ABSTRACT
The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by facilitating the exchange of information between cells engaged in physical contact. 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 to study 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 plasma membrane-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.
Competing Interest Statement
Ben Peacock, Alice Law, and Dimitri Aubert are employed by NanoFCM Co., Ltd.
