Shigeki Chiba

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

The mechanisms by which tumor microenvironments modulate nucleic acid–mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

Tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells

Recent evidence has unveiled the critical role of tumor cells with stem cell activities in tumorigenicity and drug resistance, but how tumor microenvironments regulate cancer stem/initiating cells (CSCs) remains unknown. We clarified the role of tumor-associated macrophages (TAMs) and their downstream factor milk-fat globule-epidermal growth factor-VIII (MFG-E8) in the regulation of CSC activities. Bone marrow chimeric systems and adoptive cell transfers elucidated the importance of MFG-E8 from TAMs in conferring to CSCs with the ability to promote tumorigenicity and anticancer drug resistance. MFG-E8 mainly activates signal transducer and activator of transcription-3 (Stat3) and Sonic Hedgehog pathways in CSCs and further amplifies their anticancer drug resistance in cooperation with IL-6. Thus, the pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.

Transcriptional repression by sumoylation of Epstein-Barr virus BZLF1 protein correlates with association of histone deacetylase

The transition from latent to lytic phases of the Epstein-Barr virus life cycle is triggered by expression of a viral transactivator, BZLF1, that then induces expression of the viral immediate-early and early genes. The BZLF1 protein is post-translationally modified by a small ubiquitin-related modifier-1 (SUMO-1). Here we found that BZLF1 is conjugated at lysine 12 not only by SUMO-1 but also by SUMO-2 and 3. The K12R mutant of BZLF1, which no longer becomes sumoylated, exhibits stronger transactivation than the wild-type BZLF1 in a reporter assay system as well as in the context of virus genome with nucleosomal structures. Furthermore, exogenous supply of a SUMO-specific protease, SENP, caused de-sumoylation of BZLF1 and enhanced BZLF1-mediated transactivation. Immunoprecipitation experiments proved that histone deacetylase 3 preferentially associated with the sumoylated form of BZLF1. Levels of the sumoylated BZLF1 increased as lytic replication progressed. Based on these observations, we conclude that sumoylation of BZLF1 regulates its transcriptional activity through histone modification during Epstein-Barr virus productive replication.

Transient increases in p53-responsible gene expression at early stages of Epstein-Barr virus productive replication

Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21WAF1/CIP1 in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.

CLIPR-59 regulates TNF-α-induced apoptosis by controlling ubiquitination of RIP1.

Tumor necrosis factor-α (TNF-α) has important roles in several immunological events by regulating apoptosis and transcriptional activation of cytokine genes. Intracellular signaling mediated by TNF-receptor-type 1 (TNFR1) is constituted by two sequential protein complexes: Complex-I containing the receptor and Complex-II-containing Caspase-8. Protein modifications, particularly ubiquitination, are associated with the regulation of the formation of these complexes. However, the underlying mechanisms remain poorly defined. Here, we identified CLIP-170-related 59 kDa protein (CLIPR-59) as a novel adaptor protein for TNFR1. Experimental reduction of CLIPR-59 levels prevented induction of apoptosis and activation of caspases in the context of TNF-α signaling. CLIPR-59 binds TNFR1 but dissociates in response to TNF-α stimulation. However, CLIPR-59 is also involved in and needed for the formation of Complex-II. Moreover, CLIPR-59 regulates TNF-α-induced ubiquitination of receptor-interacting protein 1 (RIP1) by its association with CYLD, a de-ubiquitinating enzyme. These findings suggest that CLIPR-59 modulates ubiquitination of RIP1, resulting in the formation of Complex-II and thus promoting Caspase-8 activation to induce apoptosis by TNF-α.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix–loop–helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL–AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL–AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL–AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL–AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL–AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Background Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5′ mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein. Methodology/Principal Findings To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth. Conclusions/Significance While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.

MFG-E8 regulates the immunogenic potential of dendritic cells primed with necrotic cell-mediated inflammatory signals.

Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity.