Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune ...system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as “find-me” signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.
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► Fas-induced apoptosis is associated with secretion of cytokines and chemokines ► Fas-driven MCP-1 and IL-8 were chemotactic for monocytes and neutrophils, respectively ► IL-8 and MCP-1 can act as “find-me” signals for apoptotic cells ► Apoptotic cells can actively communicate with cells of the immune system
Necroptosis (programmed necrosis) occurs in response to TNF, Fas, or TRAIL, as well as certain TLR ligands, when caspase activity required for apoptosis is blocked. Necroptosis is typically ...considered a highly pro-inflammatory mode of cell death, due to release of intracellular “danger signals” that promote inflammation. However, because most pro-necroptotic stimuli are intrinsically highly pro-inflammatory—due to their ability to initiate the synthesis of numerous cytokines and chemokines—the inflammatory consequences of necroptosis are complex. Here, we suggest that necroptosis might have anti-inflammatory effects in certain settings, through curbing excessive TNF- or TLR-induced inflammatory cytokine production.
Necroptosis (programmed necrosis) is typically considered a highly pro-inflammatory mode of cell death. However, because most pro-necroptotic stimuli are intrinsically pro-inflammatory, the immunological consequences of necroptosis are complex. Here, Kearney and Martin argue that necroptosis might be anti-inflammatory in certain settings, through abrupt termination of TNF- or TLR-induced cytokine production.
The NLRP3 inflammasome is involved in caspase-1-dependent maturation of IL-1β in many contexts. A two-signal model has emerged for IL-1β maturation, with LPS providing “signal I” and diverse agents ...such as ATP, Nigericin, streptolysin O, uric acid crystals, and alum salts capable of acting as “signal II.” In the absence of signal II, pro-IL-1β is upregulated but typically fails to be processed or released. What unites signal II stimuli has been debated, with the ability to promote K+ efflux suggested as a common factor, but the mechanism of IL-1β release remains unclear. Here, we show that all examined inflammasome signal II agents triggered necrosis, which was highly correlated with their ability to promote IL-1β release. IL-1β secretion occurred in tandem with the release of many additional proteins and was confined to necrotic cells. Thus, signal II agents initiate inflammation by promoting necrosis-driven IL-1β release, suggesting that IL-1β represents an inducible danger signal.
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•Diverse NLRP3 activators promote necrosis•IL-1β release is confined to necrotic cells•“Signal II” represents a necrotic trigger•IL-1β functions as a danger signal
A two-signal model has emerged for NLRP3 inflammasome-dependent IL-1β maturation, but the mechanism of IL-1β release remains unclear. Here, Cullen et al. show that all inflammasome “signal II” agents examined triggered necrosis, with IL-1β secretion confined to necrotic cells, suggesting that IL-1β represents an inducible danger signal.
Microbial infection and tissue injury are well established as the two major drivers of inflammation. However, although it is widely accepted that necrotic cell death can trigger or potentiate ...inflammation, precisely how this is achieved still remains relatively obscure. Certain molecules, which have been dubbed ‘damage-associated molecular patterns’ (DAMPs) or alarmins, are thought to promote inflammation upon release from necrotic cells. However, the precise nature and relative potency of DAMPs, compared to conventional pro-inflammatory cytokines or pathogen-associated molecular patterns (PAMPs), remains unclear. How different modes of cell death impact on the immune system also requires further clarification. Apoptosis has long been regarded as a non-inflammatory or even anti-inflammatory mode of cell death, but recent studies suggest that this is not always the case. Necroptosis is a programmed form of necrosis that is engaged under certain conditions when caspase activation is blocked. Necroptosis is also regarded as a highly pro-inflammatory mode of cell death but there has been little explicit examination of this issue. Here we discuss the inflammatory implications of necrosis, necroptosis and apoptosis and some of the unresolved questions concerning how dead cells influence inflammatory responses.
Tumor necrosis factor (TNF) is a proinflammatory cytokine that is produced and secreted by cytotoxic lymphocytes upon tumor target recognition. Depending on the context, TNF can mediate either ...pro-survival or pro-death signals. The potential cytotoxicity of T cell-produced TNF, particularly in the context of T cell-directed immunotherapies, has been largely overlooked. However, a spate of recent studies investigating tumor immune evasion through the application of CRISPR-based gene-editing screens have highlighted TNF-mediated killing as an important component of the mammalian T cell antitumor repertoire. In the context of the current understanding of the role of TNF in antitumor immunity, we discuss these studies and touch on their therapeutic implications. Collectively, we provide an enticing prospect to augment immunotherapy responses through TNF cytotoxicity.
Cancer immunotherapy aims to enhance antitumor T cell cytotoxicity. Inflammatory cytokines are a part of the cytotoxic T cell arsenal; however, their cytotoxic activity is largely overlooked in the context of immunotherapy.TNF is one such inflammatory cytokine that can signal either pro-survival or pro-death signals depending on the context.Interest in TNF in the immunotherapy field has been reinvigorated by a recent collection of loss-of-function genetic screens in tumor cells, and these have highlighted multiple components of the TNF signaling pathway that can be protective against CD8+ T cell cytotoxicity.Pharmacological targeting of several proteins identified in these preliminary screens might provide a means to reroute tumor TNF signaling towards pro-death, representing a new combinatorial strategy to exploit T cell-derived TNF and boost responses to immunotherapy.
Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I ...molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape.
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•CRISPR screening allows discovery of tumor genes that limit NK cell effector function•Disruption of IFN-γ signaling and/or MHC-I triggers sensitivity to NK cells•Evasion of T cell immunity promotes tumor vulnerability to NK cells•NK cell-targeted immunotherapies may antagonize tumor immune evasion from T cells
Freeman et al. use a series of genome-wide loss-of-function genetic screens to identify genes that limit tumor sensitivity to killing by natural killer cells. The findings highlight that natural killer cells can suppress tumor immune evasion from T cells, identifying a potential strategy to overcome resistance to checkpoint blockade therapy.
Smac-mimetics are emerging as promising anti-cancer agents and are being evaluated in clinical trials for a variety of malignancies. Smac-mimetics can induce TNF production from a subset of tumor ...cells and simultaneously sensitize them to TNF-induced apoptosis. However, TNF derived from other cellular sources, such as cytotoxic lymphocytes (CLs) within the tumor, may also contribute to the anti-tumor activity of SMs. Here, we show that CD8
T cells and NK cells potently kill tumor cells in the presence of the SM, birinapant. Enhanced CL killing occurred through TNF secretion upon tumor antigen recognition or NK-activating receptor ligation. Importantly, the perforin/granzyme route to CL-mediated tumor cell killing was dispensable for the efficacy of birinapant, emphasizing the importance of the TNF-mediated apoptosis pathway. Time-lapse microscopy revealed that birinapant sensitized tumor cells to apoptosis as bystanders and to membrane-bound TNF delivered to tumor cells within the immunological synapse. Furthermore, PD-L1 expression on tumor cells suppressed antigen-driven TNF production by CD8
T cells, which could be antagonized through PD-1 blockade. Importantly, the elevated levels of TNF produced upon PD-1 blockade further enhanced tumor cell killing when combined with birinapant. The combined anti-tumor activity of IAP antagonism and PD-1 blockade occurred independently of perforin-mediated tumor cell death. Taken together, we identify CL-derived TNF as a potent effector of birinapant mediated anti-tumor immunity and opportunity for combination therapy through co-inhibition of immune checkpoints.
Despite the clinical success of cancer immunotherapies including immune checkpoint blockade and adoptive cellular therapies across a variety of cancer types, many patients do not respond or ...ultimately relapse; however, the molecular underpinnings of this are not fully understood. Thus, a system‐level understating of the routes to tumor immune evasion is required to inform the design of the next generation of immunotherapy approaches. CRISPR screening approaches have proved extremely powerful in identifying genes that promote tumor immune evasion or sensitize tumor cells to destruction by the immune system. These large‐scale efforts have brought to light decades worth of fundamental immunology and have uncovered the key immune‐evasion pathways subverted in cancers in an acquired manner in patients receiving immune‐modulatory therapies. The comprehensive discovery of the main pathways involved in immune evasion has spurred the development and application of novel immune therapies to target this process. Although successful, conventional CRISPR screening approaches are hampered by a number of limitations, which obfuscate a complete understanding of the precise molecular regulation of immune evasion in cancer. Here, we provide a perspective on screening approaches to interrogate tumor‐lymphocyte interactions and their limitations, and discuss further development of technologies to improve such approaches and discovery capability.
Despite the clinical success of cancer immunotherapies including immune checkpoint blockade and adoptive cellular therapies, many patients do not respond or ultimately relapse. We discuss the use of CRISPR/Cas‐based approaches to identify mechanisms of tumor immune evasion and avenues to sensitize cancers to destruction by the immune system. We provide a perspective on screening approaches and review the further development of technologies to improve such approaches and discovery capability.
Tumour necrosis factor and lipopolysaccharide can promote a regulated form of necrosis, called necroptosis, upon inhibition of caspase activity in cells expressing receptor‐interacting ...serine/threonine kinase (RIPK)3. Because inhibitors of RIPK1 kinase activity such as necrostatin‐1 block necroptosis in many settings, RIPK1 is thought to be required for activation of RIPK3, leading to necroptosis. However, here we show that, although necrostatin potently inhibited tumour necrosis factor‐induced, lipopolysaccharide‐induced and polyIC‐induced necroptosis, RIPK1 knockdown unexpectedly potentiated this process. In contrast, RIPK3 knockdown potently suppressed necroptosis under the same conditions. Significantly, necrostatin failed to block necroptosis in the absence of RIPK1, indicating that its ability to suppress necroptosis was indeed RIPK1‐dependent. These data argue that RIPK1 is dispensable for necroptosis and can act as an inhibitor of this process. Our observations also suggest that necrostatin enhances the inhibitory effects of RIPK1 on necroptosis, as opposed to blocking its participation in this process.
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following ...transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
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•Loss of the INTS6 subunit of Integrator confers resistance to CDK9 inhibition•INTS6 recruits PP2A to chromatin and forms a submodule of Integrator (Int-PP2A)•Int-PP2A opposes CDK9 at the phosphorylation level to fine-tune transcription•PP2A activators synergize therapeutically with CDK9 inhibitors in cancer
Interplay between PP2A and CDK9 provides a control point for gene expression that can be exploited to inhibit tumorigenesis.
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