Immunogenic cell death in cancer therapy.

Depending on the initiating stimulus, cancer cell death can be immunogenic or nonimmunogenic. Immunogenic cell death (ICD) involves changes in the composition of the cell surface as well as the release of soluble mediators, occurring in a defined temporal sequence. Such signals operate on a series of receptors expressed by dendritic cells to stimulate the presentation of tumor antigens to T cells. We postulate that ICD constitutes a prominent pathway for the activation of the immune system against cancer, which in turn determines the long-term success of anticancer therapies. Hence, suboptimal regimens (failing to induce ICD), selective alterations in cancer cells (preventing the emission of immunogenic signals during ICD), or defects in immune effectors (abolishing the perception of ICD by the immune system) can all contribute to therapeutic failure. We surmise that ICD and its subversion by pathogens also play major roles in antiviral immune responses.

Caspase-6 Is a Key Regulator of Innate Immunity, Inflammasome Activation, and Host Defense.

Caspases regulate cell death, immune responses, and homeostasis. Caspase-6 is categorized as an executioner caspase but shows key differences from the other executioners. Overall, little is known about the functions of caspase-6 in biological processes apart from apoptosis. Here, we show that caspase-6 mediates innate immunity and inflammasome activation. Furthermore, we demonstrate that caspase-6 promotes the activation of programmed cell death pathways including pyroptosis, apoptosis, and necroptosis (PANoptosis) and plays an essential role in host defense against influenza A virus (IAV) infection. In addition, caspase-6 promoted the differentiation of alternatively activated macrophages (AAMs). Caspase-6 facilitated the RIP homotypic interaction motif (RHIM)-dependent binding of RIPK3 to ZBP1 via its interaction with RIPK3. Altogether, our findings reveal a vital role for caspase-6 in facilitating ZBP1-mediated inflammasome activation, cell death, and host defense during IAV infection, opening additional avenues for treatment of infectious and autoinflammatory diseases and cancer.

Human ADAR1 Prevents Endogenous RNA from Triggering Translational Shutdown.

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

Auto-aggressive CXCR6+ CD8 T cells cause liver immune pathology in NASH.

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer1,2. The accumulation of metabolites leads to cell stress and inflammation in the liver3, but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6+ CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6+ CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6+ CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

IL-7 signaling must be intermittent, not continuous, during CD8⁺ T cell homeostasis to promote cell survival instead of cell death.

The maintenance of naive CD8(+) T cells is necessary for lifelong immunocompetence but for unknown reasons requires signaling via both interleukin 7 (IL-7) and the T cell antigen receptor (TCR). We now report that naive CD8(+) T cells required IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induced naive CD8(+) T cells to proliferate, produce interferon-γ (IFN-γ) and undergo IFN-γ-triggered cell death. Homeostatic engagement of the TCR interrupted IL-7 signaling and thereby supported the survival and quiescence of CD8(+) T cells. However, CD8(+) T cells with insufficient TCR affinity for self ligands received prolonged IL-7 signaling and died during homeostasis. In this study we identified regulation of the duration of IL-7 signaling by homeostatic engagement of the TCR as the basis for in vivo CD8(+) T cell homeostasis.

Pore formation in regulated cell death.

The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.

Inflammasomes Coordinate Pyroptosis and Natural Killer Cell Cytotoxicity to Clear Infection by a Ubiquitous Environmental Bacterium.

Defective neutrophils in patients with chronic granulomatous disease (CGD) cause susceptibility to extracellular and intracellular infections. Microbes must first be ejected from intracellular niches to expose them to neutrophil attack, so we hypothesized that inflammasomes detect certain CGD pathogens upstream of neutrophil killing. Here, we identified one such ubiquitous environmental bacterium, Chromobacterium violaceum, whose extreme virulence was fully counteracted by the NLRC4 inflammasome. Caspase-1 protected via two parallel pathways that eliminated intracellular replication niches. Pyroptosis was the primary bacterial clearance mechanism in the spleen, but both pyroptosis and interleukin-18 (IL-18)-driven natural killer (NK) cell responses were required for liver defense. NK cells cleared hepatocyte replication niches via perforin-dependent cytotoxicity, whereas interferon-γ was not required. These insights suggested a therapeutic approach: exogenous IL-18 restored perforin-dependent cytotoxicity during infection by the inflammasome-evasive bacterium Listeria monocytogenes. Therefore, inflammasomes can trigger complementary programmed cell death mechanisms, directing sterilizing immunity against intracellular bacterial pathogens.

