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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Identification of TNFR2 and IL-33 as therapeutic targets in localized fibrosis.

Dissecting the molecular landscape of fibrotic disease, a major unmet need, will inform the development of novel treatment strategies to target disease progression and identify desperately needed therapeutic targets. Here, we provide a detailed single-cell analysis of the immune landscape in Dupuytren's disease, a localized fibrotic condition of the hand, and identify a pathogenic signaling circuit between stromal and immune cells. We demonstrate M2 macrophages and mast cells as key cellular sources of tumor necrosis factor (TNF) that promotes myofibroblast development.

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 and its immunophenotype Interestingly, we found that differentiated CD103 cancer-specific CTLs expressed the active form of TGFβ1 to continually self-regulate CD103 expression, without relying on external TGFβ1-producing cells.

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.

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.

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.

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.

Addendum: Precise tuning of gene expression levels in mammalian cells.

Following re-sequencing of the miSFIT constructs used in the paper, two of the construct variants inserted into the 3'UTR of PD-1, namely '12C' and '17A, 18G', have been found to contain additional insertions not present in the other construct variants. The data points corresponding to these constructs in Figs. 2c, f and Supplementary Fig.

Cytoskeletal Control of Antigen-Dependent T Cell Activation.

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen.

Cytoskeletal actin patterns shape mast cell activation.

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation.

Precise tuning of gene expression levels in mammalian cells.

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs).