Synthetic antigen-presenting cells reveal the diversity and functional specialisation of extracellular vesicles composing the fourth signal of T cell immunological synapses

Abstract

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of synaptic vesicles (SVs) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with SVs and steadily shed EVs (sEVs). Altogether, our data provide evidence for a higher specialisation of SVs which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the SV output, which is also accompanied by SMAPs, we propose their classification as signal four.

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

“…The flexibility of the method allows the reconstitution of complex and reductionist membrane compositions to study the effects of ligands and their signals on the secretion of tSVs and supramolecular attack particles and their components. We have tested this technology on various T cells, including preactivated TH, CTL, Tregs, and CART 15 . This protocol also works for the measurement of synaptic particle release of freshly isolated and quiescent T cells.…”

“…This protocol also works for the measurement of synaptic particle release of freshly isolated and quiescent T cells. One limitation of using freshly isolated T cells is that these quiescent populations produce a different profile of trans-synaptic particles, which correlates with their cell surface composition (see 15 for more details). synaptic transfer (NST%) metric can be used (Figure 3C panel (ii) top equation).…”