Host Cell Death and Modulation of Immune Response against Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a prevalent infectious disease affecting populations worldwide. A classic trait of TB pathology is the formation of granulomas, which wall off the pathogen, via the innate and adaptive immune systems. Some key players involved include tumor necrosis factor-alpha (TNF-α), foamy macrophages, type I interferons (IFNs), and reactive oxygen species, which may also show overlap with cell death pathways. Additionally, host cell death is a primary method for combating and controlling Mtb within the body, a process which is influenced by both host and bacterial factors. These cell death modalities have distinct molecular mechanisms and pathways. Programmed cell death (PCD), encompassing apoptosis and autophagy, typically confers a protective response against Mtb by containing the bacteria within dead macrophages, facilitating their phagocytosis by uninfected or neighboring cells, whereas necrotic cell death benefits the pathogen, leading to the release of bacteria extracellularly. Apoptosis is triggered via intrinsic and extrinsic caspase-dependent pathways as well as caspase-independent pathways. Necrosis is induced via various pathways, including necroptosis, pyroptosis, and ferroptosis. Given the pivotal role of host cell death pathways in host defense against Mtb, therapeutic agents targeting cell death signaling have been investigated for TB treatment. This review provides an overview of the diverse mechanisms underlying Mtb-induced host cell death, examining their implications for host immunity. Furthermore, it discusses the potential of targeting host cell death pathways as therapeutic and preventive strategies against Mtb infection.

Regulatory mechanisms of RIPK1 in cell death and inflammation.

Receptor Interacting Protein Kinase 1 (RIPK1) and RIPK3 are key adaptors that play critical roles in inflammatory and cell death signaling. Work in recent years have shown that their activities are tightly regulated by ubiquitination, phosphorylation and proteolysis. In addition to these post-translational modifications, the expression and activities of these kinases can further be tuned by environmental changes in pH and oxygen content. Proper control of these regulatory processes is crucial for the RIP kinases to execute their functions in immune responses and tissue homeostasis. In this review, we discuss recent advance in our understanding of the molecular mechanisms that regulate the activities of the RIP kinases. We will also discuss how the different regulatory mechanisms contribute to the functions of RIPK1 and RIPK3 in different pathophysiological settings.

An immunogenic cell injury module for the single-cell multiplexed activity metabolomics platform to identify promising anti-cancer natural products.

Natural products constitute and significantly impact many current anti-cancer medical interventions. A subset of natural products induces injury processes in malignant cells that recruit and activate host immune cells to produce an adaptive anti-cancer immune response, a process known as immunogenic cell death. However, a challenge in the field is to delineate forms of cell death and injury that best promote durable antitumor immunity. Addressing this with a single-cell chemical biology natural product discovery platform, like multiplex activity metabolomics, would be especially valuable in human leukemia, where cancer cells are heterogeneous and may react differently to the same compounds. Herein, a new ten-color, fluorescent cell barcoding-compatible module measuring six immunogenic cell injury signaling readouts are as follows: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). A proof-of-concept screen was performed to validate functional changes in single cells induced by secondary metabolites with known mechanisms within bacterial extracts. This assay was then applied in multiplexed activity metabolomics to reveal an unexpected mammalian cell injury profile induced by the natural product narbomycin. Finally, the functional consequences of injury pathways on immunogenicity were compared with three canonical assays for immunogenic hallmarks, ATP, HMGB1, and calreticulin, to correlate secondary metabolite-induced cell injury profiles with canonical markers of immunogenic cell death. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulate injury response pathways that can contribute to cancer immunogenicity.

A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Coronaviruses: Innate Immunity, Inflammasome Activation, Inflammatory Cell Death, and Cytokines.

The innate immune system acts as the first line of defense against pathogens, including coronaviruses (CoVs). Severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV are epidemic zoonotic CoVs that emerged at the beginning of the 21st century. The recently emerged virus SARS-CoV-2 is a novel strain of CoV that has caused the coronavirus 2019 (COVID-19) pandemic. Scientific advancements made by studying the SARS-CoV and MERS-CoV outbreaks have provided a foundation for understanding pathogenesis and innate immunity against SARS-CoV-2. In this review, we focus on our present understanding of innate immune responses, inflammasome activation, inflammatory cell death pathways, and cytokine secretion during SARS-CoV, MERS-CoV, and SARS-CoV-2 infection. We also discuss how the pathogenesis of these viruses influences these biological processes.

