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.
Reviewing Editor
- 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
- Received: April 9, 2019
- Accepted: August 28, 2019
- Accepted Manuscript published: August 30, 2019 (version 1)
- 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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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
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- Immunology and Inflammation
- Medicine
Background:
Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin.
Methods:
Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors.
Results:
We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01–2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 μg/mL, p=0.004).
Conclusions:
Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin.
Funding:
LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust).
Clinical trial number:
-
- Immunology and Inflammation
Trained immunity is the long-term functional reprogramming of innate immune cells, which results in altered responses toward a secondary challenge. Despite indoxyl sulfate (IS) being a potent stimulus associated with chronic kidney disease (CKD)-related inflammation, its impact on trained immunity has not been explored. Here, we demonstrate that IS induces trained immunity in monocytes via epigenetic and metabolic reprogramming, resulting in augmented cytokine production. Mechanistically, the aryl hydrocarbon receptor (AhR) contributes to IS-trained immunity by enhancing the expression of arachidonic acid (AA) metabolism-related genes such as arachidonate 5-lipoxygenase (ALOX5) and ALOX5 activating protein (ALOX5AP). Inhibition of AhR during IS training suppresses the induction of IS-trained immunity. Monocytes from end-stage renal disease (ESRD) patients have increased ALOX5 expression and after 6 days training, they exhibit enhanced TNF-α and IL-6 production to lipopolysaccharide (LPS). Furthermore, healthy control-derived monocytes trained with uremic sera from ESRD patients exhibit increased production of TNF-α and IL-6. Consistently, IS-trained mice and their splenic myeloid cells had increased production of TNF-α after in vivo and ex vivo LPS stimulation compared to that of control mice. These results provide insight into the role of IS in the induction of trained immunity, which is critical during inflammatory immune responses in CKD patients.
