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

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Abstract

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs)1. 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 between T cells and APCs, known as the immunological synapse2, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft3. 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 synapse4,5, which is characterized by central accumulation of TCRs5, adjacent to a secretory domain2, both surrounded by an adhesive ring4,5. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function4, this domain is itself largely devoid of TCR signalling activity5,6, and is characterized by an unexplained immobilization of TCR–pMHC complexes relative to the highly dynamic immunological synapse periphery4,5. 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)6 sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4)7,8 mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

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Figure 1: Antigen-induced release of TCR-enriched microvesicles at the centre of immunological synapse.
Figure 2: TCR-enriched microvesicles are post-signalling extracellular products of T-cell activation that retain pMHC-binding competence.
Figure 3: Biogenesis of TCR-enriched microvesicles is mediated by ESCRT proteins and antagonized by HIV Gag.
Figure 4: TSG101 selectively controls TCR transfer to B cells that signal in response to pMHC engagement by microvesicle-tethered TCRs.

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Acknowledgements

We thank G. Schütz for suggesting the photoactivation experiment, H. Chen for microfabrication, W. Sundquist for providing Gag–GFP, VPS4–GFP and VPS4dn–GFP constructs, P. Bieniasz for Gag–mCherry, GagΔL-mCherry, ALIX-GFP and CHMP4B–GFP constructs, the New York Structural Biology Center for electron microscopy tomography, support and instrumentation and members of the Dustin laboratory for helpful discussions and contributions of reagents. We thank J. Nance for the gift of biotinylated duramycin-linked biotin. This work was supported in part by a Cancer Research Institute fellowship and NIH grant K99AI093884 (K.C.), a Wellcome Trust Principal Research Fellowship (M.L.D.), a Kennedy Trust Senior Research Fellowship (M.L.D.) and NIH grants AI043542, AI045757, AI055037, AI088377, AI093884 and EY016586 (Nanomedicine Development Center).

Author information

K.C., M.L.D. and D.L.S. conceived the study, K.C. and J.L. designed and performed the experiments, E.W.R. performed sectioning, L.C.K. and J.T. designed and fabricated optical–electron microscopy reference grids, K.W.W. and S.G. made essential reagents, K.C. and M.L.D. wrote the manuscript. All authors edited the manuscript.

Correspondence to David L. Stokes or Michael L. Dustin.

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Supplementary information

Supplementary Information

This file contains the Supplementary Discussion, Supplementary References and Supplementary Figures 1-19. (PDF 3002 kb)

Tomogram of an extended volume of the IS center reconstructed from 4 ~150-250 nm serial sections of the IS center

AND T cells were fixed and processed for EM after 10 min interaction with supported lipid bilayers (SLB) containing MCC/I-Ek and ICAM-1. Dual-axis tomograms acquired at 14,500x magnification from 4 serial sections (~150 - 250 nm thickness) were aligned and joined to produce a segmented series encompassing ~ 5 µm3 of the IS center. Relative Z-position is indicated at top left, section number is indicated at top right, scale bar represents 1 µm. (MOV 12559 kb)

Three-dimensional model of organelle configuration and released microvesicles at the antigen-induced polarized IS

The model was constructed from the segmented tomogram in Supplementary Video 1 and is rotated 360° clockwise in the y-axis. The nuclear and plasma membranes are depicted at 50% transparency for clarity. (MOV 12168 kb)

Extracellular microvesicles predominate over plasma-membrane-tethered vesicles and membrane projections at the contact interface of the IS

a. The intracellular components of the IS are removed to reveal the contact interface between the T cell plasma membrane (green, 50% transparency) and supported lipid bilayer (SLB). MVB, multivesicular body; Cent./M.T., centrosome/microtubules. Horizontal scale bar, 250 nm. b. The plasma membrane is removed and the model rotated 90° clockwise in the x-axis to demonstrate the distribution of extracellular microvesicles, (plasma-membrane) tethered vesicle buds, and plasma membrane projections within the inter-membrane space of the T cell-SLB contact interface. Vertical scale bar, 250 nm c. Extracellular microvesicles shown without SLB and rotated 360° in the x-axis. (MOV 16482 kb)

En face segmented tomogram of the T cell IS center

Segmentation was performed on a dual-axis tomographic tilt-series acquired at 9,600x magnification from the first 50 nm section, that was cut parallel to the plane of the block face in contact with coverslips. Red asterisks indicate areas of cytoplasmic granularity within the T cell plasma membrane that surround the central cavity. SLB, supported lipid bilayer, scale bar 100 nm. (MOV 1061 kb)

En face model of the T cell IS center

The model was created from the segmented tomogram in Supplementary Video 3, and rotated around its horizontal axis and then around its vertical axis by 360° in 15° increments. The orientation of microvesicles with respect to the supported lipid bilayer (SLB) and T cell is indicated. The T cell plasma membrane (green) and microvesicles (orange) are delineated. (MOV 1353 kb)

AND T cells break IS symmetry and resume motility after IS formation, releasing TCR-enriched extracellular microvesicles

AND T cells were imaged for 60 min at 30 s intervals, after 5 min interaction with supported bilayers containing MCC/I-Ek (green) and ICAM-1(magenta). TCR (red) was imaged using fluorescently labeled H57 antibody Fab’ fragments. a-b. Central TCR accumulation, along with MCC/I-Ek, and subsequent release of TCR-enriched microvesicles from the IS center. c. Shown is the ICAM-1 distribution during IS formation at the sessile symmetrical IS, followed by asymmetric ICAM-1 polarization (white asterisk), brief radial oscillation of polarized ICAM-1 (white asterisks), and commitment to a direction for initial cell motility. d. Overlay of TCR, MCC/I-Ek and ICAM images with IRM images (grayscale) to delineate the extent of the cell contacts with the supported bilayer. Scale bar 3 µm. (MOV 1693 kb)

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Choudhuri, K., Llodrá, J., Roth, E. et al. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature 507, 118–123 (2014) doi:10.1038/nature12951

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