Clec12a is an inhibitory receptor for uric acid crystals that regulates inflammation in response to cell death.

Recognition of cell death by the innate immune system triggers inflammatory responses. However, how these reactions are regulated is not well understood. Here, we identify the inhibitory C-type lectin receptor Clec12a as a specific receptor for dead cells. Both human and mouse Clec12a could physically sense uric acid crystals (monosodium urate, MSU), which are key danger signals for cell-death-induced immunity. Clec12a inhibited inflammatory responses to MSU in vitro, and Clec12a-deficient mice exhibited hyperinflammatory responses after being challenged with MSU or necrotic cells and after radiation-induced thymocyte killing in vivo. Thus, we identified a negative regulatory MSU receptor that controls noninfectious inflammation in response to cell death that has implications for autoimmunity and inflammatory disease.

Bystander CD4 T-cell death is inhibited by broadly neutralizing anti-HIV antibodies only at levels blocking cell-to-cell viral transmission.

The progressive loss of CD4+ T cells during HIV infection of lymphoid tissues involves both the apoptotic death of activated and productively infected CD4 T cells and the pyroptotic death of large numbers of resting and abortively infected bystander CD4 T cells. HIV spreads both through cellular release of virions and cell-to-cell transmission involving the formation of virological synapses. Cell-to-cell transmission results in high-level transfer of large quantities of virions to the target cell exceeding that achieved with cell-free virions. Broadly neutralizing anti-HIV antibodies (bNAbs) binding to HIV envelope protein capably block cell-free virus spread, and when added at higher concentrations can also interdict cell-to-cell transmission. Exploiting these distinct dose-response differences, we now show that four different bNAbs block the pyroptotic death of bystander cells, but only when added at concentrations sufficient to block cell-to-cell transmission. These findings further support the conclusion that HIV killing of abortively infected bystander CD4 T cells requires cell-to-cell transfer of virions. As bNAbs attract more interest as potential therapeutics, it will be important to consider the higher concentrations of these antibodies required to block the inflammatory death of bystander CD4 T cells.

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs.

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,ß-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.

Control systems and decision making for antibody production.

This paper synthesizes recent progress toward understanding the integrated control systems and fail-safes that guide the quality and quantity of antibody produced by B cells. We focus on four key decisions: (1) the choice between proliferation or death in perifollicular B cells in the first 3 days after antigen encounter; (2) differentiation of proliferating perifollicular B cells into extrafollicular plasma cells or germinal center B cells; (3) positive selection of B cell antigen receptor (BCR) affinity for foreign antigen versus negative selection of BCR affinity for self antigen in germinal center B cells; and (4) survival versus death of antibody-secreting plasma cells. Understanding the engineering of these control systems represents a challenging future step for treating disorders of antibody production in autoimmunity, allergy and immunodeficiency.

Arabidopsis natural variation in insect egg-induced cell death reveals a role for LECTIN RECEPTOR KINASE-I.1.

In Arabidopsis (Arabidopsis thaliana), a hypersensitive-like response (HR-like response) is triggered underneath the eggs of the large white butterfly Pieris brassicae (P. brassicae), and this response is dependent on salicylic acid (SA) accumulation and signaling. Previous reports indicate that the clade I L-type LECTIN RECEPTOR KINASE-I.8 (LecRK-I.8) is involved in early steps of egg recognition. A genome-wide association study was used to better characterize the genetic structure of the HR-like response and discover loci that contribute to this response. We report here the identification of LecRK-I.1, a close homolog of LecRK-I.8, and show that two main haplotypes that explain part of the variation in HR-like response segregate among natural Arabidopsis accessions. Besides, signatures of balancing selection at this locus suggest that it may be ecologically important. Disruption of LecRK-I.1 results in decreased HR-like response and SA signaling, indicating that this protein is important for the observed responses. Furthermore, we provide evidence that LecRK-I.1 functions in the same signaling pathway as LecRK-I.8. Altogether, our results show that the response to eggs of P. brassicae is controlled by multiple LecRKs.