Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

The recognition events thatmediate adaptive cellular immunity and regulate antibody responsesdependon intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction betweenT cells andAPCs, known as the immunological synapse, which mediates efficient delivery of effectormolecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR–pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion inmicrovesicles, whereas vacuolar protein sorting 4 (VPS4)mediates scissionofmicrovesicles from theT-cell plasmamembrane.The human immunodeficiency virus polyprotein Gag co-opts this process forbuddingof virus-likeparticles.B cells bearingcognatepMHC receiveTCRs fromTcells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestratesTCR sorting and release in extracellular microvesicles. Thesemicrovesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs. The nature of the biophysical environment that governs molecular domain organization at the immunological synapse remains unclear. Confinement of pMHC, TCRs and cytoplasm (Supplementary Fig. 1) suggests that a general diffusion barrier separates TCRs and cytoplasm at the immunological synapse centre fromthe rest of theTcell. Tobetter understand the basis for the observed central confinement of pMHC, TCRs and cytoplasm at the immunological synapse, we investigated CD4 T-cell immunological synapse formation using high-resolution optical imaging by total internal reflection fluorescence microscopy (TIRFM), integrated with transmission electron microscopy (TEM) and electron tomography. T cells from TcrAND transgenic mice form TCR microclusters in response to engagement by the cognate class II pMHC molecule I-E complexedwith themoth cytochromeC peptideMCC 88-103 (MCC– I-E)6. Over approximately 10min, TCR microclusters, together with bound pMHC, are transported on the cell surface to the immunological synapse centre, where they are consolidated into an immobilized domain. To follow ultrastructural changes associated with immunological synapse formation, TcrANDT cells were fixed after 5, 10, 15 and 20min of interaction with supported lipid bilayers containing MCC– I-E and ICAM-1, and imaged first by TIRFM and then by TEM. As a control, we used the non-cognate pMHC b2m–I-E, which did not arrestmotility or induce immunological synapse formation inTcrANDT cells (Supplementary Fig. 2a). TEM time series of TcrAND T cells forming immunological synapses on antigen-containing bilayers revealed changes in cellmorphology thatwere characteristic of antigen-induced cell polarization (Supplementary Fig. 2b–d).Notably, at the 10min time point, the centre of the T-cell contact interface showed an unexpected change inmorphology, from a planar plasmamembrane in continuous close apposition with the planar bilayer (Fig. 1a) to the appearance of numerous microvesicles (Fig. 1b and Supplementary Fig. 2e), approximately 70nm in diameter (Supplementary Fig. 3a), that were contained within a central extracellular cavity (Fig. 1b). Microvesicle formation was antigen-specific, as they did not formwith bilayers containingb2m– I-E (Fig. 1a and Supplementary Fig. 2e), and could be modulated by the potency of the activating ligand, or by provision of costimulation (Fig. 1f and Supplementary Discussion). To visualize the distribution of microvesicles more clearly and verify their dissociation from the plasmamembrane, we performed dual-axis tomography (Supplementary Video 1) on four serial sections through an immunological synapse, ranging from150–250nmin thickness.Theassociated three-dimensional model (Fig. 1c–e and SupplementaryVideos 2 and 3) of the joined tomograms demonstrated that discrete extracellular microvesicles, with no connection to overlying plasmamembrane (Supplementary Fig. 4a, b), predominate in the central cavity, along with occasional membrane projections andmembrane buds of nascentmicrovesicles (Supplementary Fig. 4c–i). Comparison of the distributions of TCRs and microvesicles at the immunological synapse demonstrated that they were spatially correlated (Supplementary Fig. 3b–d). To establishwhether TCRs present at the immunological synapse centre were associated with microvesicles, we developed a novelmethod for optical–electronmicroscopy correlation, based on registration of TIRFM and corresponding TEM images of immunological synapses aligned using a microfabricated grid (Supplementary Fig. 5). Electron tomography of T-cell–bilayer interfaces confirmed the presence ofmicrovesicleswithin a roughly circular extracellular cavity at the immunological synapse centre (Fig. 1h, i and Supplementary Videos 4 and 5). Optical–electronmicroscopy correlation