Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model.

Both monocytes and macrophages are heterogeneous populations. It was traditionally understood that Ly6Chi classical (inflammatory) monocytes differentiate into pro-inflammatory Ly6Chi macrophages. Accumulating evidence has suggested that Ly6Chi classical monocytes can also differentiate into Ly6Clo pro-resolving macrophages under certain conditions, while their differentiation trajectory remains to be fully elucidated. The present study with scRNA-seq and flow cytometric analyses reveals that Ly6ChiPD-L2lo classical monocytes recruited to the allergic skin lesion sequentially differentiate into Ly6CloPD-L2hi pro-resolving macrophages, via intermediate Ly6ChiPD-L2hi macrophages but not Ly6Clo non-classical monocytes, in an IL-4 receptor-dependent manner. Along the differentiation, classical monocyte-derived macrophages display anti-inflammatory signatures followed by metabolic rewiring concordant with their ability to phagocytose apoptotic neutrophils and allergens, therefore contributing to the resolution of inflammation. The failure in the generation of these pro-resolving macrophages drives the IL-1α-mediated cycle of inflammation with abscess-like accumulation of necrotic neutrophils. Thus, we clarify the stepwise differentiation trajectory from Ly6Chi classical monocytes toward Ly6Clo pro-resolving macrophages that restrain neutrophilic aggravation of skin allergic inflammation.

A novel in silico scaffold-hopping method for drug repositioning in rare and intractable diseases.

In the field of rare and intractable diseases, new drug development is difficult and drug repositioning (DR) is a key method to improve this situation. In this study, we present a new method for finding DR candidates utilizing virtual screening, which integrates amino acid interaction mapping into scaffold-hopping (AI-AAM). At first, we used a spleen associated tyrosine kinase inhibitor as a reference to evaluate the technique, and succeeded in scaffold-hopping maintaining the pharmacological activity. Then we applied this method to five drugs and obtained 144 compounds with diverse structures. Among these, 31 compounds were known to target the same proteins as their reference compounds and 113 compounds were known to target different proteins. We found that AI-AAM dominantly selected functionally similar compounds; thus, these selected compounds may represent improved alternatives to their reference compounds. Moreover, the latter compounds were presumed to bind to the targets of their references as well. This new "compound-target" information provided DR candidates that could be utilized for future drug development.

Combination of cancer vaccine with CD122-biased IL-2/anti-IL-2 Ab complex shapes the stem-like effector NK and CD8+ T cells against tumor.

BACKGROUND: A key to success of cancer immunotherapy is the amplification and sustenance of various effector cells. The hallmark of prominent antitumor T cells is their long-term effector function. Although interleukin (IL)-2 is an attractive cytokine, several attempts have been made towards developing IL-2 modalities with improved effectiveness and safety that enhance natural killer (NK) cells or T cells in cancer models. However, whether such IL-2 modalities can simultaneously support long-term innate and adaptive immunity, particularly stem-like memory, has not been shown. To resolve this issue, we compared the antitumor cellular mechanism with two IL-2/anti-IL-2 complexes (IL-2Cxs) administered in combination with a therapeutic cancer vaccine, which we had previously established as an in vivo dendritic cell-targeting therapy. METHODS: Two types of IL-2Cxs, CD25-biased IL-2Cx and CD122-biased IL-2Cx, together with a Wilms' tumor 1-expressing vaccine, were evaluated in a leukemic model. The immunological response and synergistic antitumor efficacy of these IL-2Cxs were then evaluated. RESULTS: When CD25-biased or CD122-biased IL-2Cxs in combination with the vaccine were assessed in an advanced-leukemia model, the CD122-biased IL-2Cx combination showed 100% survival, but the CD25-biased IL-2Cx did not. We first showed that invariant natural killer T (NKT) 1 cells are predominantly activated by CD122-biased IL-2Cx. In addition, in-depth analysis of immune responses by CD122-biased IL-2Cx in lymphoid tissues and the tumor microenvironment revealed a dramatic increase in the distinct subsets of NK and CD8+ T cells with stem-like phenotype (CD27+Sca-1hi, CXCR3hi, CD127+TCF-1+T-bet+ Eomes+). Moreover, CD122-biased IL-2Cx combination therapy maintained long-term memory CD8+ T cells capable of potent antitumor protection. After the high dimensional profiling analysis of NK and CD8+T cells, principal component analysis revealed that the stem-like-NK cell and stem-like-CD8+T cell state in the combination were integrated in the same group. CONCLUSIONS: CD122-biased IL-2Cx combined with the vaccine can induce a series of reactions in the immune cascade, including activation of not only NKT1 cells, but also NK and CD8+ T cells with a stem-like memory phenotype. Since it can also lead to a long-term, strong antitumor response, the combination of CD122-biased IL-2Cx with a vaccine may serve as a potential and competent strategy for patients with advanced cancer.

Single cell transcriptomics clarifies the basophil differentiation trajectory and identifies pre-basophils upstream of mature basophils.

Basophils are the rarest granulocytes and are recognized as critical cells for type 2 immune responses. However, their differentiation pathway remains to be fully elucidated. Here, we assess the ontogenetic trajectory of basophils by single-cell RNA sequence analysis. Combined with flow cytometric and functional analyses, we identify c-Kit-CLEC12Ahi pre-basophils located downstream of pre-basophil and mast cell progenitors (pre-BMPs) and upstream of CLEC12Alo mature basophils. The transcriptomic analysis predicts that the pre-basophil population includes previously-defined basophil progenitor (BaP)-like cells in terms of gene expression profile. Pre-basophils are highly proliferative and respond better to non-IgE stimuli but less to antigen plus IgE stimulation than do mature basophils. Although pre-basophils usually remain in the bone marrow, they emerge in helminth-infected tissues, probably through IL-3-mediated inhibition of their retention in the bone marrow. Thus, the present study identifies pre-basophils that bridge the gap between pre-BMPs and mature basophils during basophil ontogeny.

Long-term blast control in high eating quality rice using multilines.

Combining genetic heterogeneity and crop homogeneity serves a dual purpose: disease control and maintaining harvest quality. Multilines, which consist of a genetically uniform mixture of plants, have the potential to suppress disease while maintaining eating quality, yet practical methods that facilitate commercial use over large geographical areas are lacking. Here, we describe effective rice multiline management based on seed mixture composition changes informed by monitoring virulent blast races in Niigata Prefecture, Japan. The most elite nonglutinous cultivar, Koshihikari, was converted into the multiline, Koshihikari BL (blast resistant lines) and planted on 94,000 ha in 2005. The most destructive rice disease, blast, was 79.4% and 81.8% less severe in leaves and panicles, respectively, during the 2005-2019 period compared to the year 2004. In addition, fungicidal application was reduced by two-thirds after the introduction of BL. Our results suggest that seed mixture diversification and rotation of resistant BL provides long-term disease control by avoiding virulent race evolution.

Single-cell transcriptomes underscore genetically distinct tumor characteristics and microenvironment for hereditary kidney cancers.

Our understanding of how each hereditary kidney cancer adapts to its tissue microenvironment is incomplete. Here, we present single-cell transcriptomes of 108,342 cells from patient specimens including from six hereditary kidney cancers. The transcriptomes displayed distinct characteristics of the cell of origin and unique tissue microenvironment for each hereditary kidney cancer. Of note, hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated kidney cancer retained some characteristics of proximal tubules, which were completely lost in lymph node metastases and present as an avascular tumor with suppressed T cells and TREM2-high macrophages, leading to immune tolerance. Birt-Hogg-Dubé (BHD)-associated kidney cancer exhibited transcriptomic intratumor heterogeneity (tITH) with increased characteristics of intercalated cells of the collecting duct and upregulation of FOXI1-driven genes, a critical transcription factor for collecting duct differentiation. These findings facilitate our understanding of how hereditary kidney cancers adapt to their tissue microenvironment.

Compromised anti-tumor-immune features of myeloid cell components in chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) is a form of myeloproliferative neoplasm caused by the oncogenic tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitors have dramatically improved the prognosis of patients with CML, several problems such as resistance and recurrence still exist. Immunological control may contribute to solving these problems, and it is important to understand why CML patients fail to spontaneously develop anti-tumor immunity. Here, we show that differentiation of conventional dendritic cells (cDCs), which are vital for anti-tumor immunity, is restricted from an early stage of hematopoiesis in CML. In addition, we found that monocytes and basophils, which are increased in CML patients, express high levels of PD-L1, an immune checkpoint molecule that inhibits T cell responses. Moreover, RNA-sequencing analysis revealed that basophils express genes related to poor prognosis in CML. Our data suggest that BCR-ABL not only disrupts the "accelerator" (i.e., cDCs) but also applies the "brake" (i.e., monocytes and basophils) of anti-tumor immunity, compromising the defense against CML cells.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Chromatin Protein PC4 Orchestrates B Cell Differentiation by Collaborating with IKAROS and IRF4.

The chromatin protein positive coactivator 4 (PC4) has multiple functions, including chromatin compaction. However, its role in immune cells is largely unknown. We show that PC4 orchestrates chromatin structure and gene expression in mature B cells. B-cell-specific PC4-deficient mice show impaired production of antibody upon antigen stimulation. The PC4 complex purified from B cells contains the transcription factors (TFs) IKAROS and IRF4. IKAROS protein is reduced in PC4-deficient mature B cells, resulting in de-repression of their target genes in part by diminished interactions with gene-silencing components. Upon activation, the amount of IRF4 protein is not increased in PC4-deficient B cells, resulting in reduction of plasma cells. Importantly, IRF4 reciprocally induces PC4 expression via a super-enhancer. PC4 knockdown in human B cell lymphoma and myeloma cells reduces IKAROS protein as an anticancer drug, lenalidomide. Our findings establish PC4 as a chromatin regulator of B cells and a possible therapeutic target adjoining IKAROS in B cell malignancies.

Network-guided analysis of hippocampal proteome identifies novel proteins that colocalize with Aß in a mice model of early-stage Alzheimer's disease.

Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by memory loss and neurotoxic amyloid beta (Aß) plaques accumulation. Numerous pharmacological interventions targeting Aß plaques accumulation have failed to alleviate AD. Also, the pathological alterations in AD start years before the onset of clinical symptoms. To identify proteins at play during the early stage of AD, we conducted proteomic analysis of the hippocampus of young AppNL-F mice model of AD at the preclinical phase of the disease. This was followed by interactome ranking of the proteome into hubs that were further validated in vivo using immunoblot analysis. We also performed double-immunolabeling of these hub proteins and Aß to quantify colocalization. Behavioral analysis revealed no significant difference in memory performance between 8-month-old AppNL-F and control mice. The upregulation and downregulation of several proteins were observed in the AppNL-F mice compared to control. These proteins corresponded to pathways and processes related to Aß clearance, inflammatory-immune response, transport, mitochondrial metabolism, and glial cell proliferation. Interactome analysis revealed several proteins including DLGP5, DDX49, CCDC85A, ADCY6, HEPACAM, HCN3, PPT1 and TNPO1 as essential proteins in the AppNL-F interactome. Validation by immunoblot confirmed the over-expression of these proteins except HCN3 in the early-stage AD mice hippocampus. Immunolabeling revealed a significant increase in ADCY6/Aß and HEPACAM/Aß colocalized puncta in AppNL-F mice compared to WT. These data suggest that these proteins may be involved in the early stage of AD. Our work suggests new targets and biomarkers for AD diagnosis and therapeutic intervention.

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice.

Dendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8- LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8- and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8- LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

Proteome and behavioral alterations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice.

CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3ß (GSK-3ß) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2ki/ki) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2ki/ki mice. The crmp2ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain.

Transcription Factor IRF8 Governs Enhancer Landscape Dynamics in Mononuclear Phagocyte Progenitors.

Monocytes and dendritic cells (DCs), mononuclear phagocytes essential for immune responses, develop from hematopoietic stem cells via monocyte-DC progenitors (MDPs). The molecular basis of their development remains unclear. Because promoter-distal enhancers are key to cell fate decisions, we analyzed enhancer landscapes during mononuclear phagocyte development in vivo. Monocyte- and DC-specific enhancers were gradually established at progenitor stages before the expression of associated genes. Of the transcription factors predicted to bind to these enhancers, IRF8, essential for monocyte and DC development, was found to be required for the establishment of these enhancers, particularly those common to both monocyte and DC lineages. Although Irf8-/- mononuclear phagocyte progenitors, including MDPs, displayed grossly normal gene expression patterns, their enhancer landscapes resembled that of an upstream progenitor population. Our results illustrate the dynamic process by which key transcription factors regulate enhancer formation and, therefore, direct future gene expression to achieve mononuclear phagocyte development.

The intracellular dynamics of hepatitis B virus (HBV) replication with reproduced virion "re-cycling".

