In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Author Correction: Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

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

Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8+ tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8+ TILs include a subpopulation of 'progenitor exhausted' cells that retain polyfunctionality, persist long term and differentiate into 'terminally exhausted' TILs. Consequently, progenitor exhausted CD8+ TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8+ T cells might be an important component of improving the response to checkpoint blockade.

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

WHIMP links the actin nucleation machinery to Src-family kinase signaling during protrusion and motility.

Cell motility is governed by cooperation between the Arp2/3 complex and nucleation-promoting factors from the Wiskott-Aldrich Syndrome Protein (WASP) family, which together assemble actin filament networks to drive membrane protrusion. Here we identify WHIMP (WAVE Homology In Membrane Protrusions) as a new member of the WASP family. The Whimp gene is encoded on the X chromosome of a subset of mammals, including mice. Murine WHIMP promotes Arp2/3-dependent actin assembly, but is less potent than other nucleation factors. Nevertheless, WHIMP-mediated Arp2/3 activation enhances both plasma membrane ruffling and wound healing migration, whereas WHIMP depletion impairs protrusion and slows motility. WHIMP expression also increases Src-family kinase activity, and WHIMP-induced ruffles contain the additional nucleation-promoting factors WAVE1, WAVE2, and N-WASP, but not JMY or WASH. Perturbing the function of Src-family kinases, WAVE proteins, or Arp2/3 complex inhibits WHIMP-driven ruffling. These results suggest that WHIMP-associated actin assembly plays a direct role in membrane protrusion, but also results in feedback control of tyrosine kinase signaling to modulate the activation of multiple WASP-family members.

Vinculin is required to maintain glomerular barrier integrity.

Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin filaments to integrin-mediated cell-matrix adhesions and to cadherin-based intercellular junctions. Here, we examined the role of vinculin in podocytes by the generation of a podocyte-specific knockout mouse. Mice lacking podocyte vinculin had increased albuminuria and foot process effacement following injury in vivo. Analysis of primary podocytes isolated from the mutant mice revealed defects in cell protrusions, altered focal adhesion size and signaling, as well as impaired cell migration. Furthermore, we found a marked mislocalization of the intercellular junction protein zonula occludens-1. In kidney sections from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy, we observed dramatic differences in the expression levels and localization of vinculin. Thus, our results suggest that vinculin is necessary to maintain the integrity of the glomerular filtration barrier by modulating podocyte foot processes and stabilizing intercellular junctions.

Reconceptualizing the hyporheic zone for nonperennial rivers and streams.

Nonperennial streams dominate global river networks and are increasing in occurrence across space and time. When surface flow ceases or the surface water dries, flow or moisture can be retained in the subsurface sediments of the hyporheic zone, supporting aquatic communities and ecosystem processes. However, hydrological and ecological definitions of the hyporheic zone have been developed in perennial rivers and emphasize the mixing of water and organisms, respectively, from both the surface stream and groundwater. The adaptation of such definitions to include both humid and dry unsaturated conditions could promote characterization of how hydrological and biogeochemical variability shape ecological communities within nonperennial hyporheic zones, advancing our understanding of both ecosystem structure and function in these habitats. To conceptualize hyporheic zones for nonperennial streams, we review how water sources and surface and subsurface structure influence hydrological and physicochemical conditions. We consider the extent of this zone and how biogeochemistry and ecology might vary with surface states. We then link these components to the composition of nonperennial stream communities. Next, we examine literature to identify priorities for hydrological and ecological research exploring nonperennial hyporheic zones. Lastly, by integrating hydrology, biogeochemistry, and ecology, we recommend a multidisciplinary conceptualization of the nonperennial hyporheic zone as the porous subsurface streambed sediments that shift between lotic, lentic, humid, and dry conditions in space and time to support aquatic-terrestrial biodiversity. As river drying increases in extent because of global change, we call for holistic, interdisciplinary research across the terrestrial and aquatic sciences to apply this conceptualization to characterize hyporheic zone structure and function across the full spectrum of hydrological states.

Assessing placement bias of the global river gauge network.

Knowing where and when rivers flow is paramount to managing freshwater ecosystems. Yet stream gauging stations are distributed sparsely across rivers globally and may not capture the diversity of fluvial network properties and anthropogenic influences. Here we evaluate the placement bias of a global stream gauge dataset on its representation of socioecological, hydrologic, climatic and physiographic diversity of rivers. We find that gauges are located disproportionally in large, perennial rivers draining more human-occupied watersheds. Gauges are sparsely distributed in protected areas and rivers characterized by non-perennial flow regimes, both of which are critical to freshwater conservation and water security concerns. Disparities between the geography of the global gauging network and the broad diversity of streams and rivers weakens our ability to understand critical hydrologic processes and make informed water-management and policy decisions. Our findings underscore the need to address current gauge placement biases by investing in and prioritizing the installation of new gauging stations, embracing alternative water-monitoring strategies, advancing innovation in hydrologic modelling, and increasing accessibility of local and regional gauging data to support human responses to water challenges, both today and in the future.

A Classification Framework to Assess Ecological, Biogeochemical, and Hydrologic Synchrony and Asynchrony.

Ecosystems in the Anthropocene face pressures from multiple, interacting forms of environmental change. These pressures, resulting from land use change, altered hydrologic regimes, and climate change, will likely change the synchrony of ecosystem processes as distinct components of ecosystems are impacted in different ways. However, discipline-specific definitions and ad hoc methods for identifying synchrony and asynchrony have limited broader synthesis of this concept among studies and across disciplines. Drawing on concepts from ecology, hydrology, geomorphology, and biogeochemistry, we offer a unifying definition of synchrony for ecosystem science and propose a classification framework for synchrony and asynchrony of ecosystem processes. This framework classifies the relationships among ecosystem processes according to five key aspects: 1) the focal variables or relationships representative of the ecosystem processes of interest, 2) the spatial and temporal domain of interest, 3) the structural attributes of drivers and focal processes, 4) consistency in the relationships over time, and 5) the degree of causality among focal processes. Using this classification framework, we identify and differentiate types of synchrony and asynchrony, thereby providing the basis for comparing among studies and across disciplines. We apply this classification framework to existing studies in the ecological, hydrologic, geomorphic, and biogeochemical literature, and discuss potential analytical tools that can be used to quantify synchronous and asynchronous processes. Furthermore, we seek to promote understanding of how different types of synchrony or asynchrony may shift in response to ongoing environmental change by providing a universal definition and explicit types and drivers with this framework.

Zero or not? Causes and consequences of zero-flow stream gage readings.

Streamflow observations can be used to understand, predict, and contextualize hydrologic, ecological, and biogeochemical processes and conditions in streams. Stream gages are point measurements along rivers where streamflow is measured, and are often used to infer upstream watershed-scale processes. When stream gages read zero, this may indicate that the stream has fully dried; however, zero-flow readings can also be caused by a wide range of other factors. Our ability to identify whether or not a zero-flow gage reading indicates a dry fluvial system has far reaching environmental implications. Incorrect identification and interpretation by the data user can lead to hydrologic, ecological, and/or biogeochemical predictions from models and analyses. Here, we describe several causes of zero-flow gage readings: frozen surface water, flow reversals, instrument error, and natural or human-driven upstream source losses or bypass flow. For these examples, we discuss the implications of zero-flow interpretations. We also highlight additional methodss for determining flow presence, including direct observations, statistical methods, and hydrologic models, which can be applied to interpret causes of zero-flow gage readings and implications for reach- and watershed-scale dynamics. Such efforts are necessary to improve our ability to understand and predict surface flow activation, cessation, and connectivity across river networks. Developing this integrated understanding of the wide range of possible meanings of zero-flows will only attain greater importance in a more variable and changing hydrologic climate.

River ecosystem conceptual models and non-perennial rivers: A critical review.

Conceptual models underpin river ecosystem research. However, current models focus on continuously flowing rivers and few explicitly address characteristics such as flow cessation and drying. The applicability of existing conceptual models to nonperennial rivers that cease to flow (intermittent rivers and ephemeral streams, IRES) has not been evaluated. We reviewed 18 models, finding that they collectively describe main drivers of biogeochemical and ecological patterns and processes longitudinally (upstream-downstream), laterally (channel-riparian-floodplain), vertically (surface water-groundwater), and temporally across local and landscape scales. However, perennial rivers are longitudinally continuous while IRES are longitudinally discontinuous. Whereas perennial rivers have bidirectional lateral connections between aquatic and terrestrial ecosystems, in IRES, this connection is unidirectional for much of the time, from terrestrial-to-aquatic only. Vertical connectivity between surface and subsurface water occurs bidirectionally and is temporally consistent in perennial rivers. However, in IRES, this exchange is temporally variable, and can become unidirectional during drying or rewetting phases. Finally, drying adds another dimension of flow variation to be considered across temporal and spatial scales in IRES, much as flooding is considered as a temporally and spatially dynamic process in perennial rivers. Here, we focus on ways in which existing models could be modified to accommodate drying as a fundamental process that can alter these patterns and processes across spatial and temporal dimensions in streams. This perspective is needed to support river science and management in our era of rapid global change, including increasing duration, frequency, and occurrence of drying.

What's in a Name? Patterns, Trends, and Suggestions for Defining Non-Perennial Rivers and Streams.

Rivers that cease to flow are globally prevalent. Although many epithets have been used for these rivers, a consensus on terminology has not yet been reached. Doing so would facilitate a marked increase in interdisciplinary interest as well as critical need for clear regulations. Here we reviewed literature from Web of Science database searches of 12 epithets to learn (Objective 1-O1) if epithet topics are consistent across Web of Science categories using latent Dirichlet allocation topic modeling. We also analyzed publication rates and topics over time to (O2) assess changes in epithet use. We compiled literature definitions to (O3) identify how epithets have been delineated and, lastly, suggest universal terms and definitions. We found a lack of consensus in epithet use between and among various fields. We also found that epithet usage has changed over time, as research focus has shifted from description to modeling. We conclude that multiple epithets are redundant. We offer specific definitions for three epithets (non-perennial, intermittent, and ephemeral) to guide consensus on epithet use. Limiting the number of epithets used in non-perennial river research can facilitate more effective communication among research fields and provide clear guidelines for writing regulatory documents.

A Comparison of Hyporheic Transport at a Cross-Vane Structure and Natural Riffle.

While restoring hyporheic flowpaths has been cited as a benefit to stream restoration structures, little documentation exists confirming that constructed restoration structures induce comparable hyporheic exchange to natural stream features. This study compares a stream restoration structure (cross-vane) to a natural feature (riffle) concurrently in the same stream reach using time-lapsed electrical resistivity (ER) tomography. Using this hydrogeophysical approach, we were able to quantify hyporheic extent and transport beneath the cross-vane structure and the riffle. We interpret from the geophysical data that the cross-vane and the natural riffle induced spatially and temporally unique hyporheic extent and transport, and the cross-vane created both spatially larger and temporally longer hyporheic flowpaths than the natural riffle. Tracer from the 4.67-h injection was detected along flowpaths for 4.6 h at the cross-vane and 4.2 h at the riffle. The spatial extent of the hyporheic zone at the cross-vane was 12% larger than that at the riffle. We compare ER results of this study to vertical fluxes calculated from temperature profiles and conclude significant differences in the interpretation of hyporheic transport from these different field techniques. Results of this study demonstrate a high degree of heterogeneity in transport metrics at both the cross-vane and the riffle and differences between the hyporheic flowpath networks at the two different features. Our results suggest that restoration structures may be capable of creating sufficient exchange flux and timescales of transport to achieve the same ecological functions as natural features, but engineering of the physical and biogeochemical environment may be necessary to realize these benefits.