Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Structure of a fully assembled γδ T cell antigen receptor.

T cells in jawed vertebrates comprise two lineages, αß T cells and γδ T cells, defined by the antigen receptors they express-that is, αß and γδ T cell receptors (TCRs), respectively. The two lineages have different immunological roles, requiring that γδ TCRs recognize more structurally diverse ligands1. Nevertheless, the receptors use shared CD3 subunits to initiate signalling. Whereas the structural organization of αß TCRs is understood2,3, the architecture of γδ TCRs is unknown. Here, we used cryogenic electron microscopy to determine the structure of a fully assembled, MR1-reactive, human Vγ8Vδ3 TCR-CD3δγε2ζ2 complex bound by anti-CD3ε antibody Fab fragments4,5. The arrangement of CD3 subunits in γδ and αß TCRs is conserved and, although the transmembrane α-helices of the TCR-γδ and -αß subunits differ markedly in sequence, packing of the eight transmembrane-helix bundles is similar. However, in contrast to the apparently rigid αß TCR2,3,6, the γδ TCR exhibits considerable conformational heterogeneity owing to the ligand-binding TCR-γδ subunits being tethered to the CD3 subunits by their transmembrane regions only. Reducing this conformational heterogeneity by transfer of the Vγ8Vδ3 TCR variable domains to an αß TCR enhanced receptor signalling, suggesting that γδ TCR organization reflects a compromise between efficient signalling and the ability to engage structurally diverse ligands. Our findings reveal the marked structural plasticity of the TCR on evolutionary timescales, and recast it as a highly versatile receptor capable of initiating signalling as either a rigid or flexible structure.

Genome-scale identification of cellular pathways required for cell surface recognition.

Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABAB receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABAB receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties.

An enhanced CRISPR repressor for targeted mammalian gene regulation.

The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

CEN-tools: an integrative platform to identify the contexts of essential genes.

An emerging theme from large-scale genetic screens that identify genes essential for cell fitness is that essentiality of a given gene is highly context-specific. Identification of such contexts could be the key to defining gene function and also to develop novel therapeutic interventions. Here, we present Context-specific Essentiality Network-tools (CEN-tools), a website and python package, in which users can interrogate the essentiality of a gene from large-scale genome-scale CRISPR screens in a number of biological contexts including tissue of origin, mutation profiles, expression levels and drug responses. We show that CEN-tools is suitable for the systematic identification of genetic dependencies and for more targeted queries. The associations between genes and a given context are represented as dependency networks (CENs), and we demonstrate the utility of these networks in elucidating novel gene functions. In addition, we integrate the dependency networks with existing protein-protein interaction networks to reveal context-dependent essential cellular pathways in cancer cells. Together, we demonstrate the applicability of CEN-tools in aiding the current efforts to define the human cellular dependency map.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1ß. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms.

The cellular signalling process is a highly complex mechanism, involving multiple players, which together orchestrate the cell's response to environmental changes and perturbations. Given the multitude of genes that participate in the process of cellular signalling, its study in a genome-wide manner has proven challenging. Recent advances in gene editing technologies, including clustered regularly-interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) approaches, have opened new opportunities to investigate global regulatory signalling programs of cells in an unbiased manner. In this review, we focus on how the application of pooled genetic screening approaches using the CRISPR/Cas9 system has contributed to a systematic understanding of cellular signalling processes in normal and disease contexts.

Transcriptional reprogramming via signaling domains of CD2, CD28, and 4-1BB.

Costimulatory signals provided to T cells during antigen encounter have a decisive role in the outcome of immune responses. Here, we used chimeric receptors harboring the extracellular domain of mouse inducible T cell costimulator (mICOS) to study transcriptional activation mediated by cytoplasmic sequences of the major T cell costimulatory receptors CD28, 4-1BB, and CD2. The chimeric receptors were introduced in a T cell reporter platform that allows to simultaneously evaluate nuclear factor κB (NF-κB), NFAT, and AP-1 activation. Engagement of the chimeric receptors induced distinct transcriptional profiles. CD28 signaling activated all three transcription factors, whereas 4-1BB strongly promoted NF-κB and AP-1 but downregulated NFAT activity. CD2 signals resulted in the strongest upregulation of NFAT. Transcriptome analysis revealed pronounced and distinct gene expression signatures upon CD2 and 4-1BB signaling. Using the intracellular sequence of CD28, we exemplify that distinct signaling motifs endow chimeric receptors with different costimulatory capacities.

A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells.

The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.

The current state and future of T-cell exhaustion research.

'Exhaustion' is a term used to describe a state of native and redirected T-cell hypo-responsiveness resulting from persistent antigen exposure during chronic viral infections or cancer. Although a well-established phenotype across mice and humans, exhaustion at the molecular level remains poorly defined and inconsistent across the literature. This is, in part, due to an overreliance on surface receptors to define these cells and explain exhaustive behaviours, an incomplete understanding of how exhaustion arises, and a lack of clarity over whether exhaustion is the same across contexts, e.g. chronic viral infections versus cancer. With the development of systems-based genetic approaches such as single-cell RNA-seq and CRISPR screens applied to in vivo data, we are moving closer to a consensus view of exhaustion, although understanding how it arises remains challenging given the difficulty in manipulating the in vivo setting. Accordingly, producing and studying exhausted T-cells ex vivo are burgeoning, allowing experiments to be conducted at scale up and with high throughput. Here, we first review what is currently known about T-cell exhaustion and how it's being studied. We then discuss how improvements in their method of isolation/production and examining the impact of different microenvironmental signals and cell interactions have now become an active area of research. Finally, we discuss what the future holds for the analysis of this physiological condition and, given the diversity of ways in which exhausted cells are now being generated, propose the adoption of a unified approach to clearly defining exhaustion using a set of metabolic-, epigenetic-, transcriptional-, and activation-based phenotypic markers, that we call 'M.E.T.A'.

Highlights from the 1st European cancer dependency map symposium and workshop.

The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution.

Cell Surface Mechanics Gate Embryonic Stem Cell Differentiation.

Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency.

CRISPR-Based Editing Techniques for Genetic Manipulation of Primary T Cells.

While clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing techniques have been widely adapted for use in immortalised immune cells, efficient manipulation of primary T cells has proved to be more challenging. Nonetheless, the rapid expansion of the CRISPR toolbox accompanied by the development of techniques for delivery of CRISPR components into primary T cells now affords the possibility to genetically manipulate primary T cells both with precision and at scale. Here, we review the key features of the techniques for primary T cell editing and discuss how the new generation of CRISPR-based tools may advance genetic engineering of these immune cells. This improved ability to genetically manipulate primary T cells will further enhance our fundamental understanding of cellular signalling and transcriptional networks in T cells and more importantly has the potential to revolutionise T cell-based therapies.

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

Investigating Cellular Recognition Using CRISPR/Cas9 Genetic Screening.

Neighbouring cells can recognise and communicate with each other by direct binding between cell surface receptor and ligand pairs. Examples of cellular recognition events include pathogen entry into a host cell, sperm-egg fusion, and self/nonself discrimination by the immune system. Despite growing appreciation of cell surface recognition molecules as potential therapeutic targets, identifying key factors contributing to cellular recognition remains technically challenging to perform on a genome-wide scale. Recently, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) knockout or activation (CRISPR-KO/CRISPRa) screens have been applied to identify the molecular determinants of cellular recognition. In this review, we discuss how CRISPR-KO/CRISPRa screening has contributed to our understanding of cellular recognition processes, and how it can be applied to investigate these important interactions in a range of biological contexts.

Large-scale datasets uncovering cell signalling networks in cancer: context matters.

Cell signaling pathways control the responses of cells to external perturbations. Depending on the cell's internal state, genetic background and environmental context, signaling pathways rewire to elicit the appropriate response. Such rewiring also can lead to cancer development and progression or cause resistance to therapies. While there exist static maps of annotated pathways, they do not capture these rewired networks. As large-scale datasets across multiple contexts and patients are becoming available the doors to infer and study context-specific signaling network have also opened. In this review, we will highlight the most recent approaches to study context-specific signaling networks using large-scale omics and genetic perturbation datasets, with a focus on studies of cancer and cancer-related pathways.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

An Antibody Screen of a Plasmodium vivax Antigen Library Identifies Novel Merozoite Proteins Associated with Clinical Protection.

BACKGROUND: Elimination of Plasmodium vivax malaria would be greatly facilitated by the development of an effective vaccine. A comprehensive and systematic characterization of antibodies to P. vivax antigens in exposed populations is useful in guiding rational vaccine design. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated antibodies to a large library of P. vivax entire ectodomain merozoite proteins in 2 Asia-Pacific populations, analysing the relationship of antibody levels with markers of current and cumulative malaria exposure, and socioeconomic and clinical indicators. 29 antigenic targets of natural immunity were identified. Of these, 12 highly-immunogenic proteins were strongly associated with age and thus cumulative lifetime exposure in Solomon Islanders (P<0.001-0.027). A subset of 6 proteins, selected on the basis of immunogenicity and expression levels, were used to examine antibody levels in plasma samples from a population of young Papua New Guinean children with well-characterized individual differences in exposure. This analysis identified a strong association between reduced risk of clinical disease and antibody levels to P12, P41, and a novel hypothetical protein that has not previously been studied, PVX_081550 (IRR 0.46-0.74; P<0.001-0.041). CONCLUSION/SIGNIFICANCE: These data emphasize the benefits of an unbiased screening approach in identifying novel vaccine candidate antigens. Functional studies are now required to establish whether PVX_081550 is a key component of the naturally-acquired protective immune response, a biomarker of immune status, or both.

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

BACKGROUND: A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. METHODOLOGY/PRINCIPAL FINDINGS: We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance. CONCLUSIONS/SIGNIFICANCE: We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00663546.