Hepatitis B virus (HBV) is a causative agent of hepatitis. Clinical outcome of hepatitis type B depends on the viral titer observed in the peripheral blood of the patient. In the chronic hepatitis patient, production of HBV virion remains low level. On the other hand, the viral load prominently increases in fulminant hepatitis patient as compared with that in the chronic hepatitis patient. We previously proposed a mathematical model describing the intracellular dynamics of HBV replication. Our model clarified that there are two distinguishable replication patterns of HBV named "arrested" and "explosive" replication. In the arrested replication, the amount of virion newly reproduced from an infected cell remains low level, while the amount of virion extremely increases in the explosive replication. Viral load is drastically changed by slight alteration of expression ratio of 3.5kb RNA to 2.4kb mRNA of HBV. Though our model provided the switching mechanism determining the replication pattern of HBV, HBV dynamics is determined by not only the expression pattern of viral genes. In this study, "recycling" of HBV virion in the replication cycle is investigated as a new factor affecting the intracellular dynamics of HBV replication. A part of newly produced virion of HBV is reused as a core particle that is a resource of HBV replication. This recycling of HBV virion lowers the threshold for the explosive replication when waiting time for the next cycle of the replication is large. It is seemingly contradicting that prominent production of HBV is caused by large recycling rate and small release rate of HBV virion from infected cell to extracellular space. But the recycling of HBV virion can contribute to the positive feedback cycle of HBV replication for the explosive replication to accumulate the core particle as a resource of HBV replication in an infected cell. Accumulation of core particle in the infected cell can be risk factor for the exacerbation of hepatitis rather than rapid release of HBV virion from the infected cell.

N-Myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system.

Ubiquitination is the posttranslational modification of a protein by covalent attachment of ubiquitin. Controlled proteolysis via the ubiquitin-proteasome system (\UPS) alleviates cellular stress by clearing misfolded proteins. In budding yeast, UPS within the nucleus degrades the nuclear proteins as well as proteins imported from the cytoplasm. While the predominantly nuclear localization of the yeast proteasome is maintained by the importin-mediated transport, N-myristoylation of the proteasome subunit Rpt2 was indicated to cause dynamic nucleo-cytoplasmic localization of proteasomes. Here, we quantitatively analyzed the ubiquitinated peptides using anti-K-ε-GG antibody in yeast cell lines with or without a mutation in the N-myristoylation site of Rpt2 and detected upregulated ubiquitination of proteins with nucleo-cytoplasmic localizations in the mutant strains. Moreover, both the protein and ubiquitinated peptide levels of two Hsp70 family chaperones involved in the nuclear import of misfolded proteins, Ssa and Sse1, were elevated in the mutant strains, whereas levels of an Hsp70 family chaperone involved in the nuclear export, Ssb, were reduced. Taken together, our results indicate that N-myristoylation of Rpt2 is involved in controlled proteolysis via regulation of the nucleo-cytoplasmic localization of the yeast proteasome.

The transcription factor IRF8 counteracts BCR-ABL to rescue dendritic cell development in chronic myelogenous leukemia.

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8(-/-) mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCR-ABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL-dysregulated genes and predicted that BCR-ABL has immune-stimulatory potential. Indeed, IRF8-rescued BCR-ABL-expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML.

Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation.

Monocytes regulate host defenses, inflammation, and tissue homeostasis. The transcription factor interferon regulatory factor-8 (IRF8) stimulates monocyte/macrophage differentiation, yet genome-wide understanding of the differentiation program initiated by IRF8 is lacking. By combining chromatin immunoprecipitation sequencing with gene expression profiling, we show that during IRF8-dependent monocyte differentiation, IRF8 binding occurs at both promoter-proximal and promotor-distal regions together with the transcription factor PU.1 and is associated with gene induction. Many of the promoter-distal IRF8 binding sites show an increase in histone H3 lysine 4 monomethylation, a signature for enhancers. However, about half the IRF8-induced genes were not bound by IRF8, suggesting the involvement of downstream transcription factors. Analysis of DNA motifs in cis-regulatory elements of these indirect IRF8 target genes predicted that Krüppel-like factor-4 (KLF4)-essential for Ly6C(+) monocyte development-is one such factor. Indeed, monocyte development in Irf8(-/-) mice is as defective as that in Klf4(-/-) chimeric mice. Moreover, Irf8(-/-) monocyte-dendritic cell progenitors do not express Klf4 messenger RNA. Introduction of KLF4 into an Irf8(-/-) myeloid progenitor cell line induced a subset of IRF8 target genes and caused partial monocyte differentiation. Taken together, our present results uncover genome-wide behavior of IRF8 and identify an IRF8-KLF4 axis that operates during monocyte differentiation.

In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis.

BACKGROUND: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. CONCLUSIONS: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.