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Acknowledgements We are grateful to our laboratory members and the Kennedy Institute of Rheumatology community for constructive scientific discussions, especially to James Felce, David Depoil, Jonathan Webber, Štefan Balint, Alexander Mørch, and Kristina Correa. We thank the technical support of Heather Rada, Kellie Johnson, and Ekaterina Zvezdova (the latter two from BioLegend). We thank Professor Catarina E. Hioe for kindly providing the HIV-1 gp120 protein. We would also like to thank all the anonymized blood donors who contributed to our study. This work was funded by Wellcome Trust Principal Research Fellowship 100262Z/12/Z, the ERC Advanced Grant (SYNECT AdG 670930), and the Kennedy Trust for Rheumatology Research (KTRR) (all three to M.L.D.). P.F.C.D was supported by EMBO Long-Term Fellowship (ALTF 1420–2015, in conjunction with the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Sklodowska-Curie Actions) and Oxford-Bristol Myers Squibb Fellowship. A.K. was supported by H2020 and the Research Council of Norway (in conjunction with Marie Sklodowska-Curie Actions 275466; to A.K.). M.F. and H.C.Y. thank the Wellcome Trust (212343/Z/18/Z) and EPSRC (EP/S004459/1). The eTIRF-SIM platform was built-in collaboration with Micron (www.micronoxford.com), an Oxford-wide advanced microscopy technology consortium supported by Wellcome Strategic Awards (091911 and 107457), and with additional funds from an MRC/EPSRC/BBSRC next-generation imaging award and the Kennedy Trust for Rheumatology Research through the Kennedy Institute Cell Dynamics Platform. We acknowledge the generous support of the Kennedy Trust for Rheumatology Research, IDRM, and Carl Zeiss GMBH for the Airyscan LSM 980 confocal microscope used in this research. Y.P., T.D., and R.F. were supported by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS), China (grant number: 2018-I2M-2-002) and UK Medical Research Council (MRC); E.S. was supported by Newton, The immunological synapse is a molecular hub that facilitates the delivery of three activation signals, namely antigen, costimulation/corepression and cytokines, from antigen-presenting cells (APC) to T cells. T cells release a fourth class of signaling entities, trans-synaptic vesicles (tSV), to mediate bidirectional communication. Here we present bead-supported lipid bilayers (BSLB) as versatile synthetic APCs to capture, characterize and advance the understanding of tSV biogenesis. Specifically, the integration of juxtacrine signals, such as CD40 and antigen, results in the adaptive tailoring and release of tSV, which differ in size, yields and immune receptor cargo compared with steadily released extracellular vesicles (EVs). Focusing on CD40L+ tSV as model effectors, we show that PD-L1 trans-presentation together with TSG101, ADAM10 and CD81 are key in determining CD40L vesicular release. Lastly, we find greater RNA-binding protein and microRNA content in tSV compared with EVs, supporting the specialized role of tSV as intercellular messengers., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Mechanical force is a fundamental regulator of cell phenotype. Myofibroblasts are central mediators of fibrosis, a major unmet clinical need characterised by the deposition of excessive matrix proteins. Traction forces of myofibroblasts play a key role in remodelling the matrix and modulate the activities of embedded stromal cells. Here, we employ a combination of unsupervised computational analysis, cytoskeletal profiling and single cell traction force microscopy as a functional readout to uncover how the complex spatiotemporal dynamics and mechanics of living human myofibroblast shape sub-cellular profiling of traction forces in fibrosis. We resolve distinct biophysical communities of myofibroblasts, and our results provide a new paradigm for studying functional heterogeneity in human stromal cells.

T cells comprise functionally diverse subtypes. Although activated via a conserved scheme of antigen recognition by their T cell receptor, they elicit heterogeneous activation and effector responses. Such functional diversity has been appreciated in gene expression studies, functional assays, and disease models. Yet, our understanding of the principles underlying T cell subtype-specific activation and antigen recognition in the immunological synapse remains limited. This is primarily due to difficulties in primary T cell visualization at high spatiotemporal resolution and the adoption of tractable transformed T cell systems for cell biological experiments that may not correctly represent primary T cell constitutional diversity. Here, we discuss recent findings regarding the architectural and dynamic diversity of the immunological synapse and state-of-the-art methodologies that can be utilized to provide clues on how biological and biophysical differences in synaptic make-up could govern functional divergences in T cell subtypes.