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.

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.

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.

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. TNF acts via the inducible TNFR2 receptor and stimulates interleukin-33 (IL-33) secretion by myofibroblasts. In turn, stromal cell IL-33 acts as a potent stimulus for TNF production from immune cells. Targeting this reciprocal signaling pathway represents a novel therapeutic strategy to inhibit the low-grade inflammation in fibrosis and the mechanism that drives chronicity.

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. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell 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). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3'UTR, demonstrating that this versatile tool can be used to tune endogenous genes.

Orchestrated control of filaggrin-actin scaffolds underpins cornification.

Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and ß-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier.

CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.

Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.