adaptive T cell response is accompanied by continuous rewiring of the T cell’s electric and metabolic state. Ion channels and nutrient transporters integrate bioelectric and biochemical signals from the environment, setting cellular electric and metabolic states. Divergent electric and metabolic states contribute to T cell immunity or tolerance. Here, we report in mice that neuritin (Nrn1) contributes to tolerance development by modulating regulatory and effector T cell function. Nrn1 expression in regulatory T cells promotes its expansion and suppression function, while expression in the T effector cell dampens its inflammatory response. Nrn1 deficiency in mice causes dysregulation of ion channel and nutrient transporter expression in Treg and effector T cells, resulting in divergent metabolic outcomes and impacting autoimmune disease progression and recovery. These findings identify a novel immune function of the neurotrophic factor Nrn1 in regulating the T cell metabolic state in a cell context-dependent manner and modulating the outcome of an immune response.
-
- Immunology and Inflammation
Antibodies are powerful tools for the therapy and diagnosis of various diseases. In addition to conventional hybridoma-based screening, recombinant antibody-based screening has become a common choice; however, its application is hampered by two factors: (1) screening starts after Ig gene cloning and recombinant antibody production only, and (2) the antibody is composed of paired chains, heavy and light, commonly expressed by two independent expression vectors. Here, we introduce a method for the rapid screening of recombinant monoclonal antibodies by establishing a Golden Gate-based dual-expression vector and in-vivo expression of membrane-bound antibodies. Using this system, we demonstrate the rapid isolation of influenza cross-reactive antibodies with high affinity from immunized mice within 7 days. This system is particularly useful for isolating therapeutic or diagnostic antibodies, for example during foreseen pandemics.
