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

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.

Model membrane systems to reconstitute immune cell signaling

“…The application of direct stochastic optical reconstruction microscopy (dSTORM) to the imaging of SLBs further demonstrated that microclusters containing either CD40L or TCR, which are initially formed in the periphery and distal area of the contact, are sorted into the cSMAC and in a later step incorporated into synaptic ectosomes (SE) and kept as discrete structures. Importantly, it is also shown that EPN-1, which binds PIP 2 leading to membrane curvature and the formation of clathrin invaginations, also forms ring-like structures in the immediate periphery of the cSMAC [47]. This suggests that the cSMAC might comprise different domains participating in the compartmentalization of endocytosis, exocytosis, and EV budding at the IS.…”

“…Similar to GUVs, liposomes have proven useful in the reconstitution of lipid/protein biological scaffolds but have also allowed reconstitution of EVs with immunostimulant properties. Furthermore, they have aided the study of cellular processes such as ion channels and membrane fusion events during viral infection [47]. SUVs are also used as delivery platforms for drugs and vaccines as they can be coated with sensitive compounds and antigen for a more targeted delivery with improved stability in vivo [48,49].…”

“…A similar partition of the cSMAC was proposed by Griffiths et al for CD8 + cytotoxic T lymphocytes (CTL), in which a signaling domain surrounds an inner secretory region specialized in the delivery of cytotoxic signals from intracellular stores [106]. Because TCR + EVs are mostly originated from the PM and delivered in a polarized fashion at the cSMAC-as evidenced by both correlative fluorescence-electron microscopy and dSTORM on glass-SLBs-we have coined the more appropriate term SE to refer to these effector EVs [47,87]. As we have recently shown using a combination of both planar SLB and BSLB methods, PM-derived SEs incorporate a number of surface-bound ligands, such as ICOS and CD40L, which are centripetally recruited in microclusters to the synaptic cleft [47,107].…”

“…Because TCR + EVs are mostly originated from the PM and delivered in a polarized fashion at the cSMAC-as evidenced by both correlative fluorescence-electron microscopy and dSTORM on glass-SLBs-we have coined the more appropriate term SE to refer to these effector EVs [47,87]. As we have recently shown using a combination of both planar SLB and BSLB methods, PM-derived SEs incorporate a number of surface-bound ligands, such as ICOS and CD40L, which are centripetally recruited in microclusters to the synaptic cleft [47,107]. In a comparable way to CTLs, the synaptic release of EVs might also include vesicles stored in multivesicular bodies and containing effectors, such as CD40L and FasL [108][109][110][111][112].…”

Inducible Polarized Secretion of Exosomes in T and B Lymphocytes

“…Microvesicles or ectosomes budding from the Th cell plasma membrane and accumulating at the IS have been described [85] (Figure 2, right side panel). These shedding vesicles were enriched in TCR and capable to trigger B-lymphocyte signaling via pMHC-II stimulation [85,86]. Thus, it appears that distinct types of EV from Th lymphocytes are secreted at the IS.…”