Not All T Cell Synapses Are Built the Same Way.

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.

Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.

Cytoskeletal actin dynamics are crucial for the activation of T-cells. Immortalised Jurkat T-cells have been the model system of choice to examine and correlate the dynamics of the actin cytoskeleton and the immunological synapse leading to T-cell activation. However, it has remained unclear whether immortalised cellular systems, such as Jurkat T-cells can recapitulate the cytoskeletal behaviour of primary T-cells. Studies delineating the cytoskeletal behaviour of Jurkat T-cells in comparison to primary T-cells are lacking. Here, we employ live-cell super-resolution microscopy to investigate the cytoskeletal actin organisation and dynamics of living primary and immortalised Jurkat T-cells at the appropriate spatiotemporal resolution. Under comparable activation conditions, we found differences in the architectural organisation and dynamics of Jurkat and primary mouse and human T-cells. Although the three main actin network architectures in Jurkat T-cells were reminiscent of primary T-cells, there were differences in the organisation and molecular mechanisms underlying these networks. Our results highlight mechanistic distinctions in the T-cell model system most utilised to study cytoskeletal actin dynamics.

Extended mechanical force measurements using structured illumination microscopy.

Quantifying cell generated mechanical forces is key to furthering our understanding of mechanobiology. Traction force microscopy (TFM) is one of the most broadly applied force probing technologies, but its sensitivity is strictly dependent on the spatio-temporal resolution of the underlying imaging system. In previous works, it was demonstrated that increased sampling densities of cell derived forces permitted by super-resolution fluorescence imaging enhanced the sensitivity of the TFM method. However, these recent advances to TFM based on super-resolution techniques were limited to slow acquisition speeds and high illumination powers. Here, we present three novel TFM approaches that, in combination with total internal reflection, structured illumination microscopy and astigmatism, improve the spatial and temporal performance in either two-dimensional or three-dimensional mechanical force quantification, while maintaining low illumination powers. These three techniques can be straightforwardly implemented on a single optical set-up offering a powerful platform to provide new insights into the physiological force generation in a wide range of biological studies. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Spatiotemporally Super-Resolved Volumetric Traction Force Microscopy.

Quantification of mechanical forces is a major challenge across biomedical sciences. Yet such measurements are essential to understanding the role of biomechanics in cell regulation and function. Traction force microscopy remains the most broadly applied force probing technology but typically restricts itself to single-plane two-dimensional quantifications with limited spatiotemporal resolution. Here, we introduce an enhanced force measurement technique combining 3D super-resolution fluorescence structural illumination microscopy and traction force microscopy (3D-SIM-TFM) offering increased spatiotemporal resolution, opening-up unprecedented insights into physiological three-dimensional force production in living cells.

Simultaneous Quantification of the Interplay Between Molecular Turnover and Cell Mechanics by AFM-FRAP.

Quantifying the adaptive mechanical behavior of living cells is essential for the understanding of their inner working and function. Yet, despite the establishment of quantitative methodologies correlating independent measurements of cell mechanics and its underlying molecular kinetics, explicit evidence and knowledge of the sensitivity of the feedback mechanisms of cells controlling their adaptive mechanics behavior remains elusive. Here, a combination of atomic force microscopy and fluorescence recovery after photobleaching is introduced offering simultaneous quantification and direct correlation of molecule kinetics and mechanics in living cells. Systematic application of this optomechanical atomic force microscopy-fluorescence recovery after photobleaching platform reveals changes in the actin turnover and filament lengths of ventral actin stress fibers in response to constant mechanical force at the apical actin cortex with a dynamic range from 0.1 to 10 nN, highlighting a direct relationship of active mechanosensation and adaptation of the cellular actin cytoskeleton. Simultaneous quantification of the relationship between molecule kinetics and cell mechanics may thus open-up unprecedented insights into adaptive mechanobiological mechanisms of cells.

Super-Resolved Traction Force Microscopy (STFM).

Measuring small forces is a major challenge in cell biology. Here we improve the spatial resolution and accuracy of force reconstruction of the well-established technique of traction force microscopy (TFM) using STED microscopy. The increased spatial resolution of STED-TFM (STFM) allows a greater than 5-fold higher sampling of the forces generated by the cell than conventional TFM, accessing the nano instead of the micron scale. This improvement is highlighted by computer simulations and an activating RBL cell model system.

Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

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.

Quantifying Immune Cell Force Generation Using Traction Force Microscopy.

Immune cells rely on the generation of mechanical force to carry out their function. Consequently, there is a pressing need for quantitative methodologies that permit the probing of the spatio-temporal distribution of mechanical forces generated by immune cells. In this chapter, we provide a guide to quantify immune cell force generation using traction force microscopy (TFM), with a specific focus on its application to the study of the T-cell immunological synapse.

Intracellular trafficking of HLA-E and its regulation.

Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

T-cell trans-synaptic vesicles are distinct and carry greater effector content than constitutive extracellular vesicles.

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.

Quantitative Methodologies to Dissect Immune Cell Mechanobiology.

Mechanobiology seeks to understand how cells integrate their biomechanics into their function and behavior. Unravelling the mechanisms underlying these mechanobiological processes is particularly important for immune cells in the context of the dynamic and complex tissue microenvironment. However, it remains largely unknown how cellular mechanical force generation and mechanical properties are regulated and integrated by immune cells, primarily due to a profound lack of technologies with sufficient sensitivity to quantify immune cell mechanics. In this review, we discuss the biological significance of mechanics for immune cells across length and time scales, and highlight several experimental methodologies for quantifying the mechanics of immune cells. Finally, we discuss the importance of quantifying the appropriate mechanical readout to accelerate insights into the mechanobiology of the immune response.

Quantifying cell-generated forces: Poisson's ratio matters.

Quantifying mechanical forces generated by cellular systems has led to key insights into a broad range of biological phenomena from cell adhesion to immune cell activation. Traction force microscopy (TFM), the most widely employed force measurement methodology, fundamentally relies on knowledge of the force-displacement relationship and mechanical properties of the substrate. Together with the elastic modulus, the Poisson's ratio is a basic material property that to date has largely been overlooked in TFM. Here, we evaluate the sensitivity of TFM to Poisson's ratio by employing a series of computer simulations and experimental data analysis. We demonstrate how applying the correct Poisson's ratio is important for accurate force reconstruction and develop a framework for the determination of error levels resulting from the misestimation of the Poisson's ratio. In addition, we provide experimental estimation of the Poisson's ratios of elastic substrates commonly applied in TFM. Our work thus highlights the role of Poisson's ratio underpinning cellular force quantification studied across many biological systems.

Astigmatic traction force microscopy (aTFM).

Quantifying small, rapidly progressing three-dimensional forces generated by cells remains a major challenge towards a more complete understanding of mechanobiology. Traction force microscopy is one of the most broadly applied force probing technologies but ascertaining three-dimensional information typically necessitates slow, multi-frame z-stack acquisition with limited sensitivity. Here, by performing traction force microscopy using fast single-frame astigmatic imaging coupled with total internal reflection fluorescence microscopy we improve the temporal resolution of three-dimensional mechanical force quantification up to 10-fold compared to its related super-resolution modalities. 2.5D astigmatic traction force microscopy (aTFM) thus enables live-cell force measurements approaching physiological sensitivity.

Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Quantitative Bio-Imaging Tools to Dissect the Interplay of Membrane and Cytoskeletal Actin Dynamics in Immune Cells.

Cellular function is reliant on the dynamic interplay between the plasma membrane and the actin cytoskeleton. This critical relationship is of particular importance in immune cells, where both the cytoskeleton and the plasma membrane work in concert to organize and potentiate immune signaling events. Despite their importance, there remains a critical gap in understanding how these respective dynamics are coupled, and how this coupling in turn may influence immune cell function from the bottom up. In this review, we highlight recent optical technologies that could provide strategies to investigate the simultaneous dynamics of both the cytoskeleton and membrane as well as their interplay, focusing on current and future applications in immune cells. We provide a guide of the spatio-temporal scale of each technique as well as highlighting novel probes and labels that have the potential to provide insights into membrane and cytoskeletal dynamics. The quantitative biophysical tools presented here provide a new and exciting route to uncover the relationship between plasma membrane and cytoskeletal dynamics that underlies immune cell function.

Single cell force profiling of human myofibroblasts reveals a biophysical spectrum of cell states.

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.

Author Correction: Cellular census of human fibrosis defines functionally distinct stromal cell types and states.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cellular census of human fibrosis defines functionally distinct stromal cell types and states.

Fibrotic disorders are some of the most devastating and poorly treated conditions in developed nations, yet effective therapeutics are not identified for many of them. A major barrier for the identification of targets and successful clinical translation is a limited understanding of the human fibrotic microenvironment. Here, we construct a stromal cell atlas of human fibrosis at single cell resolution from patients with Dupuytren's disease, a localized fibrotic condition of the hand. A molecular taxonomy of the fibrotic milieu characterises functionally distinct stromal cell types and states, including a subset of immune regulatory ICAM1+ fibroblasts. In developing fibrosis, myofibroblasts exist along an activation continuum of phenotypically distinct populations. We also show that the tetraspanin CD82 regulates cell cycle progression and can be used as a cell surface marker of myofibroblasts. These findings have important implications for targeting core pathogenic drivers of human fibrosis.

Self-Maintaining CD103+ Cancer-Specific T Cells Are Highly Energetic with Rapid Cytotoxic and Effector Responses.

Enrichment of CD103+ tumor-infiltrating T lymphocytes (TIL) is associated with improved outcomes in patients. However, the characteristics of human CD103+ cytotoxic CD8+ T cells (CTL) and their role in tumor control remain unclear. We investigated the features and antitumor mechanisms of CD103+ CTLs by assessing T-cell receptor (TCR)-matched CD103+ and CD103- cancer-specific CTL immunity in vitro and its immunophenotype ex vivo Interestingly, we found that differentiated CD103+ cancer-specific CTLs expressed the active form of TGFß1 to continually self-regulate CD103 expression, without relying on external TGFß1-producing cells. The presence of CD103 on CTLs improved TCR antigen sensitivity, which enabled faster cancer recognition and rapid antitumor cytotoxicity. These CD103+ CTLs had elevated energetic potential and faster migration capacity. However, they had increased inhibitory receptor coexpression and elevated T-cell apoptosis following prolonged cancer exposure. Our data provide fundamental insights into the properties of matured human CD103+ cancer-specific CTLs, which could have important implications for future designs of tissue-localized cancer immunotherapy strategies.