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

AbstractCell communication through extracellular vesicles is an emerging topic in biology, including communication between cells of the immune system. Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and intercellular adhesion molecule-1 (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 their ability to incorporate 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. Our results demonstrate selective enrichment of CD40 ligand (CD40L) and inducible T-cell costimulator (ICOS) in SE in response to addition of CD40 and ICOS ligand (ICOSL), respectively, to SLB presenting TCR ligands and ICAM-1. TCR triggering mobilized intracellular CD40L to the T cells surface at the IS, where it engaged CD40 to enable sorting into SE. SEs were enriched in tetraspanins and bone marrow stromal cell antigen 2 (BST-2) by immunofluorescence and TCR signalling and endosomal sorting complexes required for transport by proteomics. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2 microclusters. CD40L in SE retains the capacity to induce dendritic cell (DC) maturation and cytokine production. SE enabled helper T cells to release effectors physically linked to TCR.One Sentence SummaryTCR and CD40L microclusters can be linked in synaptic ectosomes (extracellular vesicles) that are released in the immunological synapse by helper T cells and induce dendritic cell maturation and cytokine production.

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

eLife ◽

10.7554/elife.47528 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

David G Saliba ◽

Pablo F Céspedes-Donoso ◽

Štefan Bálint ◽

Ewoud B Compeer ◽

Kseniya Korobchevskaya ◽

...

Keyword(s):

T Cells ◽

Lipid Bilayers ◽

Super Resolution ◽

Synaptic Cleft ◽

Cell Receptor ◽

Cd40 Ligand ◽

Dendritic Cell Maturation ◽

Helper T Cells ◽

Supported Lipid Bilayers ◽

Selective Enrichment

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.

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Glycan-Modified Melanoma-Derived Apoptotic Extracellular Vesicles as Antigen Source for Anti-Tumor Vaccination

Cancers ◽

10.3390/cancers11091266 ◽

2019 ◽

Vol 11 (9) ◽

pp. 1266 ◽

Cited By ~ 5

Author(s):

Horrevorts ◽

Stolk ◽

Ven ◽

Hulst ◽

Hof ◽

...

Keyword(s):

T Cell ◽

Dendritic Cell ◽

Extracellular Vesicles ◽

T Cell Responses ◽

Intercellular Adhesion Molecule ◽

Antigen Delivery ◽

Vaccine Strategy ◽

Cell Responses ◽

High Mannose

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

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Bystander T-Cells Support Clonal T-Cell Activation by Controlling the Release of Dendritic Cell-Derived Immune-Stimulatory Extracellular Vesicles

Frontiers in Immunology ◽

10.3389/fimmu.2019.00448 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 10

Author(s):

Marthe F. S. Lindenbergh ◽

Daniëlle G. J. Koerhuis ◽

Ellen G. F. Borg ◽

Esther M. van ‘t Veld ◽

Tom A. P. Driedonks ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dendritic Cell ◽

Extracellular Vesicles ◽

T Cell Activation ◽

Cell Activation

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Calcium Signaling in T Cells Is Induced by Binding to Nickel-Chelating Lipids in Supported Lipid Bilayers

Frontiers in Physiology ◽

10.3389/fphys.2020.613367 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tommy Dam ◽

Victoria Junghans ◽

Jane Humphrey ◽

Manto Chouliara ◽

Peter Jönsson

Keyword(s):

T Cells ◽

T Cell ◽

Calcium Signaling ◽

Lipid Bilayers ◽

Cell Activation ◽

Molecular Mechanisms ◽

Cell Receptor ◽

Model Systems ◽

Supported Lipid Bilayers ◽

Ligand Free

Supported lipid bilayers (SLBs) are one of the most common cell-membrane model systems to study cell-cell interactions. Nickel-chelating lipids are frequently used to functionalize the SLB with polyhistidine-tagged ligands. We show here that these lipids by themselves can induce calcium signaling in T cells, also when having protein ligands on the SLB. This is important to avoid “false” signaling events in cell studies with SLBs, but also to better understand the molecular mechanisms involved in T-cell signaling. Jurkat T cells transfected with the non-signaling molecule rat CD48 were found to bind to ligand-free SLBs containing ≥2 wt% nickel-chelating lipids upon which calcium signaling was induced. This signaling fraction steadily increased from 24 to 60% when increasing the amount of nickel-chelating lipids from 2 to 10 wt%. Both the signaling fraction and signaling time did not change significantly compared to ligand-free SLBs when adding the CD48-ligand rat CD2 to the SLB. Blocking the SLB with bovine serum albumin reduced the signaling fraction to 11%, while preserving CD2 binding and the exclusion of the phosphatase CD45 from the cell-SLB contacts. Thus, CD45 exclusion alone was not sufficient to result in calcium signaling. In addition, more cells signaled on ligand-free SLBs with copper-chelating lipids instead of nickel-chelating lipids and the signaling was found to be predominantly via T-cell receptor (TCR) triggering. Hence, it is possible that the nickel-chelating lipids act as ligands to the cell’s TCRs, an interaction that needs to be blocked to avoid unwanted cell activation.

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