Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells.

David G Saliba,Pablo F Céspedes-Donoso,Sarah Bonham,Kseniya Korobchevskaya,Maria-Laura Tognoli,Štefan Bálint,Viveka Mayya,Ewoud B Compeer,Benedikt M Kessler,Michael L Dustin,Eric O'Neill,Roman Fischer,Tao Dong,Salvatore Valvo,Yanchun Peng,Audun Kvalvaag

doi:10.7554/elife.47528

David G Saliba, Pablo F Céspedes-Donoso + Show 14 more

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

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Abstract

Planar supported lipid bilayers (PSLB) 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 signaling 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.

Highlights

Immune response communication depends on intercellular interactions of surface receptors expressed on T cells and antigen presenting cells (APC) via immunological synapses (IS), kinapses or stabilized microvilli (Cai et al, 2017; Mayya et al, 2018)
Due to challenges with fluorescent protein tagging of CD40L, we detected it in the IS using an anti-CD40L monoclonal antibodies (mAb), which has the caveat that it competes with CD40, but detects recruitment of CD40L to the IS (Papa et al, 2017)
We allowed T cells to form IS with bead supported lipid bilayers (BSLB) at a 1:1 ratio and disrupted the conjugates to allow analysis of material transferred from the T cell to the BSLB

Summary

Introduction

Immune response communication depends on intercellular interactions of surface receptors expressed on T cells and antigen presenting cells (APC) via immunological synapses (IS), kinapses or stabilized microvilli (Cai et al, 2017; Mayya et al, 2018). The IS is a platform for signal integration, and enables polarized delivery of effector function These include the polarized delivery of cytokines (Huse et al, 2006), nucleic acid containing exosomes (Mittelbrunn et al, 2011), and TCR enriched extracellular vesicles that bud directly into the synaptic cleft from the T cell side of the IS (Choudhuri et al, 2014). We define TCR enriched extracellular vesicles that are formed in and simultaneously exported across the IS as synaptic ectosomes (SE